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ECOLOGY AND CONSERVATION OF EUMAEUS ATALA POEY 1832 (LEPIDOPTERA; LYCAENIDAE)

SANDRA E. KOI

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

2013

I dedicate this work to HaShem and all those Light beings who have played a significant role in my life: family and friends, human and other.

ACKNOWLEDGMENTS

I extend my heartfelt appreciation to my major professor, Dr. Jaret Daniels, who provided me with encouragement, materials, equipment and his expert guidance, giving me everything necessary to accomplish a complete and detailed life history and biological monograph of the Atala butterfly. I thank my committee members, Dr.

Jacqueline Miller for sharing her in-depth and accomplished knowledge of tropical lepidoptera and excellent editing of my sometimes hurried and rough writing, and Dr.

Christine Miller for her fresh ideas about monitoring behavior and keen advice in designing my experiments, terminology and scientific ethics. I also thank the leaders in

Lepidoptera biology with whom I have been privileged to work, in conferences and/or in the field: Dr. Thomas Emmel, Dr. Donald Hall, Dr. Robert Michael Pyle, Dr. Marc Minno, and Dr. Dean and Sally Jue.

I thank Dr. George Casella (z”l) for his superb statistical contribution to my experiment design, and Jonathan Colburn for his thoughtful feedback and help with statistical analyses. I thank Dr. Daniel Hahn for his generosity with laboratory materials, helpful practical advice, use of equipment and the expertise of his postdocs, Gian-Carlo

Lopez and Caroline Williams. Dr. James Maruniak is acknowledged for his kind assistance with pathogen identification and Dr. Lyle Buss for his beetle and ant identifications as well as guidance using automontage. Dr. James Nation has been a great source friendship, support and information. Drs. Drion Boucias and Verena Lietze are thanked for help identifying possible pathogens. I thank Dr. Oscar Liburd for the use of his larger balance to weigh plant material.

I am very appreciative of Dr. Deborah Matthews-Lott for guidance regarding preservation protocols for genitalic dissections and Dr. Charlie Covell is deeply thanked

for giving me access to his historical documents and for sharing his many memories of the early days of lepidoptera studies. I thank Jim Schlachta for his assistance in acoustic monitoring at McGuire Center for Lepidoptera and Biodiversity.

I am deeply indebted to Susan Wright and the Biocontrol labs at our USDA/ARS facility for permission to use the environmental chambers to document life stage development of the Atala under controlled conditions. The help and guidance of USDA scientists Dr. Paul Skelley and Lou Soma for teaching me how to use the SEM camera.

Drs. Richard Mankin, Seth McNeill, and Mirian Hay-Roe were very helpful setting up experiments to monitor Atala pupae and larvae for putative stridulation. Dr. James

Hayden has been very helpful and often seemed to be the only other living soul on campus when questions arose on weekends and holidays.

I am grateful to Ga-Eun Lee for months of dedicated help in all aspects of Atala rearing, as well as Matt Thom, Lukasz Barczak, and Marissa Streifel for help rearing the colony, and volunteers Jane Fowler, Bethene Wilkinson, Ryan Huether, Catherine

White, Debby Gluckman, Michelle Gray, Ashleigh Price, Erin Kalinowski and Carlos

Iglesias for temporary volunteer help.

I thank Christine Eliazar, Debbie Hall, Nancy Sanders, Ruth Brumbaugh, and all of the staff in McGuire Center and the Entomology/Nematology Department for their ongoing kind assistance in the myriad of details associated with graduate school!

I thank Bud and Jackie Klein at Duck Lake Coontie Farm in Dade City for a substantial host plant donation and Botanics Wholesale in Homestead, Florida for donating a non-native cycad species for herbivory choice tests.

I thank with love and appreciation the close friends who literally fortified me materially, spiritually, emotionally and intellectually during the years this project has been in process: Dr. Joshua Feingold and Laurie Flebotte, Dr. Sarah Meltzoff, Dr. Robin

Sherman, Ericka Helmick and Dr. Thomas Chouvenc, and Thomas and Lisset Genung.

I thank many associates for their friendship: Dr. Elane Nuehring, Janice Malkoff,

Barbara DeWitt and members of the North American Butterfly Association in Palm

Beach, Broward and Miami-Dade County, as well as biologists and staff in the city, county, state and federal parks in the tri-county area, who have helped collect data over many years. I thank the rabbis and members of Temple Adath Or in Fort Lauderdale,

Temple Beth El in Bradenton, Congregation P’nai Or in Tampa, and Temple Shir

Shalom in Gainesville for their support, friendship and spiritual nourishment as life’s journeys led me from place to place.

I thank my friends and colleagues Marilyn Griffiths, Dr. Tighe Shomer and Susan

Shapiro from Fairchild Tropical Botanic Garden for their massive contributions to as yet unpublished research, as well as Martin Feather, Mary Collins, and Georgia Tasker. I also thank the biologists and staff at Montgomery Botanical Center for sharing their on- going research about cycads native to Central and South America and their specialized herbivores as well as our Caribbean and Floridian species.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 10

LIST OF FIGURES ...... 11

LIST OF OBJECTS ...... 15

LIST OF ABBREVIATIONS ...... 16

ABSTRACT ...... 17

CHAPTER

1 HISTORY AND LITERATURE REVIEW OF EUMAEUS ATALA POEY 1832 (LEPIDOPTERA: LYCAENIDAE: THECLINAE) ...... 19

Taxonomy ...... 19 Description ...... 21 Historical and Current Range and Distribution ...... 23 Conservation Status ...... 42 Larval Host Plant: Zamia integrifolia (Zamiaceae: Cycadales) ...... 44 Conclusions ...... 49

2 NEW AND REVISED LIFE HISTORY OF THE HAIRSTREAK EUMAEUS ATALA (LEPIDOPTERA: LYCAENIDAE) WITH NOTES ON CURRENT CONSERVATION STATUS ...... 65

Introduction ...... 65 Materials and Methods...... 68 Biological Rearing: Livestock and Progeny ...... 68 Immature Measurements ...... 72 Adult Biology and Reproductive Behavior ...... 73 Results ...... 74 Oogenesis, Oocyte Development, Ovipositing and Ova Production ...... 74 Larval Development ...... 75 Pupal Development ...... 80 Total Development Time ...... 81 Adult Measurements and Longevity ...... 81 Adult Dissections and Ova Development ...... 84 Lifespan and Sex Ratio ...... 85 Diseases, Pathogens and Abnormalities ...... 86 Discussion ...... 86 Conservation Notes ...... 89

3 ETHOLOGY (BEHAVIORAL STUDY) OF EUMAEUS ATALA (LEPIDOPTERA: LYCAENIDAE) ...... 115

Insect Behavior ...... 116 Materials and Methods...... 117 Atala Adults ...... 118 Learned behaviors ...... 118 Perchers and patrollers ...... 119 Lekking, hilltopping and canopy dwelling ...... 121 Group feeding ...... 123 Adult host plant recognition: Visual recognition...... 125 Adult host plant recognition: Tactile recognition ...... 127 Adult host plant recognition: Chemical recognition ...... 127 Female ovipositing behavior ...... 129 Mating behavior ...... 132 Male mating behavior ...... 133 Female solicitation and behavior ...... 135 Distinctive mating behaviors ...... 136 Atala adult death and dying behavior ...... 141 Adult defensive warning behavior ...... 143 Larval Behavior ...... 144 Neonate larval behavior ...... 144 Second to final larval instar and pre-pupal behavior ...... 147 Larval host plant recognition and consumption behavior ...... 147 Larval dispersal and pre-pupation behavior ...... 148 Larval cannibalism ...... 149 Larval death ...... 151 Pupal Communications...... 152 Conclusion ...... 153

4 PLASTICITY IN LIFE-HISTORY TRAITS OF EUMAEUS ATALA POEY (LEPIDOPTERA: LYCAENIDAE) ...... 184

Introduction ...... 184 Materials and Methods...... 189 Results ...... 191 Discussion ...... 196 Conclusion ...... 198

5 IPM AND ATALA BUTTERFLY HOST PLANT CHOICE ...... 213

Introduction ...... 213 Beneficial Insect Associations with Cycads ...... 216 Chemecology of Cycads and Insect Associates ...... 217 Integrated Pest Management ...... 220 Host Plant Choice Tests with the Atala Butterfly ...... 222 Materials and Methods...... 224

Adult Choice Test I ...... 224 First Generation Adult Choice Test I ...... 225 First Generation Adult Choice Test II ...... 225 Larval Choice Test I ...... 225 Larval Choice Test II ...... 227 Results ...... 228 Adult Choice Test I ...... 228 First Generation Adults from “Adult Choice” Test I ...... 229 Larval Host Plant Choice Test I ...... 229 Larval Host Plant Choice test II ...... 230 First Generation Adults from “Larval Host Plant Choice” II ...... 230 Discussion ...... 230 Conservation Concerns: Encounters between Wildlife and Humans ...... 231 Conclusion: Hand-Management and IPM Practices for Atala Butterflies ...... 235

APPENDIX: CONSUMPTION DATA FOR EUMAEUS ATALA LARVAE IN A CAPTIVE COLONY ...... 253

LIST OF REFERENCES ...... 266

BIOGRAPHICAL SKETCH ...... 295

LIST OF TABLES

Table page

1-1 Species and subspecies of Eumaeus genera and site of first collection ...... 54

1-2 Chart showing some of the incongruent or missing biological data...... 55

2-1 The average age of female ovipositing and mating for males and females ...... 94

2-2 Atala development time from ovipositing to adult emergence...... 94

2-3 The average weight of larvae at first eclosure ...... 94

2-4 Mean weight (g) of Atala pupae by sex and age at weighing...... 95

2-5 Pupal development time is not significantly influenced by sex...... 96

2-6 Brood size did not significantly alter larval survival...... 97

3-1 Brood size does not reflect survival to adulthood in Eumaeus atalas...... 155

4-1 Temperature, photoperiod and relative humidity were programmed ...... 200

4-2 Development times of immature life stages of Eumaeus atala...... 201

4-3 Wing chord length of E. atala female and male butterflies ...... 202

4-4 Development times for life stages in environmental chambers programmed ... 203

4-5 Life table of brood reared in environmental chamber ...... 204

4-6 Life table of brood reared in environmental chamber ...... 204

4-7 Life table of brood reared in environmental chamber ...... 204

4-8 Life table of brood reared in environmental chamber ...... 205

5-1 Development time varied between groups ...... 238

A-1 Number of Atala larvae in broods and mean coontie plant consumption (g) .... 263

A-2 Number of broods containing designated number of larvae ...... 263

LIST OF FIGURES

Figure page

1-1 Males exhibit a “Caribbean blue”-to-“Sea green” dorsal forewing color ...... 56

1-2 Females exhibit a “Royal blue” splash across the dorsal forewing ...... 56

1-3 Individual variation in species-recognized ventral wing patterns are distinct ...... 57

1-4 Wing scales have a heterogeneous appearance ...... 58

1-5 Distribution of Atala by Florida Wildlife Commission...... 59

1-6 The New River in Broward County circa 1880 ...... 60

1-7 The mouth of the Miami River in 1883 ...... 60

1-8 The pine rocklands in Everglades National Park ...... 61

1-9 Hurricane Katrina caused habitat damage to many Atala colony sites ...... 62

1-10 An Echo Moth (Seirarctia echo) on coontie (Zamia integrifolia)...... 63

1-11 European settlers manufactured “coontie flour” in vast quantities ...... 63

1-12 Commercial coontie flour mills ...... 64

1-13 The larvae of the Atala butterfly are capable of severe defoliation ...... 64

2-1 Frequency of Atala ova sizes exhibiting a bi-modal distribution ...... 98

2-2 Atala ova...... 99

2-3 Ovariole and developing larva...... 100

2-4 Automontage photographs of life stages...... 101

2-5 There was a significant difference in size and weight of pupae ...... 104

2-6 Wing chord length does not affect lifespan in individuals ...... 106

2-7 Mean proportion of survival of Atala per life stage ...... 106

2-8 Longevity was not significantly affected by pupal weight in either sex...... 107

2-9 Graph of wing chord length ...... 107

2-10 Development of Atala pupal stages ...... 108

2-11 Dorsal wing colors of Atala...... 110

2-12 Wing scales have a heterogeneous appearance ...... 111

2-13 Atalas of both sexes exhibit a strong startle and warning response ...... 112

2-14 Graph displaying number of ova found per age in females...... 113

2-15 Older individuals display worn and tattered wings ...... 113

2-16 Normal wing chord length was not significantly correlated with sex ...... 114

2-17 Sex ratio of first generation adults originating as wild larval stock...... 114

3-1 Atala butterflies congregated on the feeders ...... 156

3-2 Atala butterflies used artificial feeders...... 156

3-3 Male Atalas are perchers...... 157

3-4 Most adults received alphanumeric codes as identifiers ...... 157

3-5 Atalas rest in the canopy or perch on vegetation...... 158

3-6 Groups of wild adult Atalas congregate on nectar sources ...... 159

3-7 Females do not display aggression to conspecifics ...... 160

3-8 Leaflets of the host plant were heavily encrusted with ova ...... 160

3-9 Atala ova and anal tufts...... 161

3-10 Male Atalas use scent glands to attract females...... 162

3-11 Coral Reef Park in Miami-Dade County...... 163

3-12 An 8-day-old male (T89) forces ova from the reproductive tract ...... 164

3-13 A male Atala died with dried ova attached to his genitals ...... 165

3-14 A 35-day-old male sits on the wings of a 36 day-old female...... 166

3-15 Unusual Atala mating behavior...... 167

3-16 Coercive mating in Atalas...... 168

3-17 A 41-day-old female (P96, number obscured in photo) dies in copulo ...... 169

3-18 An exhausted female retires to the ground at a wild colony site ...... 170

3-19 Adults experienced chill coma...... 171

3-20 Defensive warning behavior in a female Atala ...... 172

3-21 Atala larvae actively avoid consuming the chorions ...... 172

3-22 Imprinting occurs in Atala larvae ...... 173

3-23 Final instar larvae and pre-pupae release silk ...... 174

3-24 Neonate behavior...... 175

3-25 Larval feeding behavior ...... 176

3-26 Larvae will spiral around the rachis of a leaf ...... 177

3-27 Voracious caterpillars devoured the leaves and stems ...... 178

3-28 Stridulation sounds emitted by ten-day-old pupae...... 179

3-29 The “stridulation” plates evident on the pupa ...... 180

3-30 Matrix showing multiple mating between Atala adults...... 181

3-31 Matrix showing multiple mating between Atala adults ...... 182

3-32 Matrices showing multiple mating between Atala adults...... 183

4-1 Percival environmental chambers ...... 205

4-2 Development rates in different environmental chambers ...... 206

4-3 Graphs of life table data showing high mortality in immature stages ...... 207

4-4 Ova development ...... 208

4-5 Larval development ...... 209

4-6 Pupal development ...... 210

4-7 Polyphenism in Atala pupae ...... 211

4-8 Seasonal polyphenism in the dorsal wing color ...... 212

5-1 Zamia encephalartoides is one of twenty non-native cycads ...... 239

5-2 Larval Host Choice I arena ...... 240

5-3 Neonate feeding behavior ...... 241

5-4 Arena for Larval Test II ...... 242

5-5 Graphical representation of test trials ...... 243

5-6 The “Adult Host Choice Test I” ...... 244

5-7 Many of the larvae feeding on non-native Z. vazquezii died ...... 245

5-8 Comparison of six-day old larvae ...... 246

5-9 Proportion of Atala surviving to the next life stage...... 247

5-10 Immature development time and duration ...... 248

5-11 Comparisons of development time per life stage ...... 249

5-12 Adult lifespan in larvae and adult choice trials...... 250

5-13 In Larval Choice Test I ...... 251

5-14 Larval Host Plant Choice Test II ...... 252

A-1 This 10-acre remnant pine rockland natural area in south Miami-Dade ...... 264

A-2 Mean wet-weight plant consumption (g) per Atala butterfly larvae ...... 265

A-3 Typical native coontie, Z. integrifolia...... 265

LIST OF OBJECTS

Object page

3-1 Eumaeus atala behaviors ...... 123

A-1 Zamia integrifolia host plant rubric ...... 262

LIST OF ABBREVIATIONS

BC Broward County

ENP Everglades National Park

FNAI Florida Natural Areas Inventory

FTBG Fairchild Tropical Botanic Garden

FWC Florida Fish and Wildlife Conservation Commission

HTBSP Hugh Taylor Birch State Park

IBWG Imperiled Butterfly Working Group

IPM Integrated Pest Management

IUCN International Union for the Conservation of Nature and Natural Resources

MDC Miami-Dade County

NABA North American Butterfly Association

PBC Palm Beach County

USFWS United States Fish and Wildlife Commission

Zi Zamia integrifolia

Zv Zamia vazquezii

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

ECOLOGY AND CONSERVATION OF EUMAEUS ATALA POEY 1832 (LEPIDOPTERA; LYCAENIDAE)

Sandra E. Koi

December 2013

Chair: Jaret Daniels Major: Entomology and Nematology

Southeast Florida is part of the Caribbean archipelago and a biodiversity hotspot for conservation priorities, with many endangered species precinctive to the Lower

Peninsula. The tropical butterfly Eumaeus atala Poey 1832 (Lepidoptera: Lycaenidae), once considered extinct, has made a significant population increase in southeast Florida during the past thirty years, but the few published papers that mention life history traits contain numerous discrepancies and conjectures. Regardless of the insect’s tentative recovery, it is considered “Imperiled” by the State of Florida because of its unpredictable crash-eruption cycles, isolated colonies located in fragmented and endangered pine rockland habitats, as well as its high vulnerability to stochastic weather events. A lack of knowledge about its biology and life history complicates recovery efforts. Relocation projects have been developed by the author in conjunction with Miami-Dade County authorities and other non-governmental agencies to restore Atala butterfly colonies in historically occupied pine rockland natural areas, as well as in domestic gardens. This captive-rearing intensive was initiated to fill in critical gaps of information relating to its overall biology and ecology, understand potential constraints to conservation and recovery, and ultimately construct best management practices to strengthen successful

reintroductions of the butterfly serving to increase understanding of other pine rockland remnant-reliant butterfly taxa, such as Bartram’s Hairstreak (Strymon acis).

CHAPTER 1 HISTORY AND LITERATURE REVIEW OF EUMAEUS ATALA POEY 1832 (LEPIDOPTERA: LYCAENIDAE: THECLINAE)

Taxonomy

Lycaenids comprise 30-40% of butterfly taxa, containing nearly 6000 species worldwide, and are often associated with specialized environments and/or host plants

(New 1993). The genus Eumaeus Hübner [1819] is located in the Eumaeini tribe in the

Theclinae subfamily of Lycaenid hairstreak butterflies. The Atlas of Neotropical

Lepidoptera (Robbins & Lamas, 2004) describes the genus as containing approximately six species and twelve subspecies, the true taxonomic status of which are still somewhat in question. The genus is geographically distributed in Central America

(Mexico, Belize, Honduras, Panama, Costa Rica, Guatemala), South America (Ecuador,

Peru, Bolivia, Columbia, Brazil); E. atala is found in the Caribbean Archipelago (Cuba, the Bahamas, Cayman Islands, Cayman Brac and southeast Florida) (Smith et al.,

1994; Robbins & Lamas, 2004) (Table 1-1).

The south Florida and Caribbean species Eumaeus atala Poey 1832 is one of the species that may or may not be a true subspecies. First described in 1832 by Felipe

Poey, the butterfly identified as “E. atala” was located throughout the Caribbean. A red- labeled syntype (number 7771) is located in Havana (Warren et al., 2012), bearing the label “Eumesia Atala” (which is likely a misspelling of the former genus name of

“Eumenia”). The location of the original holotype is unknown. In 1926, Johann Röber published a brief comment declaring that in his opinion the south Florida denizens deserved a subspecies status based on “significant” differences he observed when compared to the Cuban form: he held that the Florida specimens were larger with bigger and glossier blue ‘seam spots’ on the hindwings. Röber suggested the subspecies

name of E. atala florida be used in order to differentiate it from the other Eumaeus species in the Caribbean Archipelago. This appellation stuck for many years, until Smith et al. (1994) placed it in synonymy (Warren et al., 2012). A holotype for a butterfly labeled “Eumaeus atala grayi” (W. Comstock & Huntington, 1943) was collected in

Miami-Dade County, Florida, and is located in the American Museum of Natural History

(Warren et al., 2012). Calhoun (1997); Robbins and Lamas (2004) reiterate the opinion of Miller et al. (1994) in their extensive checklists of Florida butterflies and skippers, i.e., that E. atala atala and E. atala florida are the same species.

The Theclinae subfamily has been through many revisions and changes, numerous new species descriptions and deletions. Nicolay (2000) noted that the last published review of the Colombian Theclinae was interspersed with erroneously labeled photographs, accounts of familiar species wearing “new specific names” with a few placed in a new genus. The south Florida species, E. atala, has received and lost its subspecies status several times since Röber first argued for its designation as a subspecies in 1926 (Röber 1926; Hall et al. 2000; Robbins and Lamas 2004c).

Consequently, the insects’ shifting conservation status has reflected this changing taxonomic designation (see below).

The butterfly species in any of the Eumaeus genera are not known to migrate, although Rutkowski (1995) observed Atala butterflies in an apparent ‘directed flight’ along the shoreline of Stafford Creek on the island of North Andros. In southeast

Florida, females especially may disperse in search of host plants, and in isolated fragmented habitats, both sexes may disperse in search in search of mates or nectar resources. It is known that even small lycaenids are capable of surviving wind-

generated dispersal, and may rely on winds to enhance migration (Robbins & Small,

1981). I have seen individual Atala butterflies flying a half mile away from known sites in

Davie, Florida and have documented several apparent self-established sites since

2004. The question underlying the taxonomic status of E. atala in southeast Florida, the

Caribbean and Cuba remains unanswered; looking at a map, it is not unfathomable that

Atala butterflies from the islands may be carried to locations in Florida by trade winds, especially since many of the “re-discoveries” have occurred on barrier islands. There have been three undocumented reports of Atala butterflies on or flying between the barrier islands in Biscayne National Park (National 2010; Koi, unpublished). The butterfly is commonly referred to as “the Atala.”

Description

The Atala is one of the largest hairstreaks in the United States with a wing chord length from 1.4 mm to 2.7 mm (Chapter 2), but averages 2.10 (male) to 2.14 (female) cm wing chord lengths. Wing chord length was measured from the basal sclerite and resilin patch of the forewing attachment to the thorax in a straight line to the apex of the wing. The left forewings of the adults reared in this captive colony were measured immediately after emergence as soon as the wings hardened. There is no significant size dimorphism between the sexes. Measurements of hundreds of specimens collected from the 1920’s to the present, housed in the McGuire Center for Lepidoptera and

Biodiversity at the University of Florida, Gainesville, show the same range in wing chord length for the two sexes (Koi, unpublished).

The wings of the Atala butterfly are a rich deep black with three C-shaped rows of curved aquamarine spots on the ventral hind wing. There is no consistent difference in the configurations of the spots between the sexes, but individuals can be identified by

the distribution patterns of the spots. Males exhibit a heterogeneous dusting of green-to-

Caribbean-blue iridescence on the dorsal surface of the forewings that varies with season and has a heritable component (Figure 1-1). Color often extends from the dorsal fore wing onto the dorsal hind wing in males. Females exhibit a ‘splash’ of royal blue across the top of the dorsal forewing that does not vary in color, but does vary considerably in the extent to which it covers the forewing (Figure 1-2). There is a deep red spot on the anal edge of the hind wing in both sexes; it is not the same color as the orange-red abdomen. Unlike most hairstreaks, Atala butterflies do not have the characteristic “tail” on the hindwings, although other species in the Theclinae subfamily do (Figure 1-3).

The wing scales of the Atala butterflies are heterogeneous, with flat scales interspersed among curved scales, giving the wings a slightly uneven appearance; the color is caused by ultrastructure in the scales themselves (Figure 1-4). Flat scales lie beneath the curved scales above, forming a double layer, forming iridescent light- reflecting patches, which are found not only on the hind wing, but on the thorax, femur, and around the eyes, in varying degrees of density between individuals.

Red-orange scales on the abdomen are flat and do not reflect light, but provide a thick covering saturated with the neurotoxins ingested from the toxic host plant as a larva (see below). Females deposit round anal tuft scales and “spicules” on the eggs as she oviposits, presumably to aid in protection of the ova from predation because the scales contain sequestered toxins ingested from the host plant as a larvae (Rothschild,

1992; Blum & Hilker, 2008).

The highly sculpted domed spherical ovum has a flat bottom where it is adhered to the substrate of the host plant, and is typical of lycaenid eggs; ova measure between

0.75 mm to 1.25 mm in diameter (Chapter 2). The eggs are creamy-white in color, exhibit a three-leaved micropyle, and are clear on the underside, allowing visualization of the embryo if removed from the substrate (Chapter 2).

Neonate larvae are pale green with light yellow spots when first eclosed, but as toxins from the host plant are ingested, larvae develop bright red color and display two rows of bright lemon-yellow spots on the dorsal side (Chapter 2). The bright colors of the adults and larvae indicate aposematic warning coloration (Healy, 1910; Rothschild et al., 1986; Bowers & Laren, 1989; Rothschild, 1992; Bowers, 1993; Nash et al., 1992;

Nishida, 2002).

The pupae maintain the bright yellow spots for the first few days after pupation, but become brown with varying amounts of black speckles as they mature (Chapter 2).

Pupae exhibit seasonal polymorphism, from light brown with small black speckles to darker brown with enlarged black spots that almost merge in places (Chapter 4). Just before eclosure, the pharate adult insect is visible through the pupal case; the wing pads turn black and the abdomen becomes red. Rothschild et al. (1986) described the droplets visible on the exterior of the pupa as a “bitter-tasting” fluid, but did not identify the substance (or reveal who tasted it!) It may be the chemical cycasin which is found throughout the insect’s body and permeates the male’s pheromone (Rothschild et al.

1986).

Historical and Current Range and Distribution

Historically, the butterfly was documented as abundant throughout southeast

Florida. Scudder (1875) described it as occurring in “great abundance.” It was called

the “most conspicuous insect in semitropical Florida” by Schwartz (1888); Schwartz also noted the butterfly as occurring in the Keys. Scudder (1875) documented the butterfly as occurring as far north as Lake Worth, in Palm Beach County, although Schwartz

(1888) said it was no longer there. Scudder (1875) also mentioned that he saw the butterfly on Key Biscayne, but Schwartz (1888) questioned that comment as he located no host plants on the island, possibly due to prior harvesting the host plant. Struttman shows a distribution primarily in south Florida, including parts of the west coast of

Florida and confirmed records of strays in Illinois and Mississippi (Mather & Mather,

1976; Struttman, undated) (Figure 1-5). It is likely that the insects in Mississippi and

Illinois were either brought via plant material or assisted by humans. Recent distribution and range data indicates that the insect has moved further north, even without human intervention, and can be found as far north as Martin County (Koi, unpublished).

Records on the west coast of Florida, which I have been able to track down, have been deployed by humans, however (Koi, unpublished).

The Atlantic Coastal Ridge, which outlined the eastern seaboard of the Florida peninsula from Palm Beach to Homestead (Richardson, 1976; Steinberg, 1982; Snyder et al., 1990) was a highland area dominated by pine rocklands, tropical hardwood hammocks rich with understory plants such as Saw Palmetto (Serenoa repens),

Beautyberry (Callicarpa americana), Melanthera (Melanthera parvifolia) and the Atala’s host plant, Zamia integrifolia (Zamiaceae: Cycadales) (Small, 1926; MacAllister, 1938;

Avery, 1983; Austin, undated). The pine rockland ridge was surrounded by tropical hardwood hammocks and ringed by freshwater rivers, such as the New River in

Broward County (Figure 1-6) and the Miami River in Miami-Dade County (Figure 1-7).

Between the Everglades and the Atlantic Ocean lay the primary original habitat for the butterfly (Figure 1-8). The original dry land was a mere 6-10 miles shore-to-glades, with dunes on the east side and hammocks on the west, ribboned with waterways (Snyder et al., 1990). The water flowed so swiftly from Lake Okeechobee to the rivers and into

Florida Bay, that there were waterfalls in places and the early explorers handled their canoes with difficulty (Willoughby, 1898).

The Atala was still fairly common in the early twentieth century (Healy, 1910;

Grossbeck, 1917). In 1931, Holland still recorded that the Atalas were swarming in

Miami. By 1951, however, Klots stated that it was “probably extinct” as few known collections or records had been made of the insect for years. By 1956, Young wrote that anyone finding the butterfly should publish a note in the Journal of the Lepidopterists’

Society because of its extreme rarity (ten years later, Richard Funk reported collecting an Atala in 1960, see below).

There were Atalas collected occasionally during this time frame, however, but they were in private collections and unknown to the general public. McGuire Center for

Lepidoptera and Biodiversity has a handful of Atalas in its collection from southeast

Florida dating from 1919 to the 1930’s. The butterfly was last collected in Royal Palm

Hammock in Everglades National Park (ENP) located in south Homestead, Miami-Dade

County, Florida, in 1933, and two males were collected in Miami in 1937 (Comstock and

Huntington, 1943). The first record of it in ENP was documented in 1919, in both Royal

Palm and Long Pine Key, and it was reported in every month except May and October

(Lenczewski, 1980). It had not been documented there for many years thereafter (Sue

Perry, pers. communication), until re-introductions were instituted again in the mid-

2000’s by the park biologists, and myself (Koi, unpublished). Kimball (1965) documented two collections made on August 20 and September 5, 1958 (by G. & B.

Klopfer).

As of 2005, McGuire had Atala butterflies in its collection from 1910, 1919, 1922,

1929, 1930, 1933, 1934, 1937, 1940, 1962, 1964, 1980, 1981, 1982, 1983, 1984, and

1986. It is interesting that the time frame in which the Atala was listed as “probably extinct” (Klots, 1951) that there were no Atala butterfly specimens in the McGuire collection. Equally fascinating is that there are records from the 1960’s after Hurricane

Donna, as well as one male collected on June 5, 1960 (Funk, 1966). There are also verified sightings of all life stages in Crandon Park, on Key Biscayne in Miami in 1977

(Jacqueline Miller, pers. comm.). And it was found at Hugh Taylor Birch State Park in

Broward County in 1959, a very interesting story described below.

This is not the only time a species thought to be extirpated or extinct has been found again, lost again and found again. Recently, the Miami Blue (Hemiargus thomasi) was thought to be gone after Hurricane Andrew, but was re-discovered in Bahia Honda

State Park in the Florida Keys in 1999 by Jane Ruffin (Ruffin, 2000). For many possible reasons, including human interference, it disappeared again for quite some time and was re-discovered yet again on Boca Grande in the Marquesas Islands, southwest of

Key West, in 2006 by Paula Cannon (Cannon, 2010). Because none have been seen since 2010 in the Keys, it was emergency-listed by the U.S. Fish and Wildlife shore are continuing to be monitored by the state and federal biologists (Jim Duquesnel, pers. comm.).

Likewise, it is apparent that the Atala was indeed very rare, but not extinct or extirpated as surmised by biologists and lepidopterists of the time. Of course, this does not answer the question of possible immigration from the Bahamas (Rutkowski, 1995).

By the late 1950’s and early 1960’s, lepidopterists were expressing concern about the status of the Atala as well as other species, one of which is now listed as

Federally Endangered (Schaus Swallowtail, Papilio aristodemus ponceanus) (Baggett,

1982; Rawson, 1961). I have been privileged to study the communications documented in letters between Dr. Charles V. Covell, Jr. and Dr. George W. Rawson, two key players in the earliest rescue attempts for what was then considered the nearly extinct

Atala. Dr. Covell was then employed as an entomology professor in the Biology

Department at the University of Louisville, Kentucky and Dr. Rawson was a retired veterinarian, who was volunteering at the Department of Entomology in the National

Museum of Natural History at the Smithsonian Institution in Washington, D.C. during the summer season. Rawson was living in New Smyrna Beach, Florida, during the winters, when the plan to rear and release Atalas took shape. Interspersed between their letters were copies of directives and comments from other biologists acting in unison with them, such as Dr. Bill Robertson, a research biologist at ENP and Dave Baggett, a biologist who worked for the State of Florida, authored material about rare and endangered biota (1982), including the Atala, and was an active member and former chair in the Southern Lepidopterists’ Society.

Covell and Rawson were communicating as early as 1960 about their concern for the Atala, the Schaus Swallowtail, and the disappearing natural areas that supported those species. In a paper published in 1961, Rawson told the amazing story of finding

and rearing the Atalas from a “top-secret” location in Fort Lauderdale in 1959 for re- distribution to Everglades National Park. Due to the fear that unscrupulous collectors would descend on the location and wipe out the colony, he did not disclose the actual location of the find in the paper, first located by the unnamed parents of the recently deceased Charles J. “Jack” Dempwolf, who was visiting south Florida from New Jersey.

(Mr. Dempwolf passed away while I was writing this chapter, on May 15, 2013.) The older Dempwolf had collected specimens of the Atala butterfly on February 28, 1959, and a mutual friend alerted Rawson to the insect, beginning the plan to rear and release

Atalas, deemed “probably extinct” by Klots (1951).

Rawson’s letters to Covell illumine the missing data: we now know that the location of that find was Hugh Taylor Birch State Park (HTBSP), located on a barrier island in North Lauderdale. HTBSP is still a location with ephemeral colonies, with several areas containing host plant, and occasionally appears to be a self-established colony. The location is significant considering the second ‘re-discovery’ of the butterfly that occurred twenty years later on another barrier island, Key Biscayne.

Hugh Taylor Birch had quite a large population of the host plant, Zamia integrifolia, both inside the park area as well as the beach-side. According to Rawson’s story in the letters, he agreed to rear the insects for re-introduction in an area further away from the city, where they could be reared without being exposed to the dangers from human activities, including increasing urban development. Rawson took four females from the park on July 18, 1960, which laid 50 eggs. He succeeded in rearing

35 healthy pupae, which were sent off by mail to Bill Robertson in ENP, with whom he had made prior arrangements to release the adults in Royal Palm Park. In September

1960, Rawson wrote that his plan to save “. . . Eumaeus atala from total extinction seems to be progressing favorably.” Although Robertson was on leave when the pupae arrived, another biologist released the adults as planned, near Royal Palm Hammock.

On September 10, 1960, Hurricane Donna, a powerful storm that still holds the record for sustaining a category three or higher status for nine days straight, hit south

Florida with a vengeance. Rawson published the date as September 11, but the weather archives indicate the storm made landfall on the 10th. The new Atala colony appeared to be doomed. Kimball (1965) mistakenly wrote that “C.J. Dempfer” had found the Atala colony (it was Dempwolf) and that Rawson had published his narrative in 1962

(not 1961). In February 1961, Rawson made another collection visit to HTBSP, saw two

Atalas, but took none. He returned again in May, but again only saw two individuals and did not take any. However, by June 1961, Rawson was back in rearing business with wild stock from HTBSP, and sent 44 pupae to ENP for release in Royal Palm Park.

Sightings of the butterfly continued to be extremely rare, however, at ENP or anywhere else. A colony was found in Port St. Lucie (Rawson, 1961) but apparently died out from a cold front (Hammer, pers. comm.), although colonies are extant in the area now. A male Atala collected in June 1960, collected while nectaring on flowers on a motel property in Key Largo, was reported by the aforementioned Richard Funk in

1966. It is ironic that that the last Atala seen in the Keys was actually found before

Rawson released captive bred stock in September of 1960, which may indicate the butterfly was alive and well hidden somewhere in the Keys or the southern end of ENP, possibly from the first release in early 1960.

Rawson (1961) wrote poignantly of his efforts:

While the explanation of our failure is hypothetical, it is quite evident that further attempts should not be undertaken without careful planning. In particular, potential habitats should first be thoroughly investigated to determine whether ecological conditions are suitable and that the location is safe from human interference or exploitation, or other detrimental factors. Furthermore, it is evident that a project of this kind is not likely to succeed by ‘remote control’ or without careful planning, cooperative support, and organization.

The Atala was rare but not entirely gone: a pair was collected in HTBSP in 1963

(Covell, pers. comm.). Kimball (1965) later confirmed two other locations in Broward

County and determined that HTBSP was indeed the “secret location” that Dempwolf had found earlier. The butterfly was not common, however and concern continued to grow.

In early 1972, Rawson referred to the “now extinct” Atala butterfly in a letter to Bill

Robertson, but Covell rebutted in reply that he felt that “. . . both Ponceanus and Atala are alive and well in isolated spots unknown to the community of lepidopterists.” Shortly thereafter, Robertson asked for more details: Had anyone seen the butterfly anywhere?

What was its status?

The Endangered Species Act was passed in 1973, but the Atala was not listed as scientists believed it to be extinct in Florida and few invertebrates were considered valuable at that time. The first invertebrate to be listed by the United States did not occur until the Schaus Swallowtail was listed as Threatened in April 28, 1976, and later as Endangered in August 31, 1984.

Rawson wrote to Covell in 1974 that he met a young collector who saw a specimen of Atala collected in 1973 from southeast Florida, while lamenting the “steady or gradual decline” in butterfly species and continued destruction of habitat caused by the “housing industry, cattle ranges and mosquito control.” In 1974, Harry Clench

conveyed to Covell that he thought the Caribbean Atala “. . . is the same bug there [that] is in Florida.” In 1977, Weems questioned the taxonomic status in a publication about the Florida endangered insect program, but wrote that the Atala was “recently extirpated.” If the Atala is a subspecies, it may not be particularly suitable for conservation concerns here or in the Bahamas. This question of subspecies has yet to be resolved.

A second re-discovery of the Atala took place in 1979 by a Miami-Dade County

Park naturalist, Roger Hammer, on another barrier island, Virginia Key, just north of Key

Biscayne, in Miami (no one knew about the Atala butterflies observed in 1977 by

Jacqueline and Lee Miller, both accomplished entomologists). Hammer found the red and yellow larvae on its host plant, our only native cycad, called ‘coontie’, but not knowing what the larvae were, he took some home to rear out. When the adults emerged he was astounded to see the “extinct” Atala butterfly!

Understanding the important implications of his find, Hammer took 50 potted coontie plants back to Virginia Key, “planted them” in their pots so they would not be conspicuous and get stolen, and went back three weeks later to gather up the haul of eggs and larvae. Hammer distributed the new starter colonies to various locations throughout Miami-Dade County, including ENP, pine rockland remnant habitats, such as

Navy Wells Pineland, Larry and Penny Thompson Park, and Fairchild Tropical Botanic

Garden in Coral Gables (Hammer, pers. comm.). Hammer also discussed his find behind a smoke screen to throw off potential over-collection, partially by telling people he found the larvae on Key Biscayne. Other conservation-minded people, many of them biologists and/or naturalists, got involved and soon populations of Atala were springing

up throughout southeast Florida, including back into HTBSP and other pinelands and parks in Broward County (Hammer, pers. comm.).

In May 1980, Baggett wrote to Rawson that he felt he had figured out at least one of the problems with why the colonies did not persist in the past, especially at ENP. He started by saying that the current status was excellent, but that the biggest issue seemed to be the difficulty that the females had finding the proper stage of new growth on the host plants on which to oviposit. He determined that in the wild, they only oviposited on fiddleheads or new growth, and that in “. . . virtually every area I have been which supports the plants, they are too mature, old and tough.” He also thought that perhaps the coastal plants had different qualities, chemistry or something, that made them more attractive to the Atalas than the inland plants (Covell, per. comm.).

Kilmer (1993) also wrote that there may have been something about the coastal environment that the Atala preferred, and a need for a particular unidentified microclimate has also been cited as a possible detriment to establishing persistent colonies (Baggett, 1982). It is well known that soil nutrients, particle composition, organic matter, light and water levels, etc., affect the qualities of the plants growing in them; Zamia plants have widely varying levels of chemical nutritional constituents

(Teas, 1967; Bowers, 1993; Rothschild, et al. 1986; Bowers et al., 1989; Nash et al.,

1992; Rothschild, 1992; Oberprieler, 1995a, 1995b, 1995c; Valdez & Hernández, 1995,

Castillo-Guevara & Rico-Gray, 2002, 2003; Nishida, 2002; Schneider et al., 2002) which affect the nutritional status of the plant (Jameson & Bowers, 2012).

Baggett stated that he was ready to commence with more recolonization and had brood stock “in custody” for the first stage of a “massive rear-release project.” He chose

three sites, but avoided ENP because he felt that the plants did not have enough new growth to stimulate the females to oviposit. Fuchs Hammock, in south Homestead, was one of the areas Baggett chose because it contained abundant host and nectar resources, and was not generally known to the “collecting crowd.” Baggett did not disclose the other two locations to Rawson.

The sense of intrigue thickened. By Aug. 1980, Rawson wrote to Covell that he “.

. . presume[d] that Eumaeus atala has made its appearance again in Florida,” since he had received a letter from Baggett, who was then in Jacksonville, telling him of the colony discovery. Ironically, Baggett did not disclose the location to Rawson, even though Rawson was the original ‘rescuer’! Shortly afterwards, Rawson received a letter from another friend telling him that “. . . this bug is alive and kicking in south Florida.” He stated that the insect was swarming in March, and had plenty of all life stages. The friend added, “Several people know about this, but right now it is supposed to be a big secret, so everyone denies knowing anything about it.”

By December of 1980, the Atala was indeed alive and well on Key Biscayne, where it had also been deposited, and Covell mentioned that there were at least “two other sites” mentioned when he wrote to Rawson about the colony. Again, however,

Baggett had expressed concern that “too many people knew about it” and too much collecting may have been occurring. In 1982, Baggett wrote with frustration that the

Atala, which used to be so abundant, was now living in a few isolated colonies, one of which was “. . . adjacent to current condominium construction.” He feared that it was likely to become extirpated, but he expressed hope that there were more isolated hidden colonies somewhere.

Although there had been a substantial population in Long Pine Key in ENP during 1991, he saw no adults while doing botanical surveys in the area but wrote the host plant, coontie, was fine. He knew that the Bahamian population was able to disperse even though the flight of the insect was weak (often called “moth-like”), and hoped that the Atala would disperse likewise in southeast Florida. Coontie was regarded as threatened because of commercial exploitation during the early part of the century (see below) and Baggett felt that this lack of host plant was an important detriment to re-establishing the colonies.

Gerberg and Arnett (1989) called the Atala a “threatened species,” which was not an entirely correct statement, since it had not been listed by the federal government or the State of Florida. However, many of the colonies that Hammer distributed were apparently flourishing, and a biologist located at the USDA Sub-Tropical Research

Station in Homestead was making notations of Atala sightings at locations such as

Perrine, Goulds, Castellow Hammock, and Florida City, most of which were either

Hammer’s original deployment sites in Miami-Dade County, or very close to those sites

(Landholt, 1984).

Hurricane Andrew struck south Florida on August 24, 1992, first hitting the barrier islands in South Miami at Biscayne National Park and sweeping a wide path of destruction through Homestead and up the southeast coast. It was the most devastating storm to hit Florida since Donna. No adults were seen in ENP, Biscayne National Park or Big Cypress National Preserve following the storm, although the host plants survived without serious damage (Davis et al., 1996). Andrew decimated many of the existing and persistent Atala colonies at that time (Minno, pers. comm.). Baggett had previously

mentioned the fluctuating population levels, to which he partially attributed the state of the host plants; Davis et al. (1996) reiterated that these year-to-year variations in the butterfly’s populations would complicate attempts to determine the effects of the hurricane on the colonies.

Amazingly, the butterfly apparently survived the hurricane in refugia and its survival could well be due to the parade of new colonies started by parks, private home owners, butterfly enthusiasts, and even inadvertently by nurseries, which were starting to plant the native “coontie” for use as a landscape ornamental in developments. In

1993, Minno and Emmel stated that further studies needed to be completed to determine if the Atala can be called a separate race from the Bahamian species and that it appeared to be out of danger. New (1990) stated that the Atala was vulnerable due to habitat destruction and that documentation and education were tantamount for understanding the ecology of the lycaenids. Cushman (1993) stated that additional information was needed to understand the population trend of the Atala. The IUCN listed the insect as vulnerable in 1993. Minno and Emmel (1994) reported that the Atala was sometimes regarded as a pest by nurseries and private home owners, but it was listed as a species of special concern because of “restricted distribution and cyclic fluctuations in abundance.”

During the early 1990’s numerous articles were published in the local papers urging homeowners who wanted the Atala butterfly in their gardens to take them from sites where the herbivore was not wanted (Kilmer 1992, 1993, 1994; Long, 1993;

Culbert 1994; Emmel, 2001). Since 2004, I have been actively involved in an Atala reintroduction and monitoring project, continuing to remove unwanted or erupting

populations from one site and redistributing them to other sites where the butterfly is wanted (Koi, unpublished). Minno and Minno described an “accidental” colony that was established in Vero Beach, Florida, that seemed to die out from the temperatures

(1999). Quite a few colonies were inadvertently started when people planted coontie as an ornamental in their gardens, bringing pupae, larvae or eggs from southeast Florida nurseries with the plant. Culbert’s IPM article (1994) was published in response to a developer’s request to remove the insects from landscaping coontie that had been brought from Miami to a development site in Indian River (Culbert, pers. comm.).

One of the post-Andrew restoration projects that directly involved the Atala butterfly took place on Key Biscayne. Andrew destroyed much of the barrier islands: the homes, vegetation, beaches and docks. The surrounding southeast Florida areas were heavily damaged, including Virginia Key, Key Biscayne and the islands of Biscayne

National Park (National Hurricane Center, 1998). The storm was a blessing in disguise, however, because it leveled the Australian Pines, Brazilian Pepper trees and other invasive non-native vegetation that had covered and smothered the natural coastal ecosystem for 100 years. Because the residents of the Village of Key Biscayne had incorporated and “seceded” from the City of Miami in 1991, they were able to make decisions regarding their own island’s restoration.

A decision the residents made included removal of the non-native plant debris and restoration of native coastal plants and trees (Lollar, 2004; Lush, 2007). In addition, the residents of the Village of Key Biscayne planted coontie and nectar sources for the

Atala butterfly; in 1999, a master’s student, Eileen Smith, prepared another reintroduction experiment. Crandon Park, located in approximately the middle of the

island had, at that time, twenty-three coontie plants (Smith 2000). Smith planted thirty- one additional coontie plants for the butterfly in specific areas containing nectar resources for the adults. She re-introduced another batch of Atalas, in egg, pupal and larval life stages, donated from Fairchild Tropical Botanic Garden in Coral Gables. The source of the original population found by Hammer in 1979 is still unknown. Smith’s colony persisted for several years, and has been documented sporadically since then

(Koi, unpublished). It is not known, however, if human intervention has played any part in those records of ephemeral colonies in Crandon Park on Key Biscayne, or other ephemeral colonies.

Regardless, it has been demonstrated that the Atala can expand its range on its own when suitable habitat corridors exist, based on research completed on Key

Biscayne in 2007 by Sam Wright (Lush 2007). After the Key Biscayne island restoration project had removed the non-native invasive vegetation and restored native coastal trees and plants, many of the residents of the Village of Key Biscayne, located mid-way on the island, re-planted native gardens, as well, specifically for the Atala butterfly. Then

Smith relocated Atalas to the park between 1999 and 2002. Biologists at Bill Baggs

State Park, on the southern end of the island, recorded Atalas arriving (apparently) unassisted in 2002. This is a distance of less than two miles; the atala is not a strong flier, but it is capable of navigating suitable patches to locate and self-establish additional metapopulations. Because the residents of the Village of Key Biscayne had planted nectar and host plant resources in their gardens, it is thought that the butterfly negotiated to the south end of the island without human interference.

In 2004, I received a grant from the National Fish and Wildlife Foundation,

Budweiser Conservation Scholarship Program, to re-establish sustainable wild populations of the Atala in historically documented sites. The first step required mapping the actual range and distribution of the butterfly, which I have continued to do since that time. I have recorded this ‘self-establishment’ quite a number of times in Miami-Dade,

Broward and Palm Beach counties and my data shows that most new colonies are located within four miles from the nearest known site, but usually less than two miles

(Koi, unpublished). Domestic gardens play an important role in this recovery, but it is apparent that by looking at the locations of atala activity that passageways of intact, remnant natural areas are not only also utilized, but very important to its dispersal (Koi, unpublished).

Sue Perry, the head ecologist for ENP in 2004, had simultaneously received federal permission from the National Park Service to re-introduce the Atala into the park after an absence of twelve years, which segued perfectly with my project. From 2004 until 2008, Atalas were delivered to ENP from eruption sites in Palm Beach, Broward and Miami-Dade locations. As mentioned, the butterfly has crash-eruption cycles and these years happened to be eruption years, especially in Palm Beach. Over the four year period, 10,000 butterflies were delivered to ENP in various life stages and it is through those early experiments that certain parameters were developed for transporting the insect from one site to another.

We chose the locations for deployment of the insect at ENP based on four criteria. First, we chose areas with previously documented sightings. Second, we decided on release sites previously deemed acceptable by former ENP biologists

Baggett and Robertson. Third, we picked areas that were somewhat isolated from human activities to prevent disruption of natural behaviors, and lastly, we selected areas containing host plants and native nectar sources.

In the course of those re-introductions, seven hurricanes and numerous tropical storms made landfall in Florida, including four in 2004 and Katrina and Wilma in 2005.

To repeat the comment by Davis et al. (1996), assessing the impact of the hurricanes on the Atala populations was challenging.

Many colonies were severely impacted in each hurricane, directly because of drowned larvae and pupae as well as indirectly because of the condition of the host and nectar resources, as well as the habitat in general, following the storm (Figure 1-9). All of the native butterfly populations were affected to some extent; Zebra Heliconian butterflies (Heliconius charithonia), for example, virtually disappeared for months after the storm because almost every roosting aggregation was literally turned into confetti as the powerful winds defoliated and shredded the leaves on every tree and shrub taller than a few feet high (pers. observation). Few nectar sources on trees or shrubs were usable until the plants recovered, but low-lying weeds such as Bidens alba were vitally important to the sustenance for any of the surviving lepidoptera.

Individual Atalas were documented many times after the releases in ENP, but it is not known if there is still an extant population as monitoring has not been consistent, partially because of federal budget cuts and reduced staff. It could be that deep in the remaining pine rocklands of the park, an atala colony or more have been hiding….there are colonies in domestic gardens that have persisted for five years or more since

original re-location, so I do not write off that there could be populations far isolated from the perimeters of the park.

New coontie plants were installed in ENP in 2008 to help mitigate Baggett’s comment that a lack of fresh new growth may have been a significant part of the Atala’s lack of persistence in the park. The coontie was grown from seed originating in the park to keep the genetics of the plant pure. Besides the limiting factors of host plant’s possible lack of fresh growth, another factor could be the changes in the nectar resources. Many of the nectar plants documented by Small (1913) are no longer found in the park, such as Exostema caribaeum (Jacq.), which is a very popular nectar resource in the Bahamas. Many other plants listed by Small (1913, 1915) are now rare in the park, such as Tetrazygia bicolor (Mill.) Cogn., which is also a used by the Atala for nectar. Storm surges and salt-water intrusion affected the vegetation and fresh water sources in the low-lying southern portion of the park after the series of hurricanes.

In the margin of my copy of Small (1913) is a notation made by an ethno-botanist who commented that E. caribaeum was used by the “Indians” to put out fires. A lack of this plant in the pinelands now may be a contributing factor to the sometimes detrimental intensity of the managed fires in ENP and other remnant pinelands (Salvato,

2004). At least one of our re-introductions in 2005 was accidently burned in a managed fire the day after we deployed the pupae, because the fire cache and biologists/ecologists were not in communication; that has been resolved so that future burns and future biological studies or research are recognized by all parties (Perry, pers. comm.)

Kilmer (1993) felt that the insect needed a ‘coastal environment’ to thrive, and

Castner (1986) suggested that unknown microhabitat requirements could be a contributing factor to low survival rates in some colonies. The coast of southeast Florida was once a few miles wide; landfill has expanded he territory forty to fifty miles inland and the butterfly has moved with it. Although coastal colonies such as Fairchild Tropical

Botanic Garden persist for years, there have been self-established colonies as far west as twenty-seven miles from the shore (Koi, unpublished). They are among the ephemeral populations that wink in and out, but many last for at least a season.

Fairchild, however, has crashed and required “re-seeding” twice in the past ten years

(Koi, unpublished).

Current Range. There are approximately 200 ephemeral scattered colonies or metapopulations (defined as an isolated populations separated by inhospitable habitat, which in this case, predominantly consists of heavily developed urban landscapes) located throughout Palm Beach, Broward and Miami-Dade counties, with occasional ephemeral colonies documented in St. Lucie, and Martin counties. There are populations that persist for three-to-five years but many die off or disperse almost yearly. In 2011, I recorded information about the landscape architecture, nectar and host plant resources, and habitat qualities of colony sites on my blog (http://e- atala.blogspot.com).

This information was gleaned by ‘ground-truthing’ the sites myself and then via surveys completed by property owners or managers, such as park rangers.

Most colonies in Palm Beach are in natural areas or public parks (12 of 28 sites); in Broward County, colonies are found more often in domestic gardens (35 of 63 sites).

In Miami-Dade County, the colonies that I have documented are usually in remaining natural lands and botanical gardens such as Fairchild Tropical Botanic Garden (FTBG) and Montgomery Botanical Research Station (25 sites of 41). It was significant that of the 63 sites recorded in Broward County, more than a third of the domestic gardens were certified as “wildlife friendly” by one or more of the environmentally-conscious non- governmental agencies, such as the North American Butterfly Association, Audubon,

The National or Florida Wildlife Federation, NatureScape Broward, Connect to Protect

(through FTBG) or Florida Friendly Yards via the University of Florida’s Institute for

Food and Agricultural Services. Miami-Dade boasts two very large federal parks,

Biscayne National Park and ENP; while documented observations have been verified in the latter, the former is still under question.

Conservation Status

The conservation status has fluctuated with the population cycles. In recent history, it has been considered threatened (1970), vulnerable and a species of special concern by the State of Florida (1999) and one of over 970 species in Florida of management concern (2000).

There have been some published speculations that the Florida species is a migrant blown in by trade winds from the Bahamas (Rutkowski, 1995) and/or from

Cuba. There is documented proof that small hairstreaks are capable of surviving such migratory or dispersal events (Robbins & Small, 1981). Local discussions in southeast

Florida with naturalists and botanists sometimes argue that the butterfly was introduced with the host plant during the thousands of years of travel and migration by the native

Calusa and Tequesta peoples, both of which were known to carry on trade with the

Yucatan and Caribbean communities.

The insect is known to self-establish within a few city blocks to a few miles of other colonies if host and nectar resources are contained in the new site (Koi, unpublished). Females are capable of dispersing to search for host plants (Smith, 2004) and I have found individual Atalas flying in a private home garden that was located a full

27 miles from the Atlantic shore and more than four miles from the nearest known colony site in a natural area. I have also seen them flying a mile away from a known colony site, nectaring at a local gas station’s flowering hedge.

Monitoring the metapopulations has become extremely valuable in understanding the ecology of the butterfly, but this does not yet answer the question concerning possible immigration from Cuba or the Bahamas. Currently, the Atala is listed as

“Imperiled” by the Florida Fish and Wildlife Conservation Commission through the

Imperiled Butterfly Working Group (IBWG) (Imperiled, 2008) to address issues that may affect butterfly declines in Florida. Additionally, it is now one of 78 species listed as imperiled by the State of Florida and being tracked by The Florida Natural Areas

Inventory (FNAI) (2011). FNAI became affiliated with Florida State University in 2001 and is a member of NatureServe, the international natural heritage program (Florida,

2009). FNAI is primarily associated with the U.S. Fish and Wildlife Service and the

Florida Department of Environmental Protection, Division of State Lands, but works with the Florida Fish and Wildlife Conservation Commission, Florida’s water management districts and other organizations. It collects information on the current population levels, range and distribution of imperiled species, including the atala butterfly (Florida, 2009), as scientists recognized a serious decline in species biodiversity (Emmel et al. 1993;

Deyrup and Franz 1994; Imperiled, 2011).

The Atala is still listed as a species of management concern in the South Florida

Multi-species Recovery Plan prepared by the U.S. Fish and Wildlife Service (1999), but it is not federally protected or listed. When the insect was re-discovered in 1979, its status was reviewed, but not changed. It was listed as S3, rare and vulnerable, by the

State of Florida (2005). It is generally described as “rare” but “locally abundant” in that it is not generally found in typical butterfly gardens, or on Pollard-type butterfly walking transect counts (Pollard et al., 1973), but is found occasionally erupting in natural areas to numbers in the thousands. It has been recognized as a species with a classic crash- eruption biology cycle, the causes of which have yet to be fully explored.

Many of the sites that had naturally occurring or re-seeded Atala populations still have persistent or ephemeral populations, such as Key Biscayne, Virginia Key, Hugh

Taylor Birch, Fuchs Hammock, Navy Wells, and Fairchild Tropical Botanic Garden. The range has expanded, partly by its adaptation to domestic and botanical gardens and partly by human dispersal. Introduced ephemeral colonies have been documented as far north as Martin County (Koi, unpublished). What impact, if any, changing climatic factors have on its distribution further north is unknown, but could have a potentially beneficial outcome as anthropomorphic habitat alteration increases in its traditional southern home range, but temperatures become more sub-tropical in the northern parts of Martin and Pam Beach counties (Walther et al., 2002; Algar et al., 2009; Gaston,

2009; Thomas, 2011).

Larval Host Plant: Zamia integrifolia (Zamiaceae: Cycadales)

Nearly all extant cycad species are currently listed as endangered, vulnerable, threatened, critically endangered or some combination of those designations

(Oberprieler, 1995a, 1995b, 1995c; Nagalingum et al., 2011; International, 2013). There

are eleven genera containing approximately 300 species. The genus Zamia, the Atala’s host plant, contains about 68 species (Nagalingum, 2011), and Z. integrifolia is North

America’s only native cycad. It ranges from the Florida Keys to South Georgia and is still considered “Commercially Exploited” by the State of Florida (Coile, 2000; Coile &

Garland, 2003). The International Union for the Conservation of Nature and Natural

Resources (IUCN) (Stevenson, 2010) considers it “Near Threatened.” The host plant for each of the larvae in each of the Eumaeus species use the cycad species which is indigenous, and sometimes precinctive, to the country in which it is found (Vovides

1991; Oberprieler, 1995b, 1995c; Valdez & Hernández, 1995; Constantino & Johnson

1997; Chemnick et al., 2002; Contreras-Medina et al., 2003; Castillo-Guevara et al.,

2003; Gonzáles 2004; Prado 2011; Calonje et al., 2013).

Regardless of the conservation status, there is still considerable argument about the actual relationship of the Zamia species to each other and it, like the Atala, has undergone names changes. In this case, there have been a staggering number of changes. The North American and Caribbean plant was named originally by Linnaeus, but the name was changed repeatedly from Z. integrifolia to Z. pumila, and back again, then to Z. floridana and back again to Z. pumila and is currently called Z. integrifolia once more; nomenclature was also sometimes confused with Z. angustifolia, Z. silvicola, Z. debilis, Z. media and Z. umbrosa (Small, 1913; MacAllister 1938; Avery &

Loope, 1980; Correll & Correll, 1982; Avery & Loope, 1983; Vovides, 1983; Vovides,

1991; Wunderlin, 1998; Dehgan, 2002; Broome, 2004; Schneider et al. 2002; Calonje et al, 2013; Stevenson 2010; Austin, undated). With better molecular techniques, the

taxonomic status of all of the plants in the Zamia genus is finally being clarified

(Nicolalde-Morejón et al., 2011; Calonje et al., 2013).

All cycads contain varying amounts and proportions of neurotoxic compounds, such as cycasins, macrozamins, and azoxyglucosoids (Teas, 1967; Bowers, 1993;

Rothschild, et al. 1986; Bowers et al., 1989; Nash et al., 1992; Rothschild, 1992;

Oberprieler, 1995a, 1995b, 1995c; Castillo-Guevara & Rico-Gray, 2002, 2003; Nishida,

2002; Schneider et al., 2002).

The Atala butterfly is sometimes called the “coontie hairstreak” because it is the only butterfly species that uses the cycad called “coontie” (Zamia integrifolia) as its host plant in North America. Another lepidopteran, the Echo Moth (Seirarctia echo) uses the coontie plant, but utilizes a wide variety of other plants as larval foods as well; Baggett

(1982) noted that the moth larvae may be a competitor with the Atala larvae for the host plant in places such as ENP or other remnant pinelands, where the host plant may be sparsely distributed (Figure 1-10).

The butterfly (and moth) are able to safely sequester the toxic chemicals by metabolizing the cycasin and methylazoxymethanol from the cycads and turning it into

β-glucoside (Teas, 1967; Bowers, 1993; Rothschild, et al. 1986; Bowers et al., 1989;

Nash et al., 1992; Rothschild, 1992; Oberprieler, 1995a, 1995b, 1995c; Bell, 2001;

Castillo-Guevara & Rico-Gray, 2002, 2003; Nishida, 2002; Schneider et al., 2002; Yagi,

2004; Blum & Hilker, 2008; Opitz & Müller, 2009). The ova and imagoes actually contain higher concentrations of the neurotoxins than other species that sequester poisons from their poisonous host plants, such as Danaus species (Schneider et al., 2002).

The Atala is a specialist butterfly, but has utilized other many other cycad species that are available in gardens as landscaping plants in southern Florida (Deyrup & Franz,

1994; Hubbuch, 1991; Hammer, 1995; Koi, unpublished). Schneider et al. (2002) determined that all life stages contain toxic compounds and toxic levels as high as 40% were stored in the adult wings.

The story of how the Atala butterfly journeyed from the “most conspicuous insect”

(Schwartz, 1888) to “nearly extinct” (Klots, 1951) is complex, but it actually starts in ancient pre-history with the fact that the first native inhabitants used the root of the butterfly’s host plant, “coontie,” as a source of starch. The plant, Zamia integrifolia, is the only cycad native to North America, and was found growing prolifically from the

Florida Keys to Georgia. Coontie is the common name of the plant and it was so thick and dense along the New River (now Fort Lauderdale), Florida, that the Calusa, and later the Seminoles, referred to the waterway as “coontie hatchee” (coontie river). It is probable that Fort Lauderdale was built at the location to prevent the Seminole Indians from accessing the coontie, a vitally important food source for the people (Kirk, 1976 &

1977; Knetsch, 1989 & 1999).

The toxic compounds in the coontie are water-soluble and the native peoples

(Calusa, Tequesta and later the Seminoles and Miccosukee) learned how to process the calyx (root) of the plant for flour. They grated the root, rinsing it very well, and dried the resulting starch in order to make flour for bread, soup thickeners, biscuits, etc. (Fix,

1967; Small 1913). It was a primary source of starch for the native peoples and was vital to their survival; the crystalline structure of the flour made it resistant to mold in the high humidity of south Florida.

The native peoples were semi-migratory and did little damage to the population of Zamia, processing only what they needed for themselves. When the Europeans arrived, however, they exploited the plant for both food and profit. The first true industry in southeast Florida was established on the banks of the New River, which was wide and deep, with a strong current that ran from the Everglades into the Atlantic Ocean

(Knetsch, 1999). A former Army officer from Maryland by the name of William Cooley arrived in along the “New River” settlement 1836. He devised a water-driven wheel on the river that allowed him to process the root for flour at an exceedingly high production rate (Kirk, 1976 & 1977). Cooley sold the valuable flour to the U.S. Army and the

Seminoles, and distributed it to the new settlements that were being established along the U.S. coastline from Key West to Boston. He shipped it to the gourmet markets in

Europe, as well (Kirk, 1976 & 1977, Knetsch, 1989 & 1999).

This first processing industry depleted the wild coontie all the way to the

Everglades themselves and production stopped only after the mill was damaged and finally destroyed during the Seminole Wars (Kirk, 1976 & 1977, Knetsch, 1989 & 1999).

In the meantime, however, new settlers moving into south Florida learned how to make the flour, and mini-coontie mills were set up throughout the area (Figure 1-11). By the

1920’s several commercial coontie mills were established in southeast Florida (Figure

1-12) and production continued until the wild stock was depleted (Kirk, 1976 & 1977).

The fact that the coontie plant is very slow growing is another factor in the apparent demise of the Atala; the early settlers, of course, did not consider that this very extensive and profusely growing plant could be completely eradicated by over- harvesting and made no attempts to plant for future use. Cycads are dioecious,

requiring not only male and female plants to successfully reproduce, but also have specialist obligate weevil pollinators associated with each of the species of cycads as well (Norstog & Fawcett, 1989; Tang, 1990; Vovides, 1991; Oberprieler, 1995a, 1995b,

1995c; Valdez & Hernández, 1995; Chemnick et al. 2002; Schneider et al., 2002;

Oberprieler, 2004; Chaves & Genaro, 2005; Prado, 2011; Stevenson et al., 1998).

Although the absence of the native pollinators has been cited as a probable cause of reproductive failure in several globally endangered cycads (Tang, 1990;

Vovides, 1991; Oberprieler, 1995a, 1995b, 1995c; Norstog & Fawcett, 1989; Chemnick et al. 2002; Schneider et al., 2002; Oberprieler, 2004; Chaves & Genaro, 2005; Prado,

2011; Stevenson et al., 1998), our native Zamia has kept its’ weevil pollinator, so this is not a factor in its decline in natural areas. Ironically, the pollinator is also an herbivore, consuming the male reproductive cones from the inside out and only ‘accidently’ pollinating the female cones (Norstog & Fawcett, 1989).

Because the Atala has utilized many of the valuable, expensive and rare non- native cycads in southeast Florida gardens, it is often unwelcome. The insect is capable of completely defoliating both the native and non-native host plants (Figure 1-13).

However, because the Atala is able to successfully complete its life cycle on non-native cycads, this has, ironically, contributed to its expanded range. Pesticides are used to control the severe herbivory, especially in developments or botanical gardens (Castner,

1986; Culbert, 1994; Deyrup & Franz, 1994; Minno & Emmel, 1993).

Conclusions

Florida, the Caribbean Archipelago, and Central and South America are significant ‘hotspot’s for biodiversity (Myers et al., 2000), but much remains to be learned about the endemic species inhabiting them. Declining lepidoptera biodiversity

and survival (Schweitzer et al., 2011; New & Sands, 2002; Minno, 2010; Minno, 2011; ) has been associated with increased anthropomorphic changes such as habitat alteration, including deforestation for development and past lumbering practices (Small,

1913, 1916, 1929; Rawson, 1961; Hardy & Dennis, 1999; Algar et al., 2009; Gaston,

2009). Increased use of fertilizers, herbicides, mosquito-control chemicals and pesticides (Salvato, 2001; Koptur, 2006; Hoang et al., 2011; Bargar, 2012) have been cited as reasons for insect declines as well as the introduction of non-native plants and animals, including ants (Schwartz, 1888; Smith, 2000, 2002; Minno, 2010). Habitat fragmentation and decreased connectively between suitable colony locations have been cited as detrimental to stable butterfly populations as well (Hanski & Singer 2001; Matter

& Roland, 2002; Haddad & Tewksbury, 2005; Imperiled 2011; Schweitzer et al., 2011).

More studies have been done on Central and South American Eumaeus species than have been published on E. atala in recent years, which underlines the need to complete not only the E. atala life history, but the life history of the other Eumaeus species as well (Constantino & Johnson, 1997; Castillo-Guevara & Rico-Gray, 2002;

Contreras-Medina et al., 2003; Gonzáles, 2004; Chaves & Genaro, 2005; Pérez-Farerra et al., 2006; Martínez-Lendech et al., 2007; Calonje, 2009; Calonje, et al. 2009; Calonje et al., 2010a, 2010b; Calonje, et al. 2013); most of these papers focused on the plant and herbivory rather than on the butterflies or weevils. Often, the butterfly or weevil associated with the particular cycad is not identified and only noted as an “herbivore” in the Eumaeus genus or a probable pollinator. The plant cannot be saved from its threatened conservation status by ignoring the insects allied with it.

Many factors, past and present, have contributed to the decline or population fluctuations of the Atala butterfly in southeast Florida. Some things, such as climate or environmental variations associated with seasons, affect the species whether in southeast Florida or in the Bahamas (Bahamians, for example, have asked me, “Where do the Atalas go?” in the winter because they witness the same seasonal fluctuations).

Hurricanes and the associated salt-water intrusion, water table changes, defoliation and unmanaged fires affect the insect, the host plants and the nectar sources (Richardson,

1976; Steinberg, 1976, 1982). In the past, as recently as 2009-2010, lower-than-usual winter temperatures seemed to affect Atala colonies in southeast Florida and most crashed (Koi, unpublished).

During the 1930’s, besides the exploitation of the host plant, south Florida was struck with successive years of powerful hurricanes (Platt et al., 2000; National, 2005), the consequent floods, and winter cold snaps in tandem. Between 1921 and 1939,

South Florida experienced over fourteen devastating hurricanes rated category 3 or higher, and over 30 tropical storms in all (National, 2005). It is no wonder that the Atala may not have survived all of those detrimental factors, except in perhaps safe refugia

“somewhere” in the peninsula or perhaps on the barrier islands. . . . or perhaps on one of the Bahamian islands awaiting good trade winds, where the insect has managed to survive stochastic weather events for hundreds or even thousands of years (hiding in limestone outcrops?)

Regarding the apparent intolerance of cold temperatures, it is of interest that

Landholt (1984) mentioned that the Atala butterfly survived night time temperatures of

34° Fahrenheit in Miami. An outdoor colony housed here on the Gainesville campus of

the University of Florida likewise survived repeated night-tie temperatures of 28°

Fahrenheit, experiencing ‘chill coma’ but managing to recover during the warmer days

(hovering around low 70’s for several hours). The colony, housed in a screenhouse, included eggs, larvae, pupae and adults. The adults lived a normal average lifespan of approximately three weeks.

Understanding life history traits of our declining or threatened butterfly species must be undertaken to expect successful results in conservation management; using insects as bioindicators has proven itself to be a valuable assay in determining the overall health of an ecosystem (Noss, 1990; Kremen, 1992a, 1992b; Schwartz et al.,

1995; McGeoch, 1998; Peck et al., 1998; Schultz et al., 2008; Henry et al., 2013).

In conclusion, the words of Chris Thomas (2011) ring true for the Atala butterfly, as well as the other pine rockland species, such as Bartram’s Scrub-Hairstreak, the

Florida Leafwing (both proposed for federal listing as endangered at this writing) and the

Florida Duskywing: these insects are confronted with intractable obstacles to dispersal in a highly altered urbanized landscape. It is impossible to restore the vast tracts of unaltered pine rockland that once belonged to them, and consequently, their risk of extinction is high. Thomas discusses the potential need to translocate these high-risk insects to areas that will be beneficial to them as climatic changes push them out of their current homes, but having “nowhere to go” without assistance from the very species that caused the damage in the first place.

It is already apparent that Atala butterflies are surviving considerably further north than was ever recorded before (Koi, unpublished) and that they are capable of adapting to domestic frontiers. I suggest that perhaps Bartram’s and Duskywings could

be relocated into safe refugia further north, as well, when their host plants and nectar sources are established before that new introduction. As plants are also moving further north (or south) as the climate changes, these butterflies could live in new suitable habitats in those areas if they could disperse across the vast areas of inhabitable parcels: the concrete, the shopping plazas, the six-to-eight lane highways, the high- rises and residential developments landscaped with non-native, invasive, non-nectar bearing ornamental plants, maintained with heavy fertilizer and pesticide use. The first thing that must be accomplished, however, is a full and complete understanding of the life history of these other pineland denizens.

I am in full agreement with Smith (2000), who stated that domestic gardens in the hands of private home owners is vital to the Atala’s survival in urbanized southeast

Florida. It is these ‘corridors’ between gardens and natural areas, botanical gardens, nurseries and parks that provide the refugia needed to survive despite stochastic weather and challenging, altered urban environments.

Table 1-1. Species and subspecies of Eumaeus genera and site of first collection. Modified from Robbins & Lamas (2004c). Eumaeus Hübner (1819)

Eumenia Godart [1824]

Eumaea Geyer [1834]

Epula Gistel 1848 repl. name.

1. …childrenae (Gray 1832) (Eumenia) -Mexico

a. …debora (Geyer [1834]) (Eumaea) - ?

2. …godartii (Boisduval 1870) (Eumenia) -“Guatemala”

a. …costaricensis Draudt 1916 -Costa Rica

3. …minyas (Hübner [1809]) (Rusticus) -?

a. …minijas (Poey 1832) (Eumenia), misspelled

b. …minyas peruviana Lathy 1926 -Peru

c. …minyas superbus Röber 1926 -Bolivia

4. …toxea (Godart [1824]) (Eumenia) -“S. America”

5. …atala (Poey 1832) (Eumenia) -Cuba

a. …atala florida (Röber 1926) -Florida, USA

b. …atala grayi ( W. P. Comstock & Huntington 1943) -Florida, USA

6. …toxana (Boisduval 1870) (Eumenia) -[Colombia]

a. …minyas f. brasiliensis Draudt 1919 –Brazil

b. …minyas obsoleta Lathy 1926 –Bolivia

c. …giganteus Röber 1927-Ecuador

d. …minijas (sp) splendidissima Bryk 1953 –Peru

e. …sara Constantino & Johnson 1977 –Colombia

7. …hagmanni Röber 1923 (nomen dubium)- Brazil

Table 1-2. Chart showing some of the incongruent or missing biological data about Eumaeus atala Poey 1832 that has been published over the past 125 years. Biological and ecological research provided in this thesis explains these discrepancies as influenced by seasonal and environmental conditions.

Author & Ova Ova Larval Pupal Pupal Season Total dev. Lifespan Wingspan year dev. size dev. dev. length observed ova to imago Schwartz 10 14 days 9-10 days May ~33-34 days 1888 days 13-15 mm 20-22 Healy 1910 (1.3-1.5 days cm) 15 mm Dethier 1941 (1.5 cm) 4-5 1.5-2 Rawson 1961 18 days 31 days +-1.5 cm 42 days 7 d. or less days mm 15-24 mm (1.5- Franz 1982 2.4 cm.) Gerberg & 1.5-1.75 in. Arnette 1989 (3.81-4.45 cm.) Deyrup & 19-24 mm (1.9-

Franz 1994 2.4 cm) 15-16 15-16 days; 10 days; 18 29-30 days Culbert 1994, 4-5 1.5-2 days and 7 d. or days or 11 Lab 75° F (4+18+10=32) 2010, 2011 days mm 13-14 more days (4+11+13=28) days (graphic) (graphic) Hall & Butler 20-24 mm (2.0- 10 d. 2000 2.4 cm) 8-10 d. on Jan.- nectar; 8- Feb., Smith 2000 4-7 10-14 12 d. on Wingspan 3.6- 15-20 days April- 40-51 days MS Thesis days days nectar & 4.8 cm May, sugar June water

Figure 1-1. Males exhibit a “Caribbean blue”-to-“Sea green” dorsal forewing color that often extends onto the hind wing. The color is both heritable and seasonal. Photo courtesy of S. Koi.

Figure 1-2. Females exhibit a “Royal blue” splash across the dorsal forewing that does not vary in color but does vary in the extent of coverage. Photo courtesy of S. Koi.

A B

C D

Figure 1-3. Individual variation in species-recognized ventral wing patterns are distinct in individuals, but not sexually dimorphic. Four examples of newly emerged adults are presented here. A and B) Females. C and D) Males. There is no sexual dimorphism on the ventral surface of the wings. Note the “sprinkled” iridescence on the anal edge of the hind wing of female B and on the entire wing surface of male D. This iridescence was a frequent observation. The forelegs, thorax, and head often had varying degrees of iridescent patterns as well. Photos courtesy of S. Koi.

A B

C D

E F

Figure 1-4. Wing scales have a heterogeneous appearance due to curved scales interspersed with flat scales. A) SEM photograph of scales from a “blue” male. B) SEM photograph of scales from a “green” male. C) SEM photograph of aquamarine scales from male ventral hindwing. D) SEM photograph of the fringed edges of a male hind wing. E) Black and blue scales from a “blue” male. F) Royal blue scales from a female. Photos courtesy of S. Koi.

Figure 1-5. Distribution of Atala by Florida Wildlife Commission. Photo courtesy of United States Geological Service, J. Struttmann, undated.

Figure 1-6. The New River in Broward County circa 1880. Photo courtesy of Historical Museum of Southern Florida.

Figure 1-7. The mouth of the Miami River in 1883, photographed by Ralph M. Munroe of Coconut Grove. Photo courtesy of Florida State Archives.

Figure 1-8. The pine rocklands in Everglades National Park, an example of the original habitat of the Atala. The lighter areas are not sand, but limestone. Coontie grows interspersed with abundant Saw Palmetto (Serenoa repens) and other understory vegetation. Photo courtesy of S. Koi.

Figure 1-9. Hurricane Katrina caused habitat damage to many Atala colony sites. A) Larvae and pupae were knocked off the coontie plants and drowned during Hurricane Katrina on August 25, 2005. B) Some larvae survived only to die searching for host plants among the debris. Photos courtesy of S. Koi.

Figure 1-10. An Echo Moth (Seirarctia echo) on coontie (Zamia integrifolia) in a remnant pine rockland natural area in Miami-Dade County. No Atalas were seen at this site at the time this photograph was taken. Photo courtesy of S. Koi.

Figure 1-11. European settlers manufactured “coontie flour” in vast quantities for themselves for selling in the late1800’s and early 1900’s in south Florida, decimating the native population of Zamia integrifolia. Photo courtesy of Historical Museum of Southern Florida.

Figure 1-12. Commercial coontie flour mills, such as the long-defunct A.B. Hurst Company in Miami, flourished in southeast Florida until the wild populations were virtually extinct. Photo courtesy of the Miami Herald.

A B

Figure 1-13. The larvae of the Atala butterfly are capable of severe defoliation of many cycads. A) South Florida’s native Zamia integrifolia. B) Zamia erosa in a botanical garden in southeast Florida completely defoliated by Atala larvae. Photo courtesy of S. Koi.

CHAPTER 2 NEW AND REVISED LIFE HISTORY OF THE HAIRSTREAK EUMAEUS ATALA (LEPIDOPTERA: LYCAENIDAE) WITH NOTES ON CURRENT CONSERVATION STATUS

Introduction

Lycaenids constitute 30-40% of all butterfly taxa with nearly 6000 species (IUCN,

2013) and are endemic to often fragile environments (New, 1993). The hairstreak

Eumaeus atala Poey 1832 (Lepidoptera: Lycaenidae) is one such insect. Known as the

Atala butterfly, it was once called “the most conspicuous insect in semitropical Florida”

(Schwartz, 1888) and yet by 1951 was considered “probably extinct” (Klots, 1951). As the human population grew and the anthropomorphic impact increased, the butterfly’s fragile pine rockland habitat was depleted (Snyder et al., 1990) and its sole host plant, commonly called coontie, was nearly eliminated from Florida. Coontie is North

America’s only native cycad, Zamia integrifolia L. (Zamiaceae: Cycadales), which was nearly destroyed for starch production in the early years of European settlement. It was listed as commercially exploited as recently as 2000 (Coile) and is still considered “Near

Threatened” by the International Union for Conservation of Nature and Natural

Resources (Stevenson, 2010).

The butterfly was absent from collections for many years and finally deemed extirpated, when a small population of the butterfly was discovered on a barrier island in

Miami-Dade County in 1979. As far as is currently known, all extant colonies originated from that population. Numerous attempts at reintroducing the butterfly have met with varying levels of success (Rawson, 1961; Franz, 1982; Deyrup, 1994; Koi, unpublished data) and its populations are currently being tracked by Florida Natural Areas Inventory

(2010) and the Imperiled Butterfly Working Group (2012), as well as myself, due to

continued threats to its diminishing habitat (Land & Cooley, undated) and the possible stochastic weather events that could potentially wipe out isolated and fragmented colonies. I have developed relocation projects in conjunction with Miami-Dade County authorities and other non-governmental agencies to restore Atala butterfly colonies in historically occupied pine rockland natural areas, as well as in domestic gardens.

Historically, the Atala and its host plant were located along waterways as well as in the pine rocklands that once flourished along southeast Florida’s Atlantic Coastal

Ridge. Most of that former habitat is now urbanized development bearing little resemblance to its former ecological richness (Snyder et al., 1990; Land & Cooley, undated; Miami-Dade County, undated). The Atala is a specialist subtropical butterfly with specific nectar requirements (Koi, 2008) as well as its specialized host plant requirements. It is currently found in introduced colonies as far north as Martin County and in specific locations from Palm Beach to the scattered self-established or reintroduced colonies in Broward and Miami-Dade Counties (Koi, unpublished). The metapopulations wink in and out of existence as seasonal or abnormal weather events affect it and it exhibits unpredictable crash-eruption cycles that have complex relationships with host plant and nectar availability that may be chaotic as well as normal seasonal fluctuations (Koi, unpublished).

Southeast Florida is part of the Caribbean Archipelago and a hotspot for biodiversity conservation priorities (Meyers et al., 2000). The natural habitat of a number of south Florida’s imperiled species live in pine rocklands and are also host plant specialists (for example, Bartram’s Hairstreak, Strymon acis, and Florida Leafwing,

Anaea floridalis, that feed on increasingly rare flora (e.g., pineland croton, Croton

linearis) (Deyrup & Franz, 1994; Salvato, 2002; Salvato, 2003; Salvato & Hennessy,

2003; Schweitzer et al., 2011). While writing this chapter, Strymon acis and Anaea floridalis were both been proposed for listing by the United States Fish and Wildlife

Service because of severely declining populations (August 14, 2013). My current research on the Atala butterfly may be of value in developing conservation management plans for those insects as well, or act as templates for reintroduction or captive rearing programs. Recent published data indicate an alarming decline in these and other pine rockland species (Deyrup & Franz, 1994; Salvato, 2002; Salvato & Hennessy, 2003;

Worth et al., 2003; Schultz et al., 2008; Schweitzer et al., 2011; Imperiled, 2012). Less than 2% of the original pine rocklands remain and most are less than 50 ha (Snyder et al., 1990; Miami-Dade County, undated), but the Atala has made use of domestic and botanical gardens where it finds food and shelter; between 100 to 200 ephemeral colonies exist in natural areas and private property during any given year, in both self- established and introduced colonies (Koi, unpublished).

Without concrete data, such as sample size or season, it is virtually impossible to develop the necessary parameters for successful reintroduction programs. This essential information, such as differences in life stage survival rates or seasonal development is needed to understand the fluctuations in populations, choose the life stage with the greatest survival rate for relocation, distinguish seasonal discrepancies in development and other factors, all of which are important considerations for conservation management and reintroduction programs. Existing published reports on the butterfly have been rare, lacking data, incorrect and largely incongruent, with disagreement concerning vital information (See Chapter 1, Table 1-2). Season and

sample size is also missing from most published reports, with partial exceptions

(Schwartz, 1888; Smith, 2002).

The objectives of this study were ultimately to support conservation management efforts and increase success of reintroduction programs by documenting previously unknown parameters of the basic life history of the Atala butterfly in a captive colony under controlled laboratory conditions and comparing these known factors with aspects of wild colonies that I have previously studied in situ.

Materials and Methods

Biological Rearing: Livestock and Progeny

Atala larvae and pupae were collected, in development stages ranging from neonates to near-emergent pupae, from wild colonies in southeast Florida according to available stock in those locations. Because the insect is multivoltine, it was possible to collect approximately 200 individuals four times during the fifteen month study. The study began in December 2011 with wild larval stock from a Miami-Dade location, and was replenished with wild larval and pupal stock from three different sites located in

Miami-Dade, Broward and Palm Beach Counties respectively in May 2012. Additional larval and pupal stock was collected from a single large erupting wild colony in Palm

Beach in July 2012, and a final collection of larvae and pupae took place at three different locations, again in Miami-Dade, Broward and Palm Beach Counties in October

2012. Neither eggs nor adults were collected at any time.

All emerged butterflies were kept in a standard laboratory setting with ambient temperatures ranging from 24-26 C ° and a relative humidity ranging from 25-42%, depending on seasonal weather variation as recorded by a Lascar EL-USB-2 data logger (Lascar Electronics, Inc., Erie, PA). Insects were housed with approximately

8:16h light: dark cycles that varied occasionally to 10:14h light: dark. Daylight lamps were programmed for 1:2 h on:off increments during the regular day period and a fan was installed to carry volatiles to improve mating and ovipositing activity.

Larvae were fed native Z. integrifolia leaves ad libitum harvested from mature plants grown on campus or in the University of Florida’s Entomology/Nematology

Department’s screenhouse or greenhouse depending on weather conditions and plant growth. Because previous experience had shown that Atala larvae may refuse to eat cut fronds (Koi, unpublished), fresh material was cut bi-weekly and the stems of the individual leaves were stored in fresh water. Fronds were then placed into a florist’s water tube (Aquapic, Syndicate Sales, Kokomo, Indiana) to ensure that fresh leaf material was presented to the larvae for consumption, which increases larval growth

(Scriber, 1979a). During slower growing seasons, larval food was supplemented with leaves from plants on campus sites and those fronds were washed in a 1% bleach solution to destroy mealybug, scale insects and other unwanted pests. Plant material was rinsed and air-dried and then stored with stems in fresh water, before feeding larvae. This cleaning procedure was unnecessary when using greenhouse stock.

Larvae were housed in standard plastic “shoeboxes” from time of hatch until the wandering stage commenced at approximately ten days of age. At that point the larvae, in their boxes, were placed into 24” x 24” x 12” fabric cages (LiveMonarch Foundation,

Castle Cage Size JUMBO, Boca Raton, FL) to prevent loss. Although larvae can be cannibalistic, they are also gregarious feeders and most chose to remain in clusters throughout the larval feeding stage. Larvae were thus housed with their brood-mates throughout the immature stages, which may have hatched from eggs from the same or

different females; however, larvae were reared singly if they eclosed without brood mates to determine differences in survival, development rates and longevity between single individuals and broods containing higher numbers of larvae.

Pupae were allowed to disperse to their preferred pupation site within the parameters of their fabric cages or larval boxes. Pupae were misted with deionized water from a sprayer at least once daily and otherwise left undisturbed until adult emergence. When adults emerged, they were manually transferred to the ceiling if necessary to aid in proper wing expansion as some pupal clusters in the cage obstructed proper eclosure. This obstruction of earlier pupae by later pupae may occur in wild populations, as well, because the pre-pupal larvae usually wander to pupation sites ‘en masse.’

At adult emergence, the butterflies were transferred to a 6’ x 6’ x 6’ fabric flight cage (LiveMonarch Foundation, Greenhouse Castle Cage, Boca Raton, FL) with congeners to mimic a more natural environment and allow for normal behaviors. Adult cages were equipped with cut coontie fronds (housed in waterpics) for ovipositioning that were hung from the ceiling via wire loops approximately 15 cm. from the roof to make them more apparent to females. Males and females were housed together to encourage normal mating behaviors.

In addition to occasional seasonal native flowers (primarily Bidens alba), the adults were provided with deionized water, to avoid potential chemicals in tap water, and Gatorade® (The Gatorade Company, Chicago, IL) in fruit punch, orange or watermelon-citrus flavors. The fluids were offered ad libitum from introverted centrifuge tube feeders, altered slightly from a description by Hughes et al. (1993). Water was also

continually provided via a length of dental packing cord (TIDI Products, Neena, WI) inserted into a 20 mL flask partially sealed with laboratory parafilm and filled with deionized water. Feeders were hung from the ceiling in the same manner as the ovipositing leaves, as well as installed on a short “table” for easier accessibility from the floor, where older adults of both sexes often congregated during the night.

All feeders were replenished daily and cleaned weekly. Cotton balls soaked with deionized water were installed on the ceiling of the cage and the cage was misted twice daily with deionized water from a regular spray bottle. Misting increased humidity in the cage environment as well as provided additional drinking spots for the insects. The flight cage was replaced with a clean cage monthly for disease prevention. All cages were cleaned with a 1% bleach solution and allowed to dry in the sun within an enclosed screenhouse.

Newly eclosed adults were identified with unique alphanumeric codes using a silver metallic Sharpie® brand permanent paint marker (Newell Rubbermaid Office

Products, Oak Brook, IL) on both ventral hindwings to allow immediate recognition of individuals. Biological data recorded included sex, dates of ovipositing, larval eclosure, pupal emergence and day of death. Observed matings, ovipositing and associated behaviors were documented as well as fecundity and fertility. Samples of wing cord length and male dorsal forewing colors were recorded. Females do not display strong variations in dorsal wing color, although there is a marked difference in the extent of color present. All individual adults observed in both the captive and in wild colonies display unique variations in the described and species-recognized patterns on the ventral wing surface (Chapter 1, Figure 1-3).

Because adult females were not isolated from one another, egg clutches were not identified per individual females. The adult Atala exhibits highly aggregate behavior in wild colonies and females have been observed simultaneously ovipositing on the same leaflet with another in the wild. With only one or two coontie leaves in the flight cage, the females gathered and oviposited together on the leaves without any observed stress or aggression. This is normal behavior in a site with limited host plant availability

(Koi, unpublished). Because individuals were marked, however, it was known in many cases, which female laid eggs as well as which male mated with her. Marking also allowed documentation of the time passed between mating and ovipositing as well as mating behaviors.

The total number of adults reared numbered over 5,800 individuals. Statistical analysis was completed on varying sample sizes pulled from the total lab colony based on parameters discussed in the text. For example, of 750 individuals measured for wing cord length, only measurements made by myself and only measurements associated with adults for which there was a complete life history were used in analysis.

Immature Measurements

Ova were collected from the adult flight cage daily in the afternoon and the number of ova recorded. The eggs were measured with a standard digital microcaliper under a Leica S8APO stereo microscope (Leica Microsystems, Inc., Buffalo Grove, IL) to prevent accidental damage to the egg while measurements were taken.

Larval exuviae were collected and photographed. Measurements of larvae were abandoned soon after recording was started as it was apparent that the size of the larvae did not necessarily correspond to its age and therefore the measurements could not determine age. Weight of larvae was also abandoned for the same reasons;

however, some measurements and weight were taken for specific parameters and results are discussed below.

Pupal development was categorized from pre-pupal stage to pharate adult.

Development time and size was recorded for each life stage and individuals were followed from egg and first eclosure as a larva until time of death. Some pupal weights were recorded as well.

Adult Biology and Reproductive Behavior

Adult wing cord length measurement was taken on the left forewing, from the bottom of the basal sclerite and proximal point of resilin where the forewing emerges from the thorax, to the tip of the apex of the forewing in a straight-line measurement to the nearest whole millimeter using a transparent ruler (C-Thru® Ruler Company,

Bloomfield, CT). Ten measurements taken with the ruler were also compared with those taken by the digital microcaliper; because there was less than 0.01 mm difference between the caliper and ruler measurements, and the ruler was easier, quicker and reduced insect handling time, the ruler was the chosen method of measurement.

Measurements of newly eclosed first generation adults from wild larval stock that pupated on arrival were measured for comparison.

Unmated newly emerged females were dissected to determine oocyte development stage at eclosure and subsequent oogenesis, chorion development, number of ovarioles and ova production. The number of ova remaining in the oviduct was counted at death. In addition to documenting mating behavior, the age of each individual at mating was recorded, the age at ovipositioning, the longevity of known mated individuals compared to unmated, as well as whether single or multiple matings occurred between individuals.

Results

Oogenesis, Oocyte Development, Ovipositing and Ova Production

Oocytes are fully formed at emergence and oogenesis is continual throughout the lifespan of the female. Females are capable of ovipositing by the third day after emergence and lay as few as one egg to a cluster of as many as fifty or more eggs on the host plants, generally using the side of a fiddlehead or the underside of a newly emerged leaf. However, females will lay eggs on older leaves, on the top of the leaf, the stems, and even the reproductive cones of the host plant in the wild when there is limited host plant available. Occasionally, females in the captive colony laid eggs on non-host substrate such as the walls and I have witnessed fresh females using non-host plants in wild colonies twice (the native host plant was located directly underneath in both cases). Eggs laid on non-host substrate were often fertile and hatched; those larvae survived to adulthood within normal parameters whether reared alone or with other larvae. Ovipositing occurred on the same day as mating or within a day afterwards and could occur before or without mating.

The average age of female ovipositing was 19.36 days with standard deviation of

±7.85 days (n=234). The average age of female mating was 15.45 days with a standard deviation of ±8.94 days. The age of observed mating females ranged from newly eclosed to 41 days of age. The average age of male mating was 11.98 days with a standard deviation of ±7.63 days (n=234). The range for observed male mating was newly eclosed to 38 days old. Of those observations, the mean longevity of observed mated females was 32.96 days (standard deviation ±8.83) (n= 76) and the mean longevity for observed mated males was slightly higher at 34.38 days (standard deviation of ±11.03) (n=76) (Table 2-1). The average lifespan of adults in general was

23.6 (n=1021) (see below), but there were many exceptional individuals that far outlived the average by weeks or even months.

Ova were chosen haphazardly for measurement, when there was sufficient time to do so, from the ovipositioning fronds in the adult flight cage, from five different broods on six different days during a two month period to assure a wide sample size (n=600).

The ova ranged in size from 0.75 mm to 1.25 mm and exhibited a bi-modal distribution, with the mode at 0.99 mm. (Figure 2-1). There was also a marked visual difference in the size of ova from dissected females, indicating that the size difference is an inherent part of their biology (Figure 2-2). The standard deviation in ova size was ±0.15 mm.

Ova development time varied greatly, ranging from 4 to 13 days in the lab colony.

Average development time in ova was 6.6 days with a standard deviation of ±1.1

(n=1021). There was a survival rate of 36% from ova to larva in the captive colony

(n=10582) (Table 2-2).

Ova have a three-leaved micropyle and highly sculptured chorion surface, typical of Lycaenids (Figure 2-2). The underside of the egg is smooth and flat and is adhered to the leaf surface with proteins exuded by the females’ accessory glands. The undersurface is clear, allowing yolk or the embryo to be visualized when the egg is removed from the substrate and inverted (Figure 2-3). Larvae chew through the micropyle area and do not eat the shell, even avoiding it entirely when a group of neonates is scraping the surface of the leaves during the first instar; older larvae also avoid eggs by eating around them.

Larval Development

An advantage to female ovipositing in clusters is that larvae often eclose within a few hours of each other and line up “sardine style” to begin eating simultaneously; doing

so allows them to break down the tough ectoderm of the coontie leaves, skeletonizing the leaves. During late winter, when the host plants were not sending up new growth, the surfaces of older leaves were scraped with a surgical blade and the mash served to neonates in a petri dish, allowing the larvae to survive until they were big enough to handle tougher leaves as a group. Individuals that hatched and were reared alone chew the surface in tiny single bites and develop much more slowly than those in groups.

Newly eclosed larvae take their first “test-bite” of the substrate immediately after emerging but before lining up with siblings.

Larval development time was highly variable in the lab colony, ranging from 14 to

28 days in the lab (n=1021), but two that remained as larvae for 34 days died in eclosion failure at 54 days and were not used in analysis. The average development time was 18.4 days with a standard deviation of ±3.6 days (Table 2-2). Neonate larvae are capable of releasing silk lifelines to aid in locomotion, such as descending from a higher leaflet to another, or to adhere to a leaf.

Larval broods were reared in the same groups from the eggs from which they eclosed; individuals were reared alone when only one ovum hatched in a clutch. Five newly hatched larvae that were weighed within two hours of hatching, and reared on Z. integrifolia, had an average weight of 0.0072 g., ranging between 0.0004 g. and 0.0025 g. with a standard deviation of ±0.0009 g. (Table 2-3). They increased in weight and by five days reached an average weight of 0.3853 g. with a range between 0.0076 g. and

0.0858 g. and a standard deviation of ±0.0347 g., an increase of 3 percent. By day eleven, larvae increased their weight by 7% with an average weight of 0.5374 g. and a standard deviation of ±0.0950 g. with a range between 0.4702 g. and 0.6046 g. Larvae

showed marked differences in feeding robustness, with some growing much faster than others.

Newly hatched larvae measured between 0.05 mm and 1.5 cm, but after the first molt it became difficult to standardize a measurement protocol partly because of the high variability in the growth and weight of the larvae regardless of age, and the extreme plasticity of the larval body, so the endeavor was abandoned after the first instar (Fig 2-4B).

This report is the first record of larval stadia in the Atala, which present a 3-day,

5-day, 7-day, 9-day and sometimes 11-day molt. Being variable in almost every stage of development, occasionally there was a 4-day, 6-day, 8-day or 10-day molt evident in a clutch (Figure 2-4C). This may have been caused by several reasons: the individual may not have been as robust a feeder as cage mates, may have actually hatched later than the others and/or perhaps started out smaller than the other larvae in the brood and therefore developed more slowly than its cage mates. Exuviae were not ingested by larvae in any stadia and were avoided in much the same way as were the ova chorions.

Additionally, although the larvae were fed ad libitum, they were such extremely robust feeders that they often completely devoured even the rachis of their host plants as well as the foliage. Occasionally, the larvae would ingest parts of the silicone caps on the waterpics, as well as cannibalize cage mates, which dropped the survival rate as low as 14% in some broods. Even so, in several instances, even a brood that started with small numbers could be reduced to a single larva by the time it entered the wandering stage if one of the larvae was a particularly aggressive feeder.

The wandering stage of development occurred at approximately day ten of larval life and by the 12th or 13th day, most larvae would become “pre-pupae” in preparation for the final larval molt. The pre-pupal stage varied in different temperature, humidity and light levels (discussed in Chapter 4). Larvae would usually migrate en masse to the pupation site within the fabric cage, which could be the walls of the shoebox, or the walls or ceiling of the fabric cage. One or two larvae in the brood would sometimes disperse away from cage mates to pupate alone; larvae that were slower in development usually moved to a pupation site previously established by earlier larvae.

Pre-pupae formed large areas of silk mats underneath their aggregation, anchoring the cluster to the substrate and to each other (Figure 2-4D). The mats most likely acted as cushioning, protection, additional security against winds and possibly helped avoid potential predators because the stickiness of the webbing may make ingestion difficult, and may provide micro-habitats as well. The silk may potentially be chemically protected via the cycasins and other neurotoxins found in the host plants

(Nash et al., 1986; Bowers et al., 1989; Nash et al., 1992; Rothschild, 1992; Osborne &

Jaffe, 1998). Wild colonies exhibit the same aggregate pupal masses; this behavior may be advantageous for securing the brood against the stochastic weather patterns in southeast Florida, such as tropical storms, hurricanes. Wild colonies may be site loyal to pupation sites, as well, perhaps following chemical or silk trails from prior groups. I have photographs of pupal clusters located on the same plant identification sign every year in

Fairchild Tropical Botanic Garden.

Larvae that were under stress from food limitations due to aggressively feeding brood mates, handling (such as being moved from wild stock), larval competition for

space, food or other unknown factors, were capable of pupating after the third instar, forming exceptionally small pupae (Figure 2-5) and small adults with wing cord lengths under the mean of 2.1 cm for the captive colony. These individuals survived to adulthood and were often successful in mating as well, particularly the small males. The lifespan of small individuals was not adversely affected by size (Figure 2-6). The mean lifespan of individuals under the mean of 2.1 cm was 20.98 days (23.03 days for males and 19.06 for females). The mean wing chord length was 1.9 cm for both sexes.

Development time for small individuals did not vary significantly from the average; ovae development averaged 6.1 for both sexes. The combined mean larval development time for both sexes was 21.36 days (20.7 days for males and 22.0 days for females). The combined mean for both sexes for pupal development was 13 days

(12.7 for males and 13.2 for females). The combined mean for total development time for both sexes was 40.0 days (39.4 days for males and 41.4 for females).

Juvenile mortality is high in the larval stage and cannibalism was substantial in broods even when provided with virtually unlimited food. Cannibalism is common in otherwise herbivorous lepidoptera species (Richardson et al., 2012). There is an increase in survival to pupation in smaller groups, but it was not found to be as significant with Atala as it has in other species, such as the Riodinid Euselasia chrysippe (Allen, 2011) or others (Grasse, 1946; Denno & Benrey, 1997; Fordyce &

Nice, 2003). Of ninety broods, the number surviving to the next life stage increased as the life stage progressed from ova to larva to pupa and finally to adult eclosure (Figure

2-7.)

The total survival rate from larvae to pupae in a sample from the captive colony sample was 66.0% (n=3880 larvae). Smith (2000) recorded a survival rate of 47.4% from larvae to pupae in an introduced ‘wild’ colony in Key Biscayne (n=369).

Pupal Development

Pupal development time in the lab colony ranged from 11 to 19 days (n=1021).

The average development time was 14.4 days and the standard deviation was ±2.4d

(Table 2-2). There was a substantial difference in size and weight of pupae, but the results were similar to individuals from wild colonies, which also exhibit smaller than normal pupae under stress (Figure 2-5). Pupae were weighed as available and whenever time and equipment use permitted.

Weights of male and female pupae indicate that even though female pupae weighed more than male pupae (Table 2-4). There was no significant difference in development time (Table 2-5). Longevity was not affected significantly by pupal weight in either sex (Figure 2-8).

Pre-pupal stages occurred over several days, beginning between 10 to 14 days of larval development and usually in the fourth stadium. In the first stage, the larvae

“bunch up” in a vermiform position, expel the gut contents, and anchor themselves to the substrate. They are still be capable of re-locating before final stages begin, as evidenced by their changing positions in the cage if I handled them indirectly during this stage (I would sometimes cut the excess ends of the frond to which they were anchored in order to clean the box cage and I would find them somewhere else the next morning).

In this stage, the last larval integument begins to separate from the pupa forming internally and the skin has a semi-translucent and mottled appearance (Figure 2-9A).

The head and caudal segment turn a yellow color as the external integument further

separates from the pupa forming inside (Figure 2-9B and C). The body becomes more firmly anchored to the substrate, the silk girdle is released from the thorax to support the body, and by the second or third day, the larva has made the transformation to a pupa.

The head and caudal region are a soft yellow color in the newly formed pupae (Figure

2-9D). Pupation takes place within one or two days after the first pre-pupal stage, indicated by the shedding of the last larval skin with associated mouth parts and exuviae (Figure 2-9E). Experiments performed in environmental chambers showed that different environmental conditions caused polyphenism in the pupa (Chapter 4).

There was 91-98% survival from pupa to adult in the lab (n=1534 pupae) (Table

2). Smith (2000) reported a similar survival rate of 93% in the introduced ‘wild’ colony mentioned above. Small pupae eclosed as small adults of both sexes, with wing cord lengths ranging from 1.4 cm to 1.9 cm, and lived on average as long as adults that had an average wing cord lengths of 2.1 cm or more (n=600). Small males often successfully mated with larger females.

Total Development Time

Total development time from ova to adult ranged from 30 to 57 days in the lab

(n=1261). Average development time was 39.3 days with a standard deviation of ±3.7.

Ninety-four percent of pupae successfully eclosed as adults. Smith (2000) reported a

93% pupa-to-adult survival in the introduced ‘wild’ colony on Key Biscayne.

Development time for all life stages varied even more widely in environmental chambers programmed for seasonal experiments (Chapter 4).

Adult Measurements and Longevity

Average adult wing chord length did not significantly differ between the sexes

(n=600), but the smallest measurements were males and the largest were female. In the

captive colony, the average wing chord length was 2.2 cm for females, with a standard deviation of ±0.19 and 2.1 cm for males, with a standard deviation of ±0.15. In the captive colony, the smallest wing chord length measurement was 1.4 cm (male) and the largest was 2.5 cm (female), although one female eclosed measuring 2.7 cm in later experiments completed in environmental chambers (Koi, unpublished). Although no wing chord measurements were taken in wild colonies in prior years, photographs of deceased individuals in both captive and wild colonies placed beside common US coinage allows a visual comparison to be made (Figures 2-5C and 2-5D).

Measurements were taken of adults that eclosed from wild larval stock collected in May 2012 (Figure 2-9). Females in that brood exhibited an average wing chord length of 2.37 cm with a standard deviation of ±0.13 cm, while males showed an average wing chord length of 2.22 cm with a standard deviation of ±0.11 cm. Results from Atala reared in controlled environmental chambers are shown in Chapter 4 of this thesis show significant differences between seasonal growth rates and size. In wild colonies, as well as in the lab, seasonal availability of vigorous nectar and host plant resources may affect size.

There was no correlation of smaller adult size (wing chord length <2.0cm) and longevity (Figure 2-6). The smallest male measured in this colony (wing chord length of

1.9 cm) lived 53 days, and the smallest female measured in this colony (wing chord length of 1.6 cm) lived for 26 days, both longer lived than the mean combined longevity for both sexes of 22.91 days.

Adults emerged from the pupa primarily in the early morning, but at times this occurred later in the day. Wings were fully expanded within an hour but flight seldom took place before several hours had elapsed.

Females exhibit royal blue color on the dorsal forewing which varied in how much forewing area was covered, but not in color (Figure 2-11A). Color in both sexes is structural and may play important roles in visual recognition during courtship and mating. Male dorsal wing color varied between teal green and deep aquamarine blue that was apparent immediately after eclosure and was not due to scale wear (Figure 2-

11B). However, male dorsal wing color varied with environmental conditions (Chapter

4). Both sexes exhibit a hetergenous appearance due to curved scales interspersed with flat scales, evident under SEM microscopy (Figure 2-12).

Adults often exhibited a strong startle/warning response when first handled (to mark with identification numbers) by quickly flashing the ventral forewings open to expose the bright blue or green iridescent colors and, when possible, raising the bright red-orange abdomen in protest, possibly to advertise its toxic nature via its aposematic colors (Figure 2-13). Evasive tactics employed by flying adults included dropping suddenly to another flight level and changing flight direction erratically and unpredictably. They are capable of flying very fast on occasion, and males use elaborate aerial displays to attract females, although in general the butterfly is largely sedentary. The flight pattern has been described as “deceptively slow and lazy-looking”

(Klots, 1951) and it often flutters rather moth-like; the neurotoxins in its host plant may affect the insect’s flight muscles, as it is known that the bodies of the larvae and adults

contain high concentrations of cycasins and other toxic chemicals (Nash et al., 1986;

Bowers et al., 1989; Nash et al., 1992; Rothschild, 1992).

Adult Dissections and Ova Development

Five females were euthanized by freezing immediately after eclosion to document ovarioles and oogenesis; none had developed ova with chorions, but did possess 4-15 fully yolked oocytes, with a mean of 9.6 oocytes. After initial dissections of newly emerged adults, additional females were dissected at death to document egg production and development (n=141) (Figure 2-14). Chorions were developed between the third and seventh days even in unmated females and oogenesis was continual throughout the females’ lifetime. Females have six ovarioles; eggs from the same female often varied in size (Figure 2-2A). As many as 51 ova were dissected from one female (15 days old) that may not have oviposited at all. One 80 day old female contained no eggs and an 86 day old female contained 29 ova. Female continuous egg production and variability was evident in all age classes (n=128). Ova maturation with chorion also varied between 3 days (1 egg) to 7 days (21 eggs). A 9-day old female contained only one ovum, but another 4-day old contained 24 ova.

Ovipositing may vary between females; daily observations were made of the adult flight cages during the early morning (0800-0900h) and late afternoon (1500-

1800h), which were the regular “cleaning and feeding” times. Any mating, ovipositing or other behaviors were noted during those times. While some females were observed to oviposit often, no observations were made of others. The 86-day old female, for example, was not observed ovipositing, indicating perhaps that she had not yet deposited any ova, or was incapable of releasing them for the physiological reasons; neither were there observations of her mating. This could indicate either that she did not

mate or simply that she did not mate during observation periods. The 80-day old female with no eggs was observed ovipositing on two occasions but may have been continually ovipositing prior to her death. Of 99 observations, the average age of ovipositing was

16.27 days (standard deviation ±6.79). The oldest ovipositing observed occurred with a

41-day female, but viability was not known as the ova were inadvertently destroyed.

Lifespan and Sex Ratio

Adults lived an average of 23.6 days in the lab (female n=308; male n=292), with a very wide range from immediate death because of eclosion failure (0 days) to a female that lived for 86 days; standard deviation was ±13.4 days. The oldest male in the captive colony died at 71 days and originated as a late instar larva from the Miami-Dade

Co. summer stock and I photographed a one month-old Atala in a wild colony in

Broward Co. in 2008 (Figure 2-15) that I had observed daily. Wing chord length was not significantly correlated with sex. (Figure 2-16).

The sex ratio in the lab colony was not significantly different, with 308 females and 292 males in a sample size of 600. In first generation eclosed adults originating from wild larval stock in two locations, the sex ratio was approximately fifty-fifty in all sites but two (Figure 2-17). Lifespans were not significantly different between males and females, with males living only slightly longer than the females (Table 2-1).

There was no significant difference in development time or survival based solely on the number of larvae in the brood (Table 2-6), which was somewhat surprising as other researchers have indicated this (DeGrasse, 1946; Fordyce & Nice, 2003; Allen,

2010). There was cannibalism in all brood sizes, but not all broods.

Diseases, Pathogens and Abnormalities

Less than 1% of the individuals exhibited pupal eclosion failure or other physiological failures, such as unexpanded wings, in the captive colony (n=1282). In observations of pooled data from wild colonies in Palm Beach, Broward and Miami-

Dade Counties, between 2004-2005, I documented 4% of the wild populations with wing abnormalities, such as an inability to fully expand the wings after eclosure, and 8% of the population with eclosion failure (n=3430). Occasionally, an individual would eclose with short ‘truncated’ wings, but DNA analysis was not explored to determine if this was a result of the wingless gene being expressed and it occurred in less than .005% of the captive population (10,199 adults).

A Paeciliomyces fungus was occasionally found in ova, larvae and pupae, which is contracted in the substrate as larvae crawl from place to place (Boucias, pers. com).

In the egg, it would have been contracted via the female, which would have contacted the fungus as a larva. It has also been recorded in one Eumaeus species in South

America as well as in several other arthropod orders including Hemiptera, Coleoptera, many species of Lepidoptera and Diptera (Norstog & Fawcett, 1989; Fargues & Bon,

2004; Torres-Barragán et al., 2004; Munster et al., 2005; Ríos-Velasco et al., 2010).

One experiment with the captive colony indicated that the fungus is contagious, but more experimentation would need to be done to determine if it has a deleterious effect on wild colonies.

Discussion

There is higher mortality associated with the immature stages of life, but as the insect mature its survival rate increases dramatically. By the time the pupal stage is reached, it is highly likely to complete its life cycle to adulthood.

The development time of the embryo in the ova varied greatly (4-13 days), and may reflect on how well the ova is provisioned as well as seasonal variation. A larger egg that is 1.25 mm will presumably contain more yolk nutrients for the embryo and may influence its ability to remain in the egg; it has no need to eclose to search for food if the egg is well provisioned by the female. On the other hand, a small egg of 0.85 mm would have fewer provisions and the larva would be forced to exit in search of food.

It is noteworthy that some larvae were larger on eclosure, were much more aggressive feeders than others, and grew larger and developed much faster than brood mates. Neonate larvae measure between 0.5 and 1.5 mm. The individuals that started out as bigger neonates (1.5 mm) had a stronger start and fed more aggressively than the smaller larvae.

A survival advantage to ovipositioning in clusters is that the larvae eclose within a few days of each other providing easier host plant consumption as they concurrently scrape the surface of the leaves, which may be tougher in the fall and winter seasons, or scrape the surfaces of the cycad cones of the female plant. In addition, aggregation increases advertisement of its toxic nature by multiplying the effect of the larval aposematic coloring (Stamp, 1980: Bowers, 1993; Osborne et al., 1998). This may help warn potential predators of the sequestered neurotoxins, thereby providing increased safety (Stamp, 1980; Nash et al., 1986; Bowers et al., 1989; Nash et al., 1992;

Rothschild, 1992). There are few reports of observed predation (Schwartz, 1888; Smith,

2000 & 2002) except for many ant species and Assassin Bugs (Reduviidae). Pharaoh ants (Monomorium pharaonis) invaded the lab where the rearing cages were installed, but they did not attack living larvae or butterflies in the lab colony. Schwartz (1888)

complained that he could not rear the butterfly from the egg because of the same ant species, and because the ants would carry away the eggs in the cages, the newly laid eggs and fronds were placed in a location that the ants could not enter. The ants disemboweled deceased adults, however, and it was important to remove dead animals as soon as possible in the morning, and throughout the day. The ants would systematically peel away the red scales from the abdomen of dead adults, which presumably contain the neurotoxins from the host plant (Nash et al., 1986; Bowers et al., 1989; Nash et al., 1992; Rothschild, 1992), and cut open the abdomen to remove the fat bodies and internal organs of both males and females; they were particularly destructive with female carcasses, sometimes leaving nothing behind but a wing

(sometimes a mangled wing on which not even the number could be read).

Pupal development varied widely in this study, ranging from as few as 6 to as long as 29 days. Interestingly, if the larval state was longer, with five stadia, the pupal development took less time. Pupal size and weight also varied significantly, but there is no correlation with sex or longevity of emerged adults.

Total development time from ova to adult emergence was 28-57 days. Insects reared in controlled environmental chambers show increased range of development in all life stages partially associated with seasonal factors (Chapter 5, this thesis).

Wing chord length in this study cannot be directly compared to wingspans recorded in previously published reports, but the assumption is that wingspans would be approximately twice the length of wing chord measurements and therefore would be in agreement with this report. Lifespan is arguably the greatest discrepancy shown between published literature and this research. Although it could be rightly argued that

the captive lab colony was living in optimal conditions that affected its lifespan in a positive manner, results from this study demonstrate that the insect is capable of a much longer lifespan than previously known.

Although previously published descriptions of Atala life stages may not be congruent, my data suggests that each researcher recorded a ‘snapshot’ of the life of the Atala in different climes, life stages, environmental conditions so that previously reported data are not incorrect, merely incomplete. Much of the previously published information consists of puzzle pieces of a life cycle that do not seem to belong to the same butterfly; my research shows the complete picture, and how all these discrepancies describe the insect. I think the story of the Atala’s life history is comparable to the story of several individuals describing an elephant behind a curtain from different locations on the beast, isolated from the total animal and each other: one declares it is a long and narrow flexible cylinder. Another person states that it is a big, round and wide like a barrel. A third determines it is a thick cylinder with heavy nails.

Yet a fourth describes a long, narrow cylinder with a tufted end. It t is not until the whole animal is in view that the descriptions make sense. The life history of the Atala butterfly can be regarded in a more complete perspective now.

Conservation Notes

Factors That May Contribute to Crash-Eruption Cycle. Factors that may contribute to the Atala butterfly’s crash-eruption cycle are not necessarily directly associated with host or nectar resources, as noted by Smith (2000), and which I have also recognized in field work. There are plenty of times when there is a lot of new growth on the host plants and lots of nectar sources, but no Atalas are seen (Koi, unpublished), factors that Baggett (1982) mentioned. Ants were also mentioned by

Smith (2000, 2002) and mentioned as interfering with his ability to captive rear the insect by Schwartz (1888.) Poaching and collecting has been cited as possible detriments to populations (Baggett, 1982; Williams, 1996; Yamaguchi, 1995), and although I have certainly been approached by collectors, it is doubtful that the Atala is in danger of over-collecting in most colony sites during the past ten years (Koi, unpublished). Mosquito control spraying is certainly a factor to be considered (Bargar,

2012; Hoang et al., 2011), but as long as there are extant metapopulations in enough density and distribution, this is a necessary evil in urban southeast Florida.

The widely variable physiological life history adaptations that E. atala displays are beneficial for an insect that lives in a highly stochastic environment such as south

Florida. There are extreme temperature events, and as has occurred in the past few years, those extremes are becoming even more pronounced. Cold has been thought to be a factor but Landholt (1984) indicated that the Atala seemingly survived winter temperatures in the 30’s in Miami, and reported anecdotal information suggesting the insect experiences a “diapause” that is more likely a quiescent state; I also documented the butterfly surviving several consecutive nights of temperatures of 28°F (2.22°C). The saving factor in both may have been increased day temperatures that allowed the insect to recover from chill coma.

Migration has not been documented, although Rutkowski (1995) noted what appeared to be directed flight occurring in the Bahamas. Migratory or dispersal patterns have not been established for the Atala in southeast Florida, but could potentially account for seeming crash-eruption cycles.

Hurricanes cause tremendous destruction to biotic systems as well as economic damage (Rawson, 1961; Covell & Rawson, 1973; Davis et al., 1996). Droughts follow floods with no apparent predictability. All of this affects the host plants and nectar sources of the butterflies that have evolved to live in this very harsh environment.

Other factors potentially influencing population fluctuations include the fact that both sexes of the Atala multiple-mate, and both have the potential to live much longer than previously documented, continuing to mate until death (Chapter 3). Because of this, and the overlapping generations, there is greater gene flow than previously considered, as well. Females exhibit continuous egg production and will oviposit until death, laying up to 50 ova at one time.

In addition to a harsh natural environment, more than half of this butterfly’s extant populations now live in domestic gardens or remnant natural areas that are widely isolated and fragmented (Koi, unpublished), surrounded by miles of concretized urban developments filled with non-native ornamental plants that provide no nectar, or non- native invasive plants and animals (ants, reptiles and amphibians) competing with its host plant and natural nectar sources (Hanski, 1999; Hardy & Dennis, 1999; Smith,

2000 & 2002; New et al., 2002; Schultz et al., 2008; Imperiled, 2011). It must also develop resistance to pesticides, herbicides, unnatural fertilizers, pollution and mosquito control spraying (Hoang et al., 2011; Bargar, 2012). Domestic and public botanical gardens are a vital link in the survival of this butterfly (Smith, 2000; Koi, 2008) and continued support of educational programs, re-introduction endeavors and monitoring of wild and semi-wild colonies is an important step in maintaining viability of a species

(Rawson, 1961; Covell & Rawson, 1973; Kremen 1992a & 1992b; Emmel & Minno,

1993; New, 1993; Deyrup & Franz, 1994; Cornell & Hawkins, 1995; Hanski, 1999;

Hardy & Dennis, 1999; Schultz et al., 2008; Algar et al., 2009; Imperiled, 2011).

There is increasing awareness of the need to protect cycads worldwide

(Oberprieler, 1995a, 1995b, 1995c, 2004; Chemnick et al., 2002; Schneider et al.,

2002; Pérez-Farerra et al., 2006; Nagalingum et al., 2011; Calonje et al., 2013) and in an urban environment, the value of ornamental non-native cycads can be exponential.

Control of herbivory in an Integrated Pest Management (IPM) manner (Culbert, 1994) is vital to sustaining the urban populations to protect both the cycad and the butterfly.

Eggs can be fairly easily removed from the slick waxy surface of most Zamia plants with a fingernail or metal spatula, which is helpful for those who wish to remove the ova to reduce the population to control eruptions or who wish to protect their cycads from herbivory damage. Detached eggs should be frozen overnight to humanely dispatch of unwanted future generations, although eggs that land in the substrate will be found quickly by predatory ants or anoles. Neonate larvae that hatch in the substrate are so small that they will be unlikely to survive long enough to find the host plant again before predators find them (especially since they do not harbor toxins until they have ingested plant material).

Lastly, there is a growing population of human inhabitants who are completely disconnected with the natural environment and the role that insects play in maintaining the health of the planet. Once asked “What good are they?” regarding ants, E.O. Wilson snapped back, “What good are you!?” A great reply that makes people think about what they are bringing to the world.

It is important to acknowledge the wide variability in the butterfly’s life history, partly to place prior work into a perspective that upholds seeming discrepancies made by previous scientists and publications. Understanding the life stages of the Atala butterfly will help scientists, biologists and homeowners manage the populations and colonies in pine rocklands, parks, and private or botanical gardens that host the butterfly

(Schultz et al., 2008). Conserving these elements is mandatory for human existence and recognizing the value of all of the earth’s biota is one of the most important steps in bringing the balance back to humanity.

Table 2-1. The average age of female ovipositing and mating for males and females (n=234). Longevity for mated females (n= 76) was not significantly shorter than mean longevity for mated males (n=76).

Average Age of Average Age of Average Age of Lifespan of Lifespan of Female Ovipositing Female Mating Male Mating Mated Females Mated Males Average 19.36 15.45 11.98 32.96 34.38 St. dev. ±7.85 ±8.94 ±7.63 ±8.83 ±11.03

Table 2-2. Atala development time from ovipositing to adult emergence in a captive colony housed in laboratory temperatures of 24-26 C° (n=1021).

Ova Larvae Pupae Total development time to adult Time (days) 4-13 14-28 11-19 30-57 Mean dev. time (days) 6.6 18.4 14.4 39.3 St. dev. (days) ±1.1 ±3.6 ±2.4 ±3.7 Survival to next stage n=10582 36% 58% 94%

Table 2-3. The average weight of larvae at first eclosure in the laboratory, 5 days and 11 days.

Neonate larvae Five-day-old larvae Eleven-day-old larvae Mean weight (g) 0.0072 0.3853 0.5374 Range (g) 0.0004-0.0025 0.0076-0.0858 0.4702-0.60466 St. dev. ±0.0009 ±0.0347 ±0.0950

Table 2-4. Mean weight (g) of Atala pupae by sex and age at weighing. Female pupal Female (n) Male pupal weight Male (n) Age of pupa (days) weight Mean weight (g) 0.4251 6 0.2679 1 5 Range (g) 0.3626-0.4648 St. dev. ±0.0387

Mean weight (g) 0.4102 5 0 6 Range (g) 0.3455-0.4676 St. dev. ±0.0515

Mean weight (g) 0.41842 5 0.34827 10 7 Range (g) 0.3850-0.4414 0.2441-.4580 St. dev. ±0.0515 ±0.0773

Mean weight (g) 0.3995 16 0.3954 6 8 Range (g) 0.3564-0.4409 0.3442-.4308 St. dev. ±0.0232 ±0.0314

Mean weight (g) 0.4124 4 0.3422 1 9 Range (g) 0.4026-0.4276 St. dev. ±0.0108

Mean weight (g) 0.3799 10 0 10 Range (g) 0.3217-0.4304 St. dev. ±0.0333

Mean weight (g) 0.4049 5 0.3049 2 11 Range (g) 0.3865-0.4345 0.2121-.3976 St. dev. ±0.0196 ±0.1312

Mean weight (g) 0 0.3786 1 12 Range (g) St. dev.

Mean weight (g) 0.3617 8 0.3461 5 13 Range (g) 0.3349-0.3940 0.2841-.4052 St. dev. ±0.0205 ±0.0514

Table 2-4. Continued Female pupal Female (n) Male pupal weight Male (n) Age of pupa (days) weight Mean weight (g) 0.3639 2 0.3629 2 14 Range (g) 0.3413-0.3865 0.3267-.3990 St. dev. ±0.0320 ±0.0511

Mean weight (g) 0.3469 3 0.3202 1 15 Range (g) 0.3910-0.3307 Standard deviation ±0.0386

Table 2-5. Pupal development time is not significantly influenced by sex.

Mean Pupal Development Time (days) Observed Expected Chi square female 38.8947 39.1238 0.9587 male 39.3529 39.1238 3.8415 p -value >0.05

Table 2-6. Brood size did not significantly alter larval survival. 11 21 31 41 51 61 71 81 Brood size 1 to to to to to to to to to 91 to 101 to 111 to 121 to 131 to 141 to 151 to (larvae) 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Mean survival larva-to- pupa (%) 0.75 0.63 0.76 0.49 0.78 0.50 0.61 0.14 0.62 0.81 0.46 0.45 0.31 0.77 n/a 0.42 St. dev. 0.31 0.31 0.25 0.41 0.23 0.28 0.05 0.00 0.20 0.09 0.23 0.00 0.35 0.13 n/a 0.30

Mean survival pupa-to- imago (%) 0.95 0.89 0.95 1.00 0.97 0.98 0.95 1.00 0.50 0.38 0.98 0.88 1.00 0.96 n/a 0.96 St. dev. 0.17 0.11 0.05 0.00 0.03 0.07 0.06 0.00 0.17 0.07 0.05 0.00 0.00 0.05 n/a 0.05

Number of broods 20 17 13 4 6 7 2 1 4 3 5 1 2 2 0 2 n=10582 ova produced n=3860 larvae (36% survival rate) which produced n=2244 pupae (58% survival rate) which produced n=2115 adults (94% survival rate)

25 n=600 20

10 Frequency Ova SizeFrequency of 5

0 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 Diameter of Atala Ova (mm)

Figure 2-1. Frequency of Atala ova sizes exhibiting a bi-modal distribution. (n=600).

Figure 2-2. Atala ova. A) Visually apparent size differences in ova from a dissected female. B) SEM photograph of an Atala ova showing highly sculptured chorion ultrastructure and micropyle. Photos courtesy of S. Koi.

Figure 2-3. Ovariole and developing larva. A) Newly formed ova (right) and developing oocytes (left) in one of six ovarioles from a three-day old female. B) The embryo is visible through the clear proteinaceous exudate on the underside of the ova, which is produced by the females’ accessory glands, allowing the eggs to adhere to the host plant’s leaves, cones or stems. Note the red- orange scales on the upper surface of the egg, which are released from the female’s abdomen as she oviposits, and that may act as protection against predators. Photos courtesy of S. Koi.

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Figure 2-4. Automontage photographs of life stages. A) Two-hour-old newly hatched larvae measure between 0.5 and 1.5 mm in length. Note just-hatching larva (arrow). B) Nine-day old larvae from the same brood, and which eclosed on the same day, exhibit high variability in size. The smaller larva successfully pupated three days later than its brood mates but developed into a smaller pupa and subsequent adult. C) Five normal larval instar exuviae. Stadia were variable, between three and five, with most larvae pupating at the fourth instar. The first instar occurs at three days. D) Silk mats may form an extensive anchor to the substrate beneath an aggregation of Atala pupae. Photos courtesy of S. Koi. B

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Figure 2-4. Continued.

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Figure 2-4. Continued.

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Figure 2-5. There was a significant difference in size and weight of pupae not correlated with sex. A) Pupae from the captive colony, approximately four days old. B) Compare with a similar photo of pupae from a wild colony in Fort Lauderdale taken in 2006, approximately two days old. C) Photographs of deceased individuals from the captive colony placed beside common U.S. coins show marked size differences. D) Deceased individuals collected in 2006 from a wild colony in Broward County after Hurricane Katrina beside common currency display the same size differences as the captive colony. Photos courtesy of S. Koi.

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Figure 2-5. Continued

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2.0

1.9

1.8

1.7

1.6 Wing chord(cm) length Wing

1.5 0 5 10 15 20 25 30 35 40 45 50 55 60 Lifespan (days)

Figure 2-6. Wing chord length does not affect lifespan in individuals measured below the combined mean of 2.1 cm for females and males (n=66).

1.2

1.0

0.8

0.6

0.4 Proportoin Survival Proportoin 0.2

0.0 Ova Larvae Pupae Life Stage

Figure 2-7. Mean proportion of survival of Atala per life stage (n=10,917 ova).

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0.500 0.450 0.400 0.350 0.300 Female pupal weight 0.250 (n=20) 0.200 Male pupal weight 0.150 (n=17)

0.100 Pupal Weight (g) Weight Pupal 0.050 0.000 0 20 40 60 Lifespan (days)

Figure 2-8. Longevity was not significantly affected by pupal weight in either sex.

3.00

2.50

2.00

1.50 Female winglength 1.00 Male winglength 0.50

0.00

3 1 1 1 1 1 2 2 2 2 2 2 2 3 1 N=30

average

Figure 2-9. Graph of wing chord length of emerged adults collected as wild late larval stock and that completed final instar in the lab, pupating immediately after relocation. Numbers refer to site locations (1 and 2 are in Broward County and 3 is located in Miami-Dade County). The last column is mean values of all 30 insects.

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A B

C D

Figure 2-10. Development of Atala pupal stages; the head region is on the right. A) The dorsal surface of the larva begins to disengage from the larval integument, leaving a mottled appearance. B) The ventral surface displays extensive webbing and the head region and caudal end turn yellow by the second day. C) By the second day, the silk girdle has formed and the larval skin is further disengaged (note the clear prolegs). D) On the third or fourth day, the pupa is fully formed. E) Automontage of 4-day old pupa showing silk girdle around the thoracic segment and mat attached to the host plant leaf, with cast-off larval skin at the caudal end of the pupa. It is shown in a head-down position. The pupa is facing down, head to the right. F) Pharate adults can be visualized through the pupal integument at 18 days, usually eclosing between the 10th and 20th days. Note the black wing pads and red abdomen. The pupa is facing down, head to the right. Photos courtesy of S. Koi.

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E F

Figure 2-10. Continued.

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Figure 2-11. Dorsal wing colors of Atala. A) Female dorsal wing color is royal blue. B) Male dorsal wing color varied on a continuum between teal green (top) to deep aquamarine blue (bottom). The thoraxes of both males have lost scales from activity and age, appearing metallic in places. Photos courtesy of S. Koi.

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A B Green

Blue

C D Red-orange

Blue Aquamarine

E F Red Aquamarine

Green Black

Figure 2-12. Wing scales have a heterogeneous appearance due to curved scales interspersed with flat scales. A) SEM photograph of scales from a “blue” male. B) SEM photograph of scales from a “green” male. C) Black and blue scales from a “blue” male. D) SEM photograph of “red-orange” scales from a female abdomen. E) Aquamarine scales clearly display the baffling ultra- structures that reflect light, causing iridescence. F) Red scales from the wing patch show a smooth, non-reflective structure. Photos courtesy of S. Koi.

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Figure 2-13. Atalas of both sexes exhibit a strong startle and warning response after being handled. This female displays her warning: “I am toxic!” Photo courtesy of Gary Bernard.

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Figure 2-14. Graph displaying number of ova found per age in females dissected at natural death (n=128). The swath of gray indicates the 95% confidence level. Zero point is altered to accommodate numerous data points in the same place at zero for the individuals dissected upon eclosion.

A B

Figure 2-15. Older individuals display worn and tattered wings. A) 68-day old male from the captive colony that died at 71 days. Note the extended proboscis, probably indicating stress from old age in this situation, not thirst or drinking. B) A month-old male from a wild colony in Broward County (2008). Photos courtesy of S. Koi.

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Figure 2-16. Normal wing chord length was not significantly correlated with sex

70 60 N=248; N F=119; N M=129 50 40 30 Males 20 Females 10

SH7 M21 SH1 SH2 SH3 SH4 SH5 SH6 M12 M13 M14 M15 M16 M17 M18 M19 M20 M22 M25

SH10 Number of Eclosed Adults Eclosed of Number Brood & Site ID

Figure 2-17. Sex ratio of first generation adults originating as wild larval stock from two locations exhibit normal 50-50 proportions (site “SH” is located in Palm Beach County and site “M” is in Miami-Dade County).

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CHAPTER 3 ETHOLOGY (BEHAVIORAL STUDY) OF EUMAEUS ATALA (LEPIDOPTERA: LYCAENIDAE)

Ethology, the species-specific analysis of an animal’s behavior, has developed into a helpful methodology for biological studies. Understanding the behavior of an animal forms a strong foundation for developing conservation programs, pest management policies and general husbandry for organizations that rear, and often deploy, captive bred insects and animals for biocontrol or re-introductions.

Insect biocontrol laboratories, for instance, must fully understand the cultural aspects of an insect’s life as well as its basic biology, not only to effectively rear the insects, but also in order to safeguard native habitats from potentially unforeseen repercussions. Unknown behaviors in the insect such as a host plant switch caused by an unexpected environmental factor could be potentially devastating to the ecosystem in which the biocontrol insect was released.

The intentional discharge of the biocontrol cactus moth, Cactoblastis cactorum, into Australian pastures in order to control an invasive introduced non-native prickly pear species (Opuntia sp.) was highly successful because there were no native cacti to attack. The same insect, however, proved to be a disastrous introduction into Caribbean ecosystems in 1957, because its switch to native cacti was not foreseen (Hight et al.,

2002). The moth was later discovered on Big Pine Key in south Florida in 1989, and has since spread to much of the southern United States, including the Carolinas. The ultimate goal of current biocontrol methodology for the introduced cactus moth is to prevent the infestation of native cacti in southwestern United States and Mexico (Hight et al., 2002).

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Grasping the importance of in-depth study and observational documentation of life stages and behavior, these factors were recorded in the Atala butterfly in order to further scientific understanding of the insects’ biological and ecological roles, crucial for successful reintroduction programs, and critical for determining the true conservation status of the insect in wild colonies. All life stages were documented; as Zalucki et al.

(2002) noted, “First instar caterpillars are not simply small versions of later instars. . . it should not be assumed that because you know the biology of the fifth instar, you know the biology of the first instar.”

Insect Behavior

While vertebrates are more often the subjects of ethology, insects exhibit a wide range of behaviors that can be catalogued and described (Immelman & Beer, 1998).

Terminology, definitions, protocols for observation and other materials are still being vetted and approved regarding insect ethology. Maintaining consistency and standardization of terms is complicated by the vast array of biota and taxa being studied worldwide. Basic terms, such as “resting,” may describe different kinds of behaviors for different insect taxa, and therefore researchers often define and give examples of the unique or associated behaviors of their study subject when presenting an ethogram, an inventory of behavioral actions (Immelman & Beer, 1998). These definitions may or may not describe the same distinctive way of presenting a behavior as it is displayed in other species.

Some of the writings of early naturalists could be called mini-ethograms as they often contained keen observations of insect behavior; for example, Schwartz (1888) commented that the Atala butterfly was “. . . so tame. . .that it is the easiest thing in the world to gather some observations on its natural history.” Knowledge of that “tameness,”

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has been essential for scientists involved in collecting the insect for research, collecting for captive rearing, or for removing the insects from an eruption site in order to re-locate them to a site in which the Atala population has ‘crashed.’ (Koi, unpublished).

Materials and Methods

Ethological observations of the imperiled butterfly Eumaeus atala were made during rearing of both a captive indoor laboratory colony and a captive outdoor colony.

The indoor colony was housed in a standard lab located in the Entomology/ Nematology

Department on the campus of the University of Florida, Gainesville. Adults of both sexes were allowed to freely interact with each other within the confines of a 6’ x 6’ x 6’ walk-in fabric mesh cage (LiveMonarch, Boca Raton, FL). The adult colony was provided with a constant supply of water and artificial nectar (Gatorade™, Pepsico, Chicago, IL) presented via slightly altered feeders developed by Hughes et al. (1993). Artificial nectar, undiluted in orange or tropical fruit punch flavors, was supplemented with occasional seasonal flowers (usually Bidens alba). The flight cage was supplied with native larval cycad host plant leaves, Zamia integrifolia, for ovipositing daily. The colony was lightly sprayed with water twice daily to simulate dew.

The outdoor captive colony was enclosed in a screen room (approximately 4’ x 8’ x 12’) within a free-standing screenhouse. The insects were provided with a variety of nectar flowers, such as Bidens alba, but no artificial nectar or additional water besides naturally occurring rain or dew was provided. Drowning in heavy downpours was a cause of mortality in the outdoor colony and has been noted by other researchers

(Zalucki et al., 2002 and references therein). Therefore, the outdoor colony was fitted with a 6’ diameter beach umbrella to provide protection from heavy rains and excess sunlight, in lieu of trees with big leaves, such as Sabal Palmetto (Sabal palmetto), to

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which they may have access in a natural South Florida environment. A tall shrub of

Sweet Almond (Aloysia virgata) provided nectar, for instance, but not shade or protection from downpours.

The outdoor colony was provided with potted host plants, Z. integrifolia, on the cement floor of the cage; eggs and larvae were counted and monitored daily, but were not touched, moved or manipulated. Adults were marked with identifying alphanumeric codes when installed into the cage, or when they emerged from the pupae of the first or second generation, but not handled thereafter.

All life stages were photographed regularly in both colonies; adult courting, mating, ovipositioning and death behaviors were recorded daily as well as observations of larval behaviors, including newly hatched larvae. Pupae were counted and monitored but were not manipulated, with one exception (one pupa that fell in a heavy rainstorm was replaced on its original frond).

Differently aged pupae from the captive indoor colony were placed into acoustic chambers to determine sound production, based on earlier reports that lacked complete documentation (i.e., data on the sound frequency, age of pupae, sample size, or reference to the original author) (Rothschild et al., 1986; Rothschild, 1992)

Atala Adults

Learned behaviors

Honeybees may have been the first insects studied that proved insects exhibited true learning behavior as well as the ability to communicate that knowledge to their sisters (Wray et al., 2011), but scientists have discovered some kind of learning in nearly every species. McNeely & Singer (2001), for example, trained Euphydryas editha

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to recognize a novel flower resource, although they were not successful training it recognize an unfamiliar host plant.

In the lab, Atalas learned from congeners very quickly where the water and nectar sources were in the flight cage (Figure 3-1). At least one savvy male discovered how to access water via the waterpic (Figure 3-2) that housed the host plant for ovipositing, even though several water and nectar feeders were available at all times. A colleague tried to rear some Colias butterflies in the lab which could not or would not adapt to using the feeders, causing her volunteers to perform hours of hand-feeding.

The Atalas, usually resting on the cage roof or walls, responded to humans that entered the flight cage by actively flying around and even landing on the helper, perhaps in anticipation that nectar and water would be renewed. Whether the insects were displaying curiosity, greetings, or simply some kind of recognition cannot be truly determined, but they did fly about and land on the helpers, often eliciting delighted giggles from the humans. They are certainly, as Schwartz mentioned, as “tame” as any butterfly with which I have ever worked.

Perchers and patrollers

In many flying insects, the males of the species may be labeled as “perchers” or

“patrollers.” Patrolling insects guard their territory by actively flying from one location to another, often in a repeated trapline pattern (often a fixed action pattern), while visually searching for mates, competing males, or prey. Perching, sitting still and attentive, occurs as the insect chooses a site in a higher location that allows it to peruse the vista for potential mates, prey or competing males. Different species of dragonflies display both kinds of behaviors (Daly et al., 1998). Mantids are an example of perchers, which exhibit a ‘stealthy’ behavior as well; Reduviidae species are perchers that ambush their

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prey while Pentotamides actively hunt for their prey by “eavesdropping” on the vibrational communications of tree hoppers (Cocroft & Hamel, 2010; Hamel & Cocroft,

2012).

Atala males exhibit perching behavior, both in the lab and in wild colonies; they are often site-loyal to their perches once chosen and will fly out to greet a female or challenge a male, returning to their perch. Atala males frequently perch on the host plant, or above the host plant on the nearest tree or higher vegetation (Figure 3-5).

Perch site loyalty has been documented in a Mexican species in the same genus,

Eumaeus toxea, as well, which perches above the host plant, Z. maritima (=furfuracea),

(Martínez-Lendech et al., 2007). Its body size and fat reserves were correlated with the butterfly’s social status as well (as the resident or an intruder congener) (Martínez-

Lendech et al., 2007). The results of the Martínez-Lendech et al. (2007) study indicated that body fat was not significantly different between winners and losers, or between resident males and intruders. They noted that there was a higher body fat content in larger males, usually the resident, and that the residents usually won the contests.

However, there was a significant difference between resident males and losers.

E. toxea defends its perching site from other males (Martínez-Lendech et al.,

2007), as does E. atala, and likewise, neither species guard their mates, eggs or prevent other matings from taking place with females with which they had previously mated. Martínez-Lendech et al. (2007) indicate that successfully mating males are larger than males which were not observed copulating.

It may be that the data from my captive colony altered that behavior somewhat in

E. atala, because there appeared to be no significant difference between the mating

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success of smaller males (wing chord length of less than the average size of 2.1 mm) and larger males (males with a wing chord length of 2.2 mm and above) (Chapter 2). I did not measure body fat, but as I did dissect over a hundred individuals, and although body fat generally decreased with size and age, it was not an absolute. Some of the young adults died with few fat reserves, possibly because of inadequate intake as a larvae, and possibly combined with an insufficient nectar uptake as a newly eclosed adult. Some older adults that died still maintained substantial fat bodies and the cause of death may have been another unknown factor.

Hernández and Benson (1998) report that small males sometimes have an advantage in these territorial contests. Small males in the captive lab colony were successful at mating and may have had the small male advantage: while other males were exhibiting territorial contests, or aggressively pursuing females, these males did not actively engage in conflicts with larger males, but simply approached and mated with females (Figure 3-4) (see also Chapter 2). This is known as the “satellite frog” technique after a study that showed successful mating of less aggressive males with females while the more aggressive males were “busy” showing off and vocalizing.

Lekking, hilltopping and canopy dwelling

Lekking, in which the males of a species congregate to await females for the purposes of mating rituals and choice was described by Bradbury (1984) and is not observed in wild colonies of Atala. Wild colonies of Atala butterflies form mixed sex- congregations around and on nectar sources as well as near or on the host plants.

Butterfly “hilltopping” is similar to lekking (see below). First described in detail by

Shields (1967), it consists of males and females meeting at a specified location, at a higher elevation than the surrounding landscape for the purpose of mating (Alcock,

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2010). Baughman & Murphy (1988) noted that “hilltopping” may be only a few feet above the surrounding landscape, but that the behavior brings together males to await females in lekking as described by Bradbury (1984). Hilltopping in great numbers occurs among members of the tribe Eumaeini in Colombia (Preito & Dahners, 2006). In

Australia, the loss of habitat was so pronounced in one area that it provoked legislation to protect a hilltopping area used by declining butterfly species, because butterflies are known to be site-loyal to hilltopping locations (NSW, 2011).

While “hilltopping” is also not known in Atala colonies, as mating interactions occur mostly in the canopy of trees or on the host plants. “Canopy resting” may be the closest approximate to hilltopping in the south Florida and Caribbean sea-level communities where the Atala lives. In the captive colony, both sexes rested on the roof

(“canopy”) of the flight cage for a large portion of the day unless they were actively flying, nectaring or mating (Figure 3-5). This is also evident in the “flight” video (Object

3-1). Although Miller and Miller recounted seeing E. toxea climbing to higher elevations flying along a stream bed in northern Mexico in 1967 and 1969, and an occasional mating pair, they did not record lekking behavior as defined by Bradbury (1984)

(Jacqueline Miller, pers. com.), which he defined as adult meeting locations for the purpose of breeding that are without nectar or host plant resources. Miller and Miller documented nectaring, but did not visualize the actual site (i.e., if nectar and/or host plants were at the location where the E. toxea butterflies finally congregated).

E. atala is considered a lowland species, and the fragmentation of its natural habitat may have impacted it in ways that have not been fully documented. More

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research and field work needs to be done to capture the effects of the changing coastal environment, altered landscapes and isolated colonies.

In wild colonies Atala adults congregate loosely in the lower canopy branches of the trees found on the site (Figure 3-5); in pine rocklands, the trees are primarily slash pines. In tropical hardwood hammocks or domestic gardens, the trees may be cypress, live oak, mahogany, fiddlewood, stoppers, dahoon holly, wild lime, satinwood or torchwood, among others. The butterflies utilize the nectar on any of the fruit trees that produce small flowers as well, such as avocado, mango or sweet almond.

Atalas are not strong fliers, possibly because the neurotoxins ingested in their host plant affect their muscle control (via disruption of neuron signaling), and the less- sclerotized wing veins found in many Lycaenids. Males are capable of robust aerial displays nonetheless (see Object 3-1) and females will disperse in search of new host plants (pers. obs.). Rutkowski (1995) reported observing directed flight of Atala adults along the shoreline in Abaco and suggested that there may be a possible Bahamian source of our south Florida Atalas, but this has not been proven. It is known that

Lycaenids are able to survive tropical winds, and may use them to disperse (Robbins &

Small, 1981). I have photographed newly emerged adults clinging tightly to a coontie leaflet two days after Hurricane Katrina (August 26, 2005), which had still had wind gusts of 21-36 mph.

Object 3-1. Eumaeus atala behaviors

Group feeding

Feeding strategies that are observed in larval aggregates (see below) are also observed in adults as they cluster around nectar sources en masse (Figures 3-1 and

Figure 3-6). Conspecific recognition plays a part in the group feeding. The heavy

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inflorescences of native palms, such as Serenoa repens and Sabal palmetto, are strong attractants for both sexes and the insects will congregate on the flowers (Figure 3-6).

This is not “lekking” behavior as described by Bradbury (1981), which occurs in areas not normally associated with the species (i.e., no resources) and wherein adult males gather in a specific spot to await the arrival of females. On the contrary, Atalas group in areas with nectar and/or host plants, which indicate immatures (eggs, larvae and pupae) are usually present as well.

In the lab, the Atala adults understood how to utilize the artificial nectar feeders the first time they were introduced to it. As soon as they were placed into the flight cage, their tarsi were placed on the rim of the feeder so that they could taste the nectar, although the insects did always drink immediately, they remembered and recognized the vials. The color of the drink often changed in the beginning as different flavors were tried and the Atalas did not have any difficulty recognizing the feeder as the source of nectar, regardless of its color. They clearly preferred some flavors to others, however, based on how quickly the vials emptied of liquid. They also sometimes gathered on the plastic wash bottle used to fill the feeders while the feeders were being filled, apparently recognizing the bottle as the source of the nectar.

There was no outright aggression between conspecifics at the nectar feeders, although the insects clustered in tight groups, especially first thing in the morning.

Heliconian butterflies (Hay-Roe & Mankin, 2004) and many other lepidopteran species exhibit pronounced aggression on nectar sources in the wild (pers. obs.), forcing smaller butterflies from the flower head.

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No acoustic study of adult Atalas was done in this research, but wing-click communication in adult Heliconians was documented by Hay-Roe and Mankin (2004) and appeared to indicate that roosting individuals have different density-tolerances for known or unrecognized individuals in the colony. Although adult Atalas do not display

‘close-contact’ behavior, as they do as larvae or pupae, the adults do exhibit a high density-tolerance for conspecifics in the wild as well as in the lab.

Adult host plant recognition: Visual recognition

Some species utilize olfactory clues more than visual clues and others rely primarily on visual clues (Tang et al., 2012). Female Atalas may visualize the larval host plant, Zamia integrifolia, before responding to other clues in an unfamiliar environment as she uses her innate “search image” to scan the environment for the host plant.

Insects have been shown to be receptive to UV reflectance patterns and leaf color

(Bernard & Remington, 1991; Briscoe et al., 2003; Sison-Magnus et al., 2006; Tang et al., 2012) as well as plant form and structure (Erlich & Raven, 1964; Feeny, 1976). Most species of butterflies have excellent color vision (Bernard & Remington, 1991; Briscoe et al., 2003; Sison-Magnus et al., 2006; Tang et al., 2012).

In the lab, female Atala butterflies did not land on or explore fronds of the proffered host plant until the leaf was hung from the ceiling so that it was “eye level” to them in the flight cage (~five feet high) and more visually apparent, even though the host plant in a natural environment is located on the ground. In a natural environment, other clues, such as plant volatiles dispensed by the wind, from an intact plant (rooted in the ground), may play an important role in host recognition. Fronds were encased in an aquapic to keep them fresh. The cage environment was quite unlike a wild habitat, filled with natural sky blues and plant greens, and may therefore have made recognition of

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the plant difficult on the ground (the cage had a black floor and white netting walls and ceiling).

The outdoor colony females, on the other hand, had no trouble visualizing the potted plants on the ground, which were set less than a foot apart. In addition, there were nectar plants and small shrubs in the cage, and the open sky above the cage, more closely approximating a natural environment.

Butterflies usually express three visual pigments, with absorbances up to ~350 nm (UV), 440 nm (blue) and 530 nm (long-wavelength) (Sison-Magnus et al., 2006), but it has also been shown that Papilio xuthus swallowtail butterflies are sensitive to the color red (Yamashita, 1995). Phoebis sennae butterflies are attracted to the color red

(Glassberg et al., 2000). Lycaenids exhibit duplicate blue opsins and at least one species, Lycaena rubidus, also has sensitivity to the violet to orange-red spectrum

(Sison-Magnus et al., 2006). It is suggested that the additional blue opsin may be an aid in species recognition of conspecifics in the “blue” Lycaenids (Sison-Magnus et al.,

2006).

Male and female butterflies may see things differently in the species studied

(Bernard & Remington, 1991; Briscoe et al., 2003; Sison-Magnus et al., 2006; Tang et al., 2012), and Atalas are no exception (Bernard, pers.com.). Although males do not need to recognize the host plant for ovipositing, they have learned to recognize it as a clue to find ovipositing females (personal obs. in the lab and field) (Figure 3-1).

Females, on the other hand, must find suitable host plants for their offspring. Heliconius butterflies use host plants and immatures as clues to find females as well (Estrada &

Gilbert, 2010). Preliminary studies by Bernard (pers. com.) indicate that male and

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female Atala butterflies have different receptors and respond to different colors and/or wavelengths. Those studies are in progress.

Adult host plant recognition: Tactile recognition

Insects utilize their antennae, setae and sensilla to recognize plant volatiles and mates (Daly et al., 1998), and I observed this behavior in the lab, especially whenever the fan would engage (every other hour during daylight). When that occurred, the Atala adult butterflies would become active and fly around the flight cage (Object 3-1).

Females responded enthusiastically by visiting the fronds of coontie when lab fans were directed toward the hanging host plants.

Touching the leaves of the host plant would activate the secondary responses to the chemical constituents. Females would repeatedly walk over the leaf surface of the fronds, actively antennating and becoming more active as the plant was recognized as a host plant. Once the female was confident that the plant was suitable, she would curve her abdomen to touch the leaf surface and commence to oviposit in a densely- packed cluster of as many as fifty eggs at a time (Figure 3-8). Females ovipositing were not easily disturbed from their concentration, although males would sometimes force mating and disrupt her. Eggs would often be displaced from her reproductive tract during coercive mating when this occurred (Figure 3-11) and it could be fatal to the males if they were inadvertently injured because the eggs prevented them from withdrawing their genital tracts (Figure 3-12).

Adult host plant recognition: Chemical recognition

Once visual image of the host plant matched the internal “search image” other factors came into the forefront of a females’ host plant probe. Predominant chemical constituents in the Atala’s larval host plant are methylazoxymethanol, azoxyglucosides

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and cycasin (Bell, 1967; Rothschild et al.,1986; Bowers & Larin, 1989; Nash et al.,

1992; Rothschild, 1992; Oberpreiler, 1995a, 1995b, 1995c). Ovipositing behaviors of the

Atala butterfly are stimulated by the chemical volatiles released by the cycad host plant.

Chemical receptors are used to recognize larval host plants by most insects, including butterflies (Minnich, 1921; Fox, 1966; Berenbaum, 1995; Nishida, 1995) and many other species (Fox, 1966). The foretarsi of female Ithomiinae for example, have groups of translucent trichoid sensilla on the foretarsi that were positively identified as chemoreceptors (Fox 1966) and in the 1920’s sensitivity to different sucrose concentrations was studied in different species of butterflies (Minnich, 1921).

Oviposition stimulants are perceived via chemoreptors located in dense sensilla on the fifth tarsomeres of swallowtail butterflies and many Pieridae species (Nishida, 1995).

These benefit the butterfly in finding suitable host plants. Drumming the forelegs on the plant surface helps release the chemicals that allow host plant recognition (Fox, 1966;

Nishida, 1995).

Atala females investigate the plant via the chemoreceptors located on the tarsomeres, as well as by volatiles released by the plant when subjected to larval herbivory (Feeny, 1976; Erlich & Raven 1964). They do not drum their feet, as do swallowtails (Nishida, 1995), but they do walk along the plant repeatedly to taste the plants in stereotypic manner. They antennate the leaf surface as well.

Similarly, danaine butterflies are known to use “leaf-scratching” on wilted leaves in order to release pyrrolizidine alkaloids used for the production of pheromones

(Boppé, 1983). Conspecifics are attracted to the group of mostly male butterflies as they jointly scratch and tear at the leaf surfaces. Neonates and young offspring of many

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aggregate-forming species display this “ganging-up” behavior, often overcoming the defenses of the food plant. Treehoppers, plant hoppers, larvae of many butterflies, including the Atala, utilize this method of group-feeding (Cocroft & Hamel, 2010; Hamel

& Cocroft, 2012). This is best described as “gregarious” behavior of both adults and larvae.

Females will also utilize other species of cycads used as ornamental landscaping plants in south Florida (Hammer, 1995; Koi, unpublished), all of which contain cocktails of varying amounts of the same and similar chemicals in different proportions

(Oberpreiler, 1995a, 1995b, 1995c). Larvae successfully complete their life cycle on all of the plants they have utilized in south Florida, but survival and development duration on at least one of them, Zamia vazquezii, is significantly decreased (Chapter 5).

Female ovipositing behavior

Females displayed no aggressive behavior with other females in the lab, even in dense aggregations while nectaring or ovipositing (Figure 3-7) nor have I observed female aggression in wild colonies. Females are born with fully provisioned oocytes but chorions are not formed until the third day, which is also the earliest noted ovipositing date after emergence in the lab colony (Chapter 2). Stamp (1980) indicates that when a population is sparse, which it often is in the fragmented metapopulations in wild colonies of Atalas, the females may lay ova in clusters to compensate for reduced time finding mates and or host plants. Atalas oviposit in large clusters, often times overlapping previously laid eggs by conspecifics (pers. obs.) (Figure 3-8).

Wild and captive females will oviposit before they are mated and will produce eggs until they die. One female in the lab colony still contained 29 ova when she died at

86 days old (see Chapter 2); another died in the process of ovipositing at 38 days of

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age. Egg clustering is common in aposematically colored larvae and adult species such as the Atala (Stamp, 1980; Bowers, 1993). Although the eggs are not aposematically colored themselves, they are provided with protective spicules and toxic anal tufts from the females, sometimes in a heavy palisade around the ova (Figure 3-9).

Egg clustering also helps neonates find each other, which facilitates feeding

(Stamp, 1980), especially on host plant cones or older, tougher fronds which females may choose when nothing else is available (pers. obs.). Hatching together has other advantages discussed below.

Atala butterflies oviposit in clusters containing eggs from a few to 50 or more on a leaflet, or on the reproductive cones of the host plant, usually the female cone although the male cone is utilized as well, when host plant leaves are not available because they have been completely consumed (pers. obs.). Ovipositing behavior in the laboratory and outdoor colonies was very similar, if not the same, as observations made of wild colonies. Sometimes, only a few eggs were laid on a frond and other times the leaves would be coated with eggs. The number seems to be partially determined by the availability of host plants, especially in pine rocklands and other natural areas where the host plants may be scattered, difficult to find or very small (Figure 3-10). Schwartz

(1888) described the female’s ovipositing as “taking a long time” and wrote that the eggs were “laid with a great effort, so that the insect has to rest for two or three minutes before going on with her work.”

The insect is multi-voltine and active populations are found year round in much of south Florida; the Atala exhibits a bi-annual ‘crash-eruption’ cycle that is not fully understood and which occurs regardless of host or nectar availability. Wild colonies

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exhibit a bi-annual “crash-eruption” cycle with two eruptions in early summer and late winter and two crash cycles in late November and early spring around March (Koi, unpublished).

The cycle is not fully understood, but over ten years of data indicate that it may be a chaotic cycle with overlapping years in different locations as ephemeral colonies die out or re-colonize sites (Koi, unpublished). The colonies appear to slow down or die off during late fall and early spring, and erupt into sometimes thousands of individuals in early winter and late spring-summer. These cycles may exhibit different patterns between different locations and/or during different years (Koi, unpublished). The initiation of summer rains, and hence new host plant growth, as well as day-length, may be associated with the population fluctuations, but this has not been fully explored.

There are population increases in some locations in winter (December-January) in southeast Florida, as well as in the summer (Koi, unpublished). There are years when the butterfly is not seen at all in places where it was once common, but it may show up a few years later. Pimental (1988) states that the time lag response between the herbivory of the host plant and the insect fluctuates, and that the insect is generally limited to consuming only 10% of its host plant resources. With the Atala butterfly larvae this may be a questionable assumption, because the larvae are capable of completely defoliating an entire bed of Zamia, as evidenced at Montgomery Biological Center or

Fairchild Tropical Botanic Garden (Koi, unpublished). These ephemeral populations are one reason the conservation status fluctuates between seemingly secure and nearly extinct in the same locations.

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This population cycle in wild colonies may be evident in the captive colony as innate behavior and may help explain the different age-mating relationships in the two observations noted above. There are hundreds of observations that have not yet been analyzed, but which may clarify this phenomenon concerning mating and age differences

Mating behavior

Both sexes multiple-mate throughout their lives with different individuals, and sometimes with two different individuals on the same day. Mating frequently occurs in communal groupings at times and pairs remain coupled for several hours, or as long as

24 hours (Object 3-1). Most mating occurs in the late afternoon, as Scott (1986) indicated for most lycaenids. Mating and ovipositing was observed less often when colony was small (fewer than 15 individuals) but once the colony size was built up to more than 20 individuals again, mating and ovipositing resumed. On one occasion, 60 mating pairs were recorded in one afternoon (Object 3-1). Communal mating behavior has also been seen in wild colonies, and pairs are often seen not only on the host plants, but on the nectar sources as well (pers. obs.).

Of 98 mating observations, females were older 53 times, males older 21 times and the pairs were the same age 14 times between the months of July and December

2012. In the months following, late December 2012 to February 2013, an additional 62 observations showed that males were older 34 times, females 17 times and the pairs were the same age 11 times.

There may have been demographic differences in the indoor colony during these two time periods, such as the numbers or ages of the individuals and/or the sex ratio.

The differences may also have been indirectly affected by season, for although these

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age-at-mating observations were of the indoor-housed butterflies, subtle changes in temperature and humidity in the building itself may have influenced their endogenous seasonal clocks.

The widest age ranges occurred between a 41-day-old female and 22-day-old male (19 days difference; she died in copulo) and conversely, between a 35-day-old male and a 1-day-old female (34 days difference) (Figure 3-17).

The average age of females observed mating was 15.45 days with a standard deviation of ±8.94 (n=102) and the average age of males observed mating was 11.98 days with a standard deviation of ±7.63 (n=102). The youngest female mating took place within hours of emergence to an equally newly emerged male in the same brood as soon as their wings were dry.

The average age of death for mated females was 32.96 days with a standard deviation of ±8.83 (n=57) and the average age of death for mated males was 34.38 days with a standard deviation of ±11.03 (n=58). There are hundreds of observations from the captive colony that have not yet been analyzed and based on other variability in the species it would not be surprising to find an even greater range in mating variability and activity.

Male mating behavior

Adult male Atalas emit pyrazines (Rothschild et al., 1992) when handled or when courting females and were capable of a less aggressive approach than usual when courting females (Object 3-1). Less intense courting behavior involved the male sidling up to the female, with androconia everted (Figure 3-11), and the caudal region of the abdomen curled strongly forward toward the female’s face while stroking her wings with his antennae.

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Males usually exhibit coercive mating, however, aggressively pursuing females.

Males also approach already mated couples with hairpencils and aedeagus everted, forcefully displaying in front of the females’ antennae and attempting to insert its aedeagus into the female (Object 3-1). Martínez-Lendech et al. (2007) also report aggressive behavior in E. toxea males

Males will approach females while the females are ovipositing on the host plant and their ova are sometimes forced out of the reproductive tract in the male’s fervor

(Figure 3-12). One male died with his genitalia thickly coated with ova when he was found dead the next day; the aedeagus and harp were still extended as though he was unable to withdraw his genitalia back into his reproductive tract because of the thick cluster of ova (Figure 3-13).

If an unpaired female is receptive, she turns so that her caudal region is accessible to the male and allows entry immediately (Object 3-1). Mating can occur immediately after the wings harden, but over 10,500 Atalas were reared in the lab during this fifteen month period and immediate mating after emergence was observed only once. Generally by the second and third day of life, however, mating occurs for both males and females.

Males also exhibit aerial displays, flying excitedly into the air, often toward other males, turning their wings dorsally to show the wing coloration and sometimes even flying upside down, doing ‘aerial loops’ that display the dorsal wing color (Object 3-1).

Wings scales of both sexes are highly structural and reflect UV light patterns, which are more visible when the insects are displaying in flight patterns, whether helping females find host plants or males find females, and other male competitors (See Chapter 2).

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Female solicitation and behavior

Females have also been observed soliciting males by approaching the males face-forward, antennating vigorously and even raising onto her back legs to use her forelegs to brush his frons and antennae. She will then turn so that her abdomen is aligned with his abdomen to join with the male. Solicitation by females in mating behavior has been observed in other species of Lepidoptera, including Pierids (Daniels

2007). In many cases, females were observed using their hind legs to ‘palpate’ the male’s abdomen when in copulo (Object 3-1). The purpose of this behavior is not known but it may assist the male in releasing spermatophores.

As mentioned earlier, Smith (2000) recorded a female using her wings aggressively in an attempt to beat down an ant that was attacking eggs. It was not clear from Smith’s description if the eggs belonged to her or another female.

Females are capable of rejecting a suitor by flying away or deflecting his approach. I witnessed a female stand on her hind legs and use her front legs to “beat” repeatedly on the head of an approaching male. He respectfully turned around and moved away.

Once a female was observed sitting on top of the folded wings of a male, which was perching on a nectar-soaked cotton swab (Figure 3-14). The cotton swabs were replaced by nectar feeders within the first week of the colony rearing (described earlier in this chapter). It is not known why she chose to sit on his wings, or if she even recognized him as another Atala.

Females do not seem to methodically reject small males (Figure 3-4) and it may actually be advantageous for some species to accept small male matings. Hernández and Benson (1998) describe a “small male” advantage in Heliconius sara

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(Nymphalidae) in which the smaller males successfully defended territory against larger males, perhaps because of increased flight agility. Pairings with small males could be an example of “kleptogamy” in which a less aggressive male “sneaks in” and mates with a female whilst other males are exhibiting territorial displays or chasing each other

(Immelman & Beer, 1989); these less dominant but successful males are sometimes referred to as “satellite” males because they act in the periphery of the territory.

Females did not outright reject males that were missing scales or otherwise “beat up” from aerial displays, territorial bouts or simple longevity (Figure 3-4). An older male, with missing scales or otherwise ‘worn’, may be attractive to females in that he has proven longevity, increasing the likelihood of improved offspring survival. Immelman and

Beer’s (1989) description of a “badge of status,” which indicates a male’s ability to hold territory, or to maintain female preferences, may explain a female’s acceptance of older, or ‘beat-up’ males. It was also shown in the lab colony that some males were much more successful at mating than others, averaging a different female every day or two.

Likewise, some females were observed mating more often than others. Age of mating did not matter in either sex, and matings of both sexes were observed until their death.

Distinctive mating behaviors

Two events were photographed in the flight cage of a group of three mating individuals, consisting of two males and one female. This incident was apparently not influenced by the number of individuals in the flight cage. The first event took place with only one other mating pair in the flight cage (Figure 3-15), while the second event was observed during a communal mating event consisting of 57 pairs located throughout the flight cage (Object 3-1). One occurrence was observed in a wild colony location, but the three individuals in copulo were the only adults observed in the site at the time. Isuspect

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that because of the coercive mating behavior exhibited by males that the two males approached the female simultaneously and consequently entered her at the same time.

In the first event, the males approached the female so aggressively that ova were forced from her body (Figure 3-16).

In the first observed triple pairing, the female was 3 days old. Both of the males were 7 days old. One of the males in the first triple-pair lived for 17 days and the second male died on an unknown day. The female of the first triple-pair lived 31 days and was observed to mate with a different male the next day. The males were not observed mating again after this event and the female was not recorded ovipositing.

In the second observed triple-pair, the female was also 3 days old. One of the males was 4 days old and the other was 11 days old at the time. The female lived for 23 days, and was not observed to mate with another male or oviposit. One of the males lived 36 days and the second male died on an unknown date. Neither male was observed mating again.

Another unusual event was photographed six times in the lab colony of two

‘paired’ males. One explanation may be that the coercive mating behavior of the males causes them to both to approach a female simultaneously, but she may fly away at the critical moment, and consequently the two males become unintentionally entangled. It may also be that the sex-ratio in the flight cage was unbalanced when these observations occurred, and the males approached each other in frustration. It could be, as noted with some other observations in other species, that the males were in such an excited state that they “mated” with the first available object which happened to be another male. Such paired males usually disengaged on their own, but the males from

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the first observed triple mating had to be manually disengaged when I noticed that they were still connected after several days and that they had been unable to drink or access food when paired.

In another instance, two males temporarily engaged for about a half an hour but disengaged themselves. I observed a male solicit another male on the floor of the flight cage, with androconia flashing. The males on the floor of the cage were generally older males (see section on Atala death behavior below). In 170 observations, male-male pairings (M-MP) were photographed 6 times; hundreds of observations in the indoor captive colony that have not yet been analyzed may indicate that this happens more often. It is not known if male-male pairing occurs in wild colonies of Atalas, although it has been documented in other species (see below).

Of the six M-MP photographed (n=12), only five of the males successfully mated with one or more females either before or after the M-MP event. One of the males successfully mated with four different females after the male-male pairing and lived 28 days. Another male was observed to pair with two different males two days apart. He successfully mated with a female the day after the first M-MP, but then paired with a different male the day after that. He then successfully mated with a female two days after the second male pairing. This male lived for 12 days. One of the male partners of that individual was not observed mating with a female after the M-MP, but he lived 62 days.

Another male in an M-MP was observed to mate with a female 12 days after the event and he lived 44 days. Seven of the nine females that mated with males that had been involved in male-male pairings were 3 days old or younger, and the remaining

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female was 6 days old. The twelve males were aged between 2 and 26 days old when observed in M-MP, but between 3 and 10 days old when mated with females.

Only two males of the male-male pairs were not observed mating with a female either before or after the single male-male event. Both of those males were close to, or older than, the average lifespan of 23 days (26 and 22 d. respectively). Those two males lived 11 and 62 days respectively after the male-male pairing.

In another M-MP, the males were 17 and 8 days old at the time of pairing. One of the males was observed successfully mating with different females twice before the male-male pairing, and he lived for 20 days. The other male in the M-MP was not observed mating with a female and lived 57 days. One M-MP occurred when they were

26 and 22 days respectively, and the males were not observed pairing with either sex again until they died at 35 and 24 days respectively. It would have been interesting to dissect the paired males to determine if there was spermatophore deposition, but unfortunately, I did not consider dissection when these events occurred.

There were two observations of male-female pairs in which one or both of the mates were found dead or dying. In one pair, both the male and female died at twenty days old and were found dead in copulo. In the other observation, a 25-day-old female died while in copulo with an 11-day old male which did not disengage until the female expired (Figure 3-17).

Two-male and one-female mating behavior has been recorded only a few times before. Odendaal et al., (1989(90)) describe two males and a female Euphydryas anicia mating in a captive colony that may have been the result of “scramble competition” wherein males aggressively approach a female and two manage to mate with her

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simultaneously. Masters (1974) describes two males and a female E. chalcedona mating in a wild colony; Perkins (1973) described two males and a female Phyciodes phaon copulating in a wild colony, which he called “pleoheterosexual mating.”

Perkins (1973) recorded inter-generic mating occurring between two males

(Speyeria mormonia erinna and Cercyonis pegala ariane), citing probable “parallel development in pre-copulatory behavior and/or chemical configuration” as possible explanations. Bhakare and Smetacek (2010) documented intergeneric mating in the wild between a male Castalius rosimon and a newly emerged female Talicada nyseus, and another between Chilades parrhasius and Zizina otis, sexes unknown.

Clark (2011) recorded inter-generic pairing between Asterocampa celtis and A. clyton. . .but more interesting than that was that the butterflies were both males. He noted that both males died the next day. Chaudhuri and Sinha (1997) documented a male-male pairing in tropical Tasar silk moths Antheraea mylitta in a captive colony and he states that both makes remained in conjunctio for 5 days and then died. Shapiro

(1988(89)) stated that males of Eucheira socialis have inbred to the point that they will mate with recently dead females as well as other males. He documented two males in copulo in the laboratory; the next day one male had died and was carried around the live male for hours before it manages to disconnect. Shapiro (1972(1973)) also documented a male Precis (=Junonia) coenia courting a female Colias eurytheme, which ignored the male’s attempts to copulate.

Benz (1973) and Johnston (1974) documented same-sex pairings between males of the same species as well. Benz (1973) commented that Zeiraphera diniana, the larch bud moth, became so excited under proper conditions eliciting mating behavior

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that males would try to mate with each other regardless of the presence or absence of females. Figures 3-30 through 3-32 graphically display a series of Atala mating events, including male-male pairings.

Atala adult death and dying behavior

Adult Atalas of both sexes, when under stress (from cold or another factor) and/or near death from old age, retired to the ground in the outside cage or to the floor in the indoor cage. This has been observed in wild colonies as well (Figure 3-18). In both captive colonies, near-death individuals crawled into tight spaces, such as the drainage holes on the bottom of a planter pot, whether inside or outdoors. The insects crept deep into the vegetation or fallen leaves in the potted plants when near death.

On occasion, the insects were not found before ants decimated all but a wing, sometimes leaving a numbered hind wing that identified the individual, but often leaving nothing but piece of a forewing providing few clues about the individual except sometimes the sex of the butterfly. There were occasionally butterflies that were never located and had to be considered “missing in action.”

An unfortunate experience occurred in the lab colony but did provide an observation confirming the death behavior of pressing into a small space to die. Nectar and water feeders were placed on top of a cardboard box that was turned upside down to form a platform on the floor of the indoor flight cage. This ‘table’ made it easier for the elderly or wing-damaged butterflies to reach the resources. The flaps of the box had been folded alternately, as one does to temporarily close a box lid for storage. The flaps were turned to the underside of the box, but apparently formed a slight opening that allowed the butterflies to scuttle into the crawl-space formed by the overlapping flaps.

Several months after installing the box as a ‘table’, I questioned whether it may have

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been a nesting site for marauding Pharaoh ants in the indoor captive colony. I removed the cardboard box, expecting to find swarming ants inside, but was sadly surprised to find nearly forty dead adult butterflies instead. They had worked themselves completely into the interior recesses of the box. I have no doubt that they were dying and that is why they sidled into the space in the first place, but it was unfortunate that I did not find them until months later and therefore could not determine their age at death to properly curate them.

To prevent this tragedy from occurring again, I installed a new ‘table’ using a small, closed Styrofoam “cooler” container normally used for ice. For unknown reasons, however, it was fortunate that the ants did not find this repository of deceased Atala butterflies or every one of the bodies would have been totally dismantled without much clue as to where they went.

In the indoor colony, I had watched an adult flatten and extend its wings like an airplane and scuttle underneath another small table sitting less than an inch above the floor. That table was also removed. It has been documented that Atala adult females are able to slink somehow into the slightest opening to oviposit on a host plant that is covered in fine netting to prevent that from occurring (Norstog & Stevenson, 1986).

Apparently this is how the female Atalas worked their way into the seemingly ‘closed’ nets Norstog and Stevenson has set up to determine pollination events of the Zamia plants at Fairchild Tropical Botanic Garden in Coral Gables, Florida.

Adults under stress or near death lose muscle control of the proboscis and it uncoils (Figures 3-16 and 3-19). Death throes include convulsions, fluttering wings and

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the relaxed proboscis. Often the wings evert forward at death and the ventral portion of the body is covered by the wings (Figure 3-17).

Dissections of the dead revealed reduced fat bodies and numerous missing scales on the thorax and upper abdomen, where the wings touch in flight, in both sexes.

Females generally had fewer ova and fewer missing scales unless they were quite younger than the average lifespan of 23 days. Males had many missing scales, tears on the apex of the forewings, and some wings on males had virtually nothing but the main wing chords intact when they died. The age of the male did not necessarily suggest how tattered the wings were as the condition of the wings were more an indication of how aggressive he was at displaying or pursuing females.

Adult defensive warning behavior

Adult butterflies contain neurotoxins ingested as a larva and are toxic enough that even large Argriope species spiders will cut them loose if one should be caught in the web (pers. obs.). Bowers and Larin (1989) indicated that the adults contained between 0.21 to 0.51 mg dry weight cycasin. It is probable that the iridescent colors of their wings and bright red abdomen act as aposematic warning coloration in the adults.

Sequestered and isolated free-floating bodies of undetermined origin or chemical make- up were also found in 24% of females dissected (to look at egg production), always between the thorax and abdomen in the adult. Based on the mass, color and location, I surmise that these masses are sequestered chemical toxins or other irritants ingested as a larva that the caterpillars were unable to expel when the gut was voided in preparation for pupation. I found masses of what appeared to be tiny bits of plastic in at least one adult (which could have been the plastic from the waterpic lids!) This will be further researched and all of the masses were saved as vouchers and to follow through

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with chemical analysis. There is a possibility, if this is true sequestration, and if the Atala adult abdomens that were used for chemical analysis by Bowers and Larin (1989) were not first dissected to remove foreign bodies, internal organs or ovae, that the chemical content may not be as high once these masses are removed.

When Atala adults were handled in order to mark them with identification numbers, both sexes often quickly displayed a warning flash by rapidly opening their wings to reveal the iridescent colors on the dorsal side of the wings. Scott (1986) showed that the spots on the hindwings register in the UV spectrum. All of the colors of the butterfly are composed of structural scales (Chapter 2) and would be very prominent to another insect. In situations wherein the butterfly was not being handled directly, they will also raise their abdomen in protest to display their toxicity (Figure 3-20).

Larval Behavior

Neonate larval behavior

Atala ova are laid in clusters in groups of a few to as many as fifty by a single female (Koi, pers. obs.) Hatching larvae chew through the micropyle and approximately about half of the chorion (Rawson, 1961); not only do the larvae not devour the whole natal egg, they will avoid the egg entirely or as much as possible when ingesting the leaf material around the eggs (Figure 3-21). Neonate larvae that eclose on non-host substrates will turn back to the egg for clues, encircling the base of the eggs as though searching for clues to tell it what it should be consuming and perhaps where the resource is located (Koi, pers. obs.). The neonate larvae will also rear upright onto their prolegs and sway side-to-side as though collecting chemical volatiles from the atmosphere (perhaps to to help them find their host plant). These behaviors both primarily occurred when ova had been laid on a non-plant substrate, such as the cage,

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as well as on non-host plant substrate, such as any small shrub that had been placed in the flight cage as a roosting site (see Chapter 5). All ‘roosting site plants’ were removed later to prevent losing track of dead butterflies in the planter bases, accidently hosting ant nests and other related mishaps.

Imprinting may be evident in the newly hatched larvae (Immelman & Beer, 1989) as the first neonate larval behavior observed after eclosion from the egg is of the larvae taking a “test-bite” of the plant substrate on which the eggs were laid (Figure 3-22).

Later experiments showed that if the plant was not acceptable, that the neonates would wander away in search of appropriate and recognizable host plants. Host plant choice tests documented that most neonates recognized their native host plant and preferred it to a non-native choice (Chapter 5), indicating a genetic component to the recognition.

Once the host plant is accepted as suitable, the larvae ‘settle in’ to feed en masse.

Neonate larvae of many species are gregarious feeders, which facilitates feeding strategies for dealing with the sometimes tough cuticle of host plants. Neonate Atala larvae are gregarious in disposition and display contact behavior for the first three or four instars. Neonates line up ‘sardine-style’ within minutes of eclosure from the egg.

This physical contact provides a tactile communication and perhaps chemical recognition, as well, which helps the new larvae stay together. This contact behavior facilitates feeding and most likely survival of neonates (Stamp, 1980). The neonate cluster may provide a more stable microhabitat as well (Stamp, 1980).

Besides enhanced feeding efficiency, aggregation may also provide passive defense and amplified advertisement of warning or aposematic coloration in some species (Bowers, 1993). Atala larvae, bright red-orange with lemon-yellow spots,

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sequester neurotoxins from the host plants and contain concentrations of cycasin ranging from ‘none-detected’ to 0.013 mg (Rothschild et al., 1986). Adults, on the other hand, contain between 0.21-0.51 mg of cycasin dry weight (Bowers & Larin, 1986).

Rothschild (1986) notes that the concentration is high for adults; this most likely contributes to the “tameness” noted by Schwartz (1888) as well as the open gregarious feeding behavior.

Another advantage to group feeding may be an increased ability to find a new feeding location, especially when the natal leaves have been devoured (Stamp, 1980).

Group cohesion may thus enhance survival (Stamp, 1980), although there was no direct relationship between group size and survival between life stages (Table 3-1 and

Chapter 2). Neonates will send out a line of silk “ballooning” to new leaves or new locations, which may be successful or not. A major disadvantage to ballooning to a new site is that the neonate most likely loses contact with its siblings, unless other neonates follow suit and end up in the same place. Another serious disadvantage is if the wind carries the neonate away from its host plant, instead of toward new growth. A neonate larva is 0.05-1 mm in length, and needs to find food quickly or it will die. Neonate feeding determines feeding in later instars, however, manifest as increased health and robustness, and thus survival (Stamp, 1980).

Ballooning does quickly remove the larvae from some dangers, such as a predator, but may not be advantageous or helpful in a heavy rainstorm. Many Lycaenids possess Newcomer’s dorsal nectary glands, ‘honeydew-producing’ organs that provide a sought-after, and placating, food resource for ants. Atala larvae do not possess such nectary glands and do not exhibit a mutualistic relationship with ants. In fact, the only

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relationship Atala butterflies in any life stage seem to have with ants is as prey (Smith,

2000, 2002). In the captive colonies (indoors and in the outdoor enclosed screenhouse) ants were a predator of neonates, as well as dying adults that were on the ground, particularly Monomorium pharaonis and Solenopsis invicta.

Second to final larval instar and pre-pupal behavior

Larvae possess a gregarious disposition throughout most of their larval life, until the fourth and sometimes fifth instar, at which point they are big enough to feed on the edges of the whole leaflet of the host plant by themselves. Occasionally an individual wanders off in a solitary fashion after the third instar to pupate alone. One or two larvae in each brood appears to be genetically programmed to wander off and pupate in the third instar (see Chapter 2), regardless of food availability or brood size. The reason for this is unknown.

Silk trails are laid by the larvae especially when seeking new host plant resources; silk mats are made when securing the larval integument in preparation for molting between instars or for pupation (Figure 3-23). The fact that larvae usually disperse to pupation sites en masse points to chemical communication, contact behavior and tactile sense via silk trails (Koi, pers. obs.). Larvae most likely communicate with each other via these semiochemicals, contact behaviors and silk trails allowing the larvae to stay together when larvae undergo simultaneous molting, or wander off together in search of a suitable pupation site.

Larval host plant recognition and consumption behavior

When host plant resources were low during the winter season in Gainesville,

Florida, I scraped the surface tissue of older leaves to make “coontie mash” for the neonates, served in a small petri dish on moist filter paper (Figure 3-24); this kept the

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neonates alive and growing until they were big enough to scrape the tougher leaves themselves. Larvae are voracious eaters and will also chew through the rachis of the host plant, often burying deeply (Figure 3-25). Larvae will work around the rachis of the plant (Figure 3-26). In addition to scraping the surface tissue of older leaves to feed the larvae, I also offered the split rachis of the older leaves to the second and third instar larvae, which they readily accepted. Understanding the feeding behavior of the larvae thus allowed me to successfully prevent mass starvation of the winter broods in the lab.

Older larvae will ingest non-plant material on occasion, as they did to the caps of the aquapics containing the rachis of the host plant (Figure 3-27), most likely attempting to reach more of the host plant. On two occasions, the larvae flooded their box-cage by eating a hole too big into the aquapic lid.

It is likely that the chewing sounds of larvae act as a vibratory communication signal between them, as well, drawing wandering larvae from devoured resources to a new feeding site. The sound of many larvae chewing is audible to human ears.

Larval dispersal and pre-pupation behavior

Site fidelity to pupation sites is often observed in persistent wild colonies (pers. obs.). Larvae disperse en masse to pupation sites in the fourth or fifth instar and cluster in a group, releasing silk to anchor themselves to the substrate and each other in preparation for final molt (Figure 3-23)(see Chapter 2). Anchoring to the substrate in an aggregation would be an evolutionary development to ensure safety of the brood in the event of the stochastic weather events found in Florida (high winds, tropical storms and hurricanes). The aggregate may also act as a micro-habitat to protect the larvae and pre-pupae from desiccation during the dry winter months. On occasion, late instar

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larvae may disperse to an already occupied pupal site and throw silk in such a way that it prevents earlier pupae from emerging.

Pre-pupae are capable of limited movement until the last larval skin is disengaged from the internal pharate adult. Although I occasionally witnessed an individual climbing to pre-pupal cluster to join other larvae already in formation, I never observed the entire group moving together to the pupation site.

Based on the fact that the pupal clusters were already in formation at the chosen site in the cage when the lab was opened in the morning, I surmise that movement to the pupation site occurs in the late evening or early morning. I once observed a pupal cluster of 38 individuals that formed overnight in a wild colony. The larvae most likely originated from the most likely dispersal site, a host plant located approximately thirty feet away from the pupation site, necessitating crossing thirty feet of lawn and a short brick decorative ‘wall’ surrounding the garden.

Larval cannibalism

Richardson et al. (2010), and references within, provide an excellent overview of cannibalism in non-carnivorous insects. Cannibalism was once attributed to laboratory conditions, but has since been documented in many species of wild, non-carnivorous, phytophagous insects, including species in Orthoptera, Blattodea, Hemiptera,

Coleoptera, Hymenoptera, Lepidoptera and Diptera taxa (Richardson et al., 2010). Most phytophagous insects that exhibit cannibalism are juveniles, and the behavior may contribute to the nutrient needs of the young, especially if food resources are low or of poor quality (Richardson et al., 2010).

Cannibalism may act as a population control mechanism, induced by density- dependent factors, such as food resources or temporal and spatial factors (Richardson

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et al., 2010). It may contribute to the health of the colony, such as preventing the spread of parasitoids or pathogens in the colony as some juveniles may attack and consume weakened individuals (Richardson et al., 2010).

Environmental factors, such as reduced humidity or high temperatures, may induce cannibalism, which may balance the age distribution of juveniles (Richardson et al., 2010). Cannibalism may act as a selective force, if it spreads pathogens, rather than eliminates them, or if it reduces the population to a sub-optimal density (Richardson et al., 2010). Much is unknown about phytophagous insect cannibalism, including whether there is a genetic basis for cannibalism, if genetic closeness influences conspecific likelihood for cannibalism and how it is affected by environmental factors such as hostplant quality (Richardson et al., 2010).

Cannibalism in Atala larvae is exhibited frequently under many different conditions. It is observed when larvae are under stress, such as when they are removed from the host plant and relocated to another plant in another location during reintroduction procedures (Koi, per. obs.). Although larval cannibalism may be a strong selective factor on brood survival depending on the number of larvae in the brood, for some species (Richardson et al., 2010), in the Atala brood size does appear to be a strong driver (Table 3-1). Brood size may provide additional food when food resources are low (Stamp, 1980), but because the larvae in the captive colony were fed ad libitum, there have been other factors, as Atala broods did not exhibit a consistent relationship between brood number and survival rates. However, attaining pupation consistently exhibited a high survival rate with few exceptions (Table 3-1) (see Chapter 2).

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It is interesting that although larvae avoid eating the chorions of their eggs, including any of their own hatched or unhatched eggs, they will commit siblicide. In most cases, it is larger larvae that ingest smaller larvae, or active larvae ingest larvae that are quiescent in molting stance. Molting is often done as a brood-group, and when larvae undergo molting together there may be less chance of siblicide.

Smith (2000) indicates that she observed a larvae consuming part of a pupae in a wild colony, but I did not observe that in the lab, nor have I observed this is wild colonies. Because the pupae are immobile, as are larvae preparing to molt, it may be that the last instar mobile larvae continue to consume the material in their path regardless of what it is.

Larval death

The warning coloration and clustering may afford increased protection from predators, such as ants, Reduviidae species or other predators (Bowers, 1993; Reader

& Hochuli, 2003; Smith, 2000, 2002; Stamp, 1980). Zalucki et al., (2002) determined that host plant quality may have a higher mortality effect than predation on neonates, and that weather events, such as heavy downpours, were also a high cause of mortality. The highest cause of mortality in the review of Zalucki et al. (2002) was simply listed as “unknown” because the death was not witnessed and in the wild as well as in the lab, that is also true. Some starvation undoubtedly occurred as more aggressive, larger larvae out-competed smaller, less aggressive larvae. (Larvae from the same brood may eclose at different times, exhibiting different body weights and sizes, and hence different levels of robustness.)

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Pupal Communications

Many species of pupae stridulate, some audibly, such as Mitoura gryneus sweadneri (Pence, 2005). Lycaenids are among the lepidoptera species that stridulate, most of which are associated with ant mutualists. Oenomaus ortignus, a fruit utilizing lycaenid in Costa Rica, pupates outside the larval fruit and is known to stridulate (Calvo,

1998). Because literature regarding Atala pupal stridulation was not consistently described, pupae and larvae at varying ages were taken to the sound chambers of R.

W. Mankin at the USDA facility by the University of Florida campus in Gainesville. Dr.

Mankin had performed numerous studies with insect sound production on plant hoppers, butterflies, termites and ants and he agreed to perform preliminary testing.

The Atala larvae and pupae were handled, prodded, tickled and squeezed in an effort to produce defensive sounds of some sort. However, not a sound was detected.

At the suggestion of Mirian Hay-Roe, who first documented wing-click communications in Heliconian butterflies with Mankin (Hay-Roe & Mankin, 2004), pupae of varying ages were installed in a sound chamber at the McGuire Center for

Lepidoptera and Biodiversity (ETS-Lindgren Acoustic Systems, Small Device Test

Enclosures, United States) with the help of the Operations Manager, J. Schlacta, to be recorded undisturbed for the weekend.

The first trial of pupae ready to emerge was set up on a Friday, and did not make an unexplained sound except for the noise of unwrapping pupal cases as the 30 adults emerged on that Sunday. The second trial, using ten-day-old pupae, seemed to make a single “click” sound to human ears, that displayed a series of very fast (0.07 seconds) and high frequency (100-500 Hz) stridulations (Object 3-1). Three ten-day-old pupae

(two females, one male) were installed a few weeks later to verify that discovery.

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This recording yielded definite proof that Atala pupae stridulate at ten days old

(Figure 3-28). The stridulating organs were photographed with an electron microscope at the USDA facility on campus (Figure 3-29) and display a typical “washboard” file and scrape shape. I hope to do more studies on this phenomenon. At ten days, the pupae are approximately half-way through their total development (range is 6-20 days, mean is

14 days) and it was only the ten-day pupae that stridulated (Object 3-1).

The reason for pupal sounds is unknown (Travassos & Pierce, 2000), although a large number of lepidoptera species are known to stridulate as larvae, which possess a mutualistic association with ants species, entailing complex chemical and vibrational communication. Atala do not have a mutualistic relationship with ants (if anything, the only relationship I saw in the lab or the outdoor colony was ant predator-Atala prey.)

Conclusion

In captive rearing, gauging an animals’ sense of well-being, for example, may be determined by the frequency of successful mating, adult longevity, healthy offspring, etc. Understanding the ethology presented in this paper of the behaviors associated with the Atala butterfly may help scientists, park biologists, natural areas managers and ecologists determine the general health and well-being of the Atala colonies in their care. It may also act as a template for those who work in butterfly conservation to better define or describe observations witnessed in other populations of imperiled species.

There is strong reason to believe that other lepidopteran species associated with the pine rockland ecosystem where wild colonies of Atala butterflies live will benefit from understanding the behaviors associated with its mating, larval survival, host plant recognition, and pupation.

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Continuing to explore the ethology of the Atala in wild colonies will help scientists understand the community relationships with other species, especially the Echo moth

(Seirarctia echo), which utilizes the same host plant. Baggett (1982), for instance, indicated that there may be a competitive and possibly detrimental relationship between the Echo moth and Atala butterfly larvae, but this has never been explored. The advantage the moth has over the butterfly may be nothing more than the moth’s ability to utilize host plants besides the coontie. In what ways the severe decline of coontie host plants during the early part of this century affected the moths’ population, and whether this was ever a driver in the moth’s host plant choices, or if it has always been extremely polyphagous, has not been explored to my knowledge. Covell (1984, 2005) lists alternative hosts for the Echo moth as Sabal Palmetto, coontie, crotons, lupines, oaks and persimmons, while Wagner (2005) adds a non-descriptive “other woody plants,” recording that the larvae may show up on virtually any green plant.

The Echo moth is not listed as a competitor with the specialist larvae of Bartram’s hairstreak (Strymon acis), which uses crotons, or White M hairstreak (Parrhasius m- album), which uses oaks, nor any other moth species that utilizes these plants, so it is interesting that Baggett (1982) felt it to be a competitor with the Atala butterfly larvae.

This possible competition between Seirarctia echo and Eumaeus atala, however, and its potential impact on both species, should be explored with rigorous field studies and laboratory follow-up to complete the ethological account of Eumaeus atala for developing a conservation management protocol.

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Table 3-1. Brood size does not reflect survival to adulthood in Eumaeus atala (N= 10582 ova; 3860 larvae; 2244 pupae; 2115 imagoes). Note that after reaching pupation, the survival rate was consistently high, except for two cases. t is not known why broods with 81-90 and 91-100 larvae had lower survival rates. Brood size 21 31 41 51 61 71 81 (number of 1 to 11 to to to to to to to to 91 to 101 to 111 to 121 to 131 to 141 to 151 to larvae) 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Mean survival larva-to-pupa (%) 0.75 0.63 0.76 0.49 0.78 0.50 0.61 0.14 0.62 0.81 0.46 0.45 0.31 0.77 n/a 0.42 St. dev. 0.31 0.31 0.25 0.41 0.23 0.28 0.05 0.00 0.20 0.09 0.23 0.00 0.35 0.13 n/a 0.30

Mean survival pupa-to-imago (%) 0.95 0.89 0.95 1.00 0.97 0.98 0.95 1.00 0.50 0.38 0.98 0.88 1.00 0.96 n/a 0.96 St. dev. 0.17 0.11 0.05 0.00 0.03 0.07 0.06 0.00 0.17 0.07 0.05 0.00 0.00 0.05 n/a 0.05

Number of broods 20 17 13 4 6 7 2 1 4 3 5 1 2 2 0 2 n=10582 ova n=3860 larvae 0.36 n=2244 pupae 0.58 n=2115 adults 0.94

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Figure 3-1. Atala butterflies congregated on the feeders filled with nectar every morning, and utilized the feeders throughout the day. Photo courtesy of S. Koi.

A B

Figure 3-2. Atala butterflies used artificial feeders. A) An Atala butterfly learned to drink water from an aquapic which is holding a leaf of the host plant. B) Atala adults drinking from a feeder filled with water. Photos courtesy of S. Koi.

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Figure 3-3. Male Atalas are perchers. A) All of these butterflies are males, perching on a non-native host plant in the flight cage that was installed for a female ovipositioning choice test. Photo courtesy of S. Koi.

Figure 3-4. Most adults received alphanumeric codes as identifiers, but small individuals were given a ‘tribal tattoo’ because their wing surfaces were too small for letters. A) Male size does not predict mating success. The female is marked “*140” and the dotted wing belongs to the male. B) The condition of the male wings does not deter female approval. The male is marked “*295” and the ‘sun’ symbol is a female. The apices of the male’s wings were torn giving the appearance of being similar in size to the female. Photos courtesy of S. Koi.

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A B

Figure 3-5. Atalas rest in the canopy. A) Adult Atalas of both sexes rested on the roof, in the “canopy” of the flight cage. In a wild colony, adults are often observed resting in the canopy above the host plants and/or nectar sources. B) A wild male, hair pencils everted, courts a female in the canopy of a Wild Lime tree (Zanthoxylum fagara); the host and nectar plants are directly below the tree in the understory. Photos courtesy of S. Koi.

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Figure 3-6. Groups of wild adult Atalas congregate on nectar sources in large numbers, up to thousands of individuals in eruption states. Here adults gather on an inflorescence of Sabal Palmetto (Sabal palmetto) flowers, a choice nectar source. Photo courtesy of Kathy Malone.

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Figure 3-7. Females do not display aggression to conspecifics and oviposit in large groups in the lab colony. Wild females have not been observed acting aggressively to other females. Photo courtesy of S. Koi.

Figure 3-8. Leaflets of the host plant were heavily encrusted with ova, which may occur in a wild colony as well. Because of the tight juxtaposition of the eggs on this leaflet, they were most likely laid by the same female. Photo courtesy of S. Koi.

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A B

Figure 3-9. Atala ova and anal tufts. A) Atala females release scales containing cycasins and other neurotoxins from anal tufts as they oviposit. Photo courtesy of Hank Poor. B) SEM photograph of the tip of the female anal tuft scales. Photo courtesy of S. Koi.

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Figure 3-10. Male Atalas use scent glands to attract females. A) A newly emerged male everts androconia (hair pencils) to attract females. B) Aedeagus and androconia partially everted in an excited male. Photos courtesy of S. Koi.

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Figure 3-11. Coral Reef Park in Miami-Dade County is typical of urban remnant pine rocklands; the small scattered host plants, circled in black, are nearly hidden in the understory, or underneath pine duff. Photo courtesy of S. Koi.

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Figure 3-12. An 8-day-old male (T89) forces ova from the reproductive tract of a 22-day-old female (T18) during coercive mating. The female’s lifespan was 38 days; the male’s lifespan was 14 days. Photo courtesy of S. Koi.

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Figure 3-13. A male Atala died with dried ova attached to his genitals from a coercive mating event such as the one pictured in Figure 3-11. Photo courtesy of S. Koi.

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Figure 3-14. A 35-day-old male sits on the wings of a 36 day-old female that is balancing on a nectar-soaked cotton swab. Photo courtesy of S. Koi.

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A B

Figure 3-15. Unusual Atala mating behavior. A) Two males and a female in copulo. This was observed twice in the lab and B) once in a wild colony. Photos courtesy of S. Koi.

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A B

Figure 3-16. Coercive mating in Atalas. A) In the second mating event concerning two males and one female, the ova were forced from the female’s body (indicated by the arrow). B) The two males from Figure 3-14 had to be manually disengaged. Notice that the proboscis of male “L 73” is uncoiled (arrow), indicating that he is stressed, possibly from dehydration, as well as whatever physical damage may have occurred. Photos courtesy of S.Koi.

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Figure 3-17. A 41-day-old female (P96, number obscured in photo) dies in copulo with an 11-day-old male (R37). The male did not disengage until the female expired. The male’s lifespan was 22 days. Photo courtesy of S. Koi.

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Figure 3-18. An exhausted female retires to the ground at a wild colony site. Numerous eggs had been laid on the coontie plants, and her age was unknown. Photo courtesy of S. Koi.

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Figure 3-19. Adults experienced chill coma after a night with 28°F temperatures and lost muscle control in the proboscis and appendages; if touched, they fell off their perches. Photo courtesy of S. Koi.

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Figure 3-20. Defensive warning behavior in a female Atala. Photo courtesy of Gary Bernard.

Figure 3-21. Atala larvae actively avoid consuming the chorions except when emerging necessitating consumption of the shell around the micropyle. Photo courtesy of S. Koi.

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A B

Figure 3-22. Imprinting occurs in Atala larvae. A) Imprinting occurs in neonate larvae when their first behavior involves tasting the substrate on which the egg was laid. The clutch of eggs displays neonate test-bites beside every ovum. B) A single individual has taken its first taste of food. Photos courtesy of S. Koi.

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Figure 3-23. Final instar larvae and pre-pupae release silk to batten down the entire brood. Photo courtesy of S. Koi.

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A B

Figure 3-24. Neonate behavior. A) Neonates consuming scraped coontie leaf mush. B) One day old larvae consuming scraped leaf material. The larvae were big enough to transfer themselves to split stems from which they consumed the pith at this point (first instar, three days old). Photos courtesy of S. Koi.

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A B

Figure 3-25. Larval feeding behavior. A) Larvae often burrow deep into the pith of the rachis. B) Larvae will work both sides of a rachis, or leaf, usually devouring everything. Photos courtesy of S. Koi.

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Figure 3-26. Larvae will spiral around the rachis of a leaf gleaning as much food as possible from the host plant. The arrows point to individuals burying into the stem. Photo courtesy of S. Koi.

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Figure 3-27. Voracious caterpillars devoured the leaves and stems of the host plants, as well as the lids of the aquapics at times. Twice, the larvae flooded their box cage by doing this, but fortunately none were drowned. Photo courtesy of S. Koi.

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Figure 3-28. Stridulation sounds emitted by ten-day-old pupae are amplified for better visualized at the bottom of the recording, directly beneath the sound recording. Both sounds were made at a frequency between 100-500 Hz and lasted for .07 seconds. Photo courtesy of J. Schlachta.

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Figure 3-29. The “stridulation” plates evident on the pupa between the 5th and 6th abdominal segments. SEM photograph courtesy of S. Koi.

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Figure 3-30. Matrix showing multiple mating between Atala adults, including male-male pairings. Females are indicated by pink circles and males by blue circles. Dates on the arrow indicate mating date. Numbers in the circle indicate individual ID numbers and their ages in days at the time of death.

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Figure 3-31. Matrix showing multiple mating between Atala adults. The center male KK was very successful mating with multiple females, indicated by pink circles. Males are indicated by blue circles. Dates on the arrow indicate mating date. Numbers in the circle indicate individual alphanumeric IDs and their ages in days at the time of death.

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Figure 3-32. Matrices showing multiple mating between Atala adults, including male-male pairings. Females are indicated by pink circles and males by blue circles. Dates on the arrow indicate mating date. Numbers in the circle indicate individual ID numbers and their ages in days at the time of death.

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CHAPTER 4 PLASTICITY IN LIFE-HISTORY TRAITS OF EUMAEUS ATALA POEY (LEPIDOPTERA: LYCAENIDAE)

Introduction

Climate change, land-use alteration, human population growth and the anthropomorphic modifications that accompany those factors are sources of significant stress to indigenous biota (Algar et al., 2009; Hanski, 1999; Hardy & Dennis, 1999;

Walther et al., 2002; Hanski et al., 2006; Gaston, 2009; Thomas, 2011; Lemes & Loyola

2012). Insects act as excellent indicator species for documenting changes in ecosystems and biodiversity, environmental degradation as well as the benefits of habitat restoration (McGeoch 1998; Kremen 1992a; Thomas 2011; Gerlach et al.,

2013); their small size, short generation time, wide distribution and immediate relationship to their environment make them more readily responsive to those elements

(Noss, 1990; McGeoch, 1998; Peck et al., 1998; Gerlach et al., 2013).

Variable life strategies may evolve that enable those taxa to persist in spite of or in response to unstable or stochastic features in their changing ecosystems, as well as determine their range and distribution (Walker, 1986; Abrams et al., 1996; Davis et al.,

1996; Gaston, 1999; Fischer & Fielder, 2002; Hanski et al., 2006; Thomas, 2011; Xu et al., 2012). Environmental challenges are rapidly increasing, particularly in urbanized areas (Hardy & Dennis, 1999; Schultz et al., 2008; Thomas, 2011), and the capacity of native invertebrate species to adapt to changing ecological factors may be the dynamic that either strengthens their fitness or drives their extirpation/extinction (Hardy & Dennis,

1999; Schultz et al., 2008; Thomas, 2011). Those that live in the highly stochastic environment of southeast Florida are subject to many extremes: drought, flooding,

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hurricanes and high-wind tropical storms, as well as urban threats such as insecticide use and fragmented remnant habitats.

Butterflies are a useful taxon for monitoring some environmental conditions

(Kremen, 1992; New, 1993; Kremen, 1994; McGeoch, 1998; Hardy & Dennis, 1999;

Gerlach et al., 2013) and Lycaenids are particularly sensitive to micro-habitats in their often specialized biomes and have complex relationships with their adult and larval food sources (New, 1993). Stress-tolerant butterflies are able to make adaptive responses to persist in spite of environmental stresses. The Atala, Eumaeus atala (Lepidoptera:

Lycaenidae), is one such insect that has thus far been able to ride on the thin edge of climate change, land-use alterations, and anthropomorphic modifications.

A tropical hairstreak, the Atala was historically a denizen of pine rockland and tropical hardwood hammocks and was once considered extinct, but has increased its populations in recent years, although it is still considered a species of conservation concern. To implement better understanding of the butterfly, life history traits were monitored under controlled temperature, light and humidity levels programmed to simulate southeast Florida seasons. Results will aid in determining captive rearing protocols, restoration of the butterfly into previously inhabited sites and optimal times of the year for re-introduction objectives. This research could act as a template for studying other imperiled southeast Florida taxa indigenous to pine rocklands, including

Florida-listed imperiled Bartram’s Scrub-Hairstreak (Strymon acis bartrami) and Florida

Leafwing (Anaea troglodyta floridalis), both of which were recently proposed for federal listing as Endangered Species (August 14, 2013).

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These climatic changes in our environment have wreaked havoc on many taxa throughout the world (Walther et al., 2002; Thomas et al 2011; Thomas 2011; Lemes &

Loyola, 2012) often coupled with land-use changes, cause significant “biotope degradation” (Dennis et al., 2004). There have been increased dispersal and range records set for many species (Blau, 1981; Gaston, 2009; Walther et al., 2002), including many Southeast Florida butterflies that are in serious decline (Schwartz et al., 1995;

Minno, 2010; Imperiled, 2011; Florida, 2011; Minno, 2012; Schweitzer et al., 2012).

Those that inhabit remnant natural areas such as pine rocklands, tropical hardwood hammocks, coastal salt marshes or other specialized niches, are particularly vulnerable

(New, 1993; Dennis et al., 2004; Minno, 2010; Schweitzer et al., 2011; Minno, 2012).

While most of these specialized species, such as the endangered butterflies

Miami Blue (Cyclargus thomasii bethunebakerii) and Schaus Swallowtail (Heraclides aristodemus ponceanus), or the imperiled butterflies Bartram’s Scrub-Hairstreak

(Strymon acis bartrami) and Florida Leafwing (Anaea troglodyta floridalis), are seemingly confined to their historical ecosystems, the imperiled Atala butterfly has expanded its traditional pine rockland domicile to include high-traffic urban parks and natural areas, as well as domestic and botanical gardens. Once thought to be extinct, it is currently monitored by the State of Florida via the Imperiled Butterfly Working Group and the Florida Natural Area Inventory, as well as by me. It is apparent that the butterfly undergoes sometimes extreme biannual crash-eruption cycles, establishes temporary ephemeral colonies, and sometimes disappears from a site for years before suddenly re-appearing in what may indicate a classic chaotic pattern. It is still unknown whether every extant population now found in southeast Florida originated with the wild colony

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that was rediscovered off Key Biscayne in 1979, or if the meta-populations are occasionally re-stocked by deliberate or accidental immigrants from the Bahamas and/or Cuba…or if remnant populations were simply overlooked for many years in unknown refugia before being discovered.

The Atala butterfly’s ability to utilize new territories in urbanized landscapes is aided in part by the increased use of the larval host plant, Zamia integrifolia, (“coontie”) as ornamental foliage, in both private homes and developments. This is both a boon for the species and a problem for management of herbivory. To its benefit, more colony locations help sustain a minimum population needed for dispersal between isolated and fragmented natural areas and for survival by supporting genetic variability. These larger range areas could prove to be critical for species survival in the event of severe hurricanes or tropical storms, which have impacted colonies in the past (Rawson, 1961;

Covell & Rawson, 1973; Platt et al., 1973; Emmel & Minno, 1993; New, 1993; Davis et al., 1996; National, 2005; Minno, 2011; Koi, unpublished).

The increased host plant use is a problem for the species because the often severe herbivory to the plant may lead to an increased need to control the herbivory for the health or aesthetics of the plant. While Integrated Pest Management (IPM) practices are designed to control the insect without damaging the environment, the insect population or the plant (Culbert, 1994; Pimentel et al., 2013; Pimentel & Hart,

2001), it is often challenging to successfully navigate without negatively impacting one or another aspect of the biotic community (Pimentel & Hart, 2001; Pimentel 2011;

Pimentel et al., 2013).

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To keep watch on or change landscaping practices may be especially difficult, particularly in sites managed by Home Owner Associations, governmental agencies

(such as the city, county or state’s department of transportation), businesses or corporations, especially those managed by “out-of-state” companies. It is also potentially detrimental for the butterfly because in urbanized areas there is an increased necessity for mosquito control practices (Salvato, 2001; Hoang et al., 2011; Bargar,

2012) to protect human populations, in addition to an increase in the use of household pesticides utilized to control yard insects such as fleas and ticks, or pests such as termites and roaches (Pimentel, 2001, 2013; Pimentel et al., 2013).

Artificial fertilizers are often required by non-native ornamentals, and homeowners may use organic fertilizers that would not normally be found in pine rocklands, a naturally nutrient-poor ecosystem (Myers, 1990; Snyder et al., 1990), to enhance growth of native plants in butterfly gardens. Park managers and natural area biologists must make educated decisions regarding fire regimes, invasive animal removal via poisons and the use of herbicides to control invasive non-native plants. All of these practices may impact the populations of imperiled butterfly species living in the remaining remnant natural areas (pinelands, hammocks, prairies, wetlands, etc.)

Re-introduction ventures in the past were limited (Rawson, 1961; Covell &

Rawson, 1973; New, 1993; Emmel & Minno, 1993; Hammer, 1995) and other colony sites, besides the single re-discovered location, were virtually unknown. Past re- establishment projects were also thwarted by hurricanes, such as Donna in 1960 and

Andrew in 1992, both of which caused significant environmental and economic damage that was difficult to access regarding the impact on imperiled butterfly populations in

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natural areas and pie rocklands (Davis et al., 1996; National, 2005; Minno, 2011; Platt et al., 1973).

New re-introductions were established in 2004 in Everglades National Park (Koi, unpublished), and expanded into domestic re-establishments, primarily through local conservation groups and butterfly clubs up to the year 2008. Currently, I am leading another re-location project with help from Citizen Science volunteers, monitored by members of local butterfly clubs in Miami-Dade, Broward and Palm Beach Counties

(Koi, unpublished).

Climate change, environmental degradation and stochastic weather events affect the ecological stability and the range and distribution of the Atala butterfly. To address potentially beneficial or deleterious effects of changing temperatures on the butterfly, as well as to explore the possibility of increased range, the effect of season was considered regarding life stage development time and mortality rate. Understanding the impact of seasonal stress to the butterfly’s overall fitness will help park managers utilize the best strategies to optimize survival.

Materials and Methods

Environmental chambers (Percival, Advanced Intellus, Environmental Controller,

Perry, IA 50220), housed at the USDA/ARS Invasive Plant Research laboratory in

Gainesville, Florida, were programmed to simulate south Florida seasonal variations in temperature, humidity and daylight, based on mean values for four seasons obtained from archived weather data for south Miami for the years 2009-2011 (NOAA).

Chambers were set to characterize the seasons based on the first day of fall, winter, spring and summer, respectively (Table 4-1). The winter chamber was re-programmed and used again for fall conditions the day after the winter experiments were completed.

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Adult E. atala butterflies reared from wild larval stock previously collected in south Florida were housed in a 6’x6’x6’ flight cage (LiveMonarch Foundation,

Greenhouse Castle, Boca Raton, FL) and allowed to oviposit freely on whole leaf fronds of their native host plant, Zamia integrifolia. Host plants were maintained in a screenhouse located on the grounds of the Entomology/Nematology Department at the

University of Florida in ambient outdoor temperatures; leaves for ovipositing were chosen haphazardly every day from the flight cage and new material installed. The flight cage was housed indoors in temperatures ranging from 24-26°C, a relative humidity of

25-42% depending on season, and approximately 8:16h light:dark cycles that varied occasionally to 10:14h light:dark. Daylight lamps were also provided to encourage mating and ovipositing, programmed for 1:2h on:off cycles during the day. A fan, similarly programmed, helped project plant volatiles and mating signals between adults.

Twenty-five to twenty-eight ova were haphazardly chosen from the flight cage ovipositing leaves and were deposited inside each environmental chamber daily. Eggs were collected for five sequential days for each chamber so that each chamber housed

~twenty-five newly laid ova as biological replicates in one-day increments. Each brood of eggs was placed within a separate fabric cage, so that each chamber contained five cages as technical replicates with each hosting 25-28 eggs, totaling ~125 individuals per chamber (Figure 1-1).

Leaflets from the host plant fronds containing the correct number of eggs were placed into a 9 cm plastic petri dish lined with grade 1 filter paper (Whatman®

International, Ltd., Filter Papers No. 1, GE Health Care Companies, Maidstone,

England) cut to size and moistened with deionized water. Petri dishes were sealed with

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cotton string until the larvae emerged in order to maintain humidity and prevent larvae from escaping. The petri dishes were placed in the appropriate chamber within thirty minutes of collection. Broods were monitored every day from egg collection until adult emergence. Development stages were recorded and individuals were counted.

Unhatched eggs were considered non-viable at 13 days post-installation, based on prior hatch rates in the lab, and removed from the brood.

Eclosed larvae were housed in clear plastic “deli boxes” measuring approximately 20 cm square lined with a paper towel (Figure 4-1) and were provided with host plant leaves from the screenhouse plant colony ad libidum. Deli dishes were further confined within 12”x12”x12” fabric cages (LiveMonarch, small castle, Boca

Raton, Florida) and placed inside the appropriate environmental chamber. Deli box lids were kept closed until the larvae reached third instar to prevent loss of larvae and moisture, at which point the dishes were opened and larvae were allowed to wander within the confines of the fabric cages. Cages were cleaned daily, larvae counted and the development stage recorded and returned to the chamber immediately after monitoring.

Emerged adults were sexed and the left forewing cord length was measured. All adults were immediately placed into glassine envelopes, euthanized in a freezer and fumigated; vouchers were deposited in the collections of the McGuire Center for

Lepidoptera and Biodiversity, University of Florida, Gainesville, Florida.

Results

Development time varied significantly for all treatments except the summer and fall chambers (Table 4-2). Summer and fall broods developed at nearly the same rates

(p-value< 0.3895) and showed the highest brood survival as well (Table 4-2). There was

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no significant difference in sex ratio between the seasons, although slightly more adults successfully emerged in warmer seasons. All development stages took significantly longer in the spring chamber than any other season (Table 4-2) (p-value <0.000) and females in particular grew larger than in the other chambers (Table 4-3).

The chambers followed the Homestead temperatures fairly well (Figure 4-2A) but for analysis, the values were adjusted using the Baskerville-Emin fomula, which compensates for possible discrepancies caused by lower on-going temperatures

(Figure 4-2B). The important factor in development is the “growing degree day” which is the amount of growth that takes place under different temperature regimes. The standard formula is maximum temperature-minimum temperature squared divided by the baseline temperature, which in normal circumstances has been determined as 50°F.

This is the temperature at which most insect development stops. However, in south

Florida, the minimum temperature for which the winter chambers were programmed was 73.4°F. The lowest values in Homestead, however, were much lower than that

(35°F). Because of that difference, I adjusted using the actual lowest temperature for

Homestead to make the comparison, substituting the actual minimum temperature for the baseline usually used.

A 99% confidence interval for life stage development in the different seasons is found in Table 4-4. Although the confidence interval from the chamber data show, for instance, that an egg will hatch between 6.27 and 6.83, there were eggs in the lab that hatched in 4 or 5 days, and conversely there were eggs in adverse colder outdoor temperatures that did not hatch for as long as 13 days. This variation was most likely an

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artifact of environmental factors as well as how well the ova were provisioned by the female (Blau, 1981; Abrams et al., 1996; Fischer & Fiedler, 2002).

Life tables (Southwood & Henderson, 2000) show a strong growth rate for all seasons (Tables 4-5, 4-6, 4-7 and 4-8), which along with the other life history traits discussed in Chapter 2 of this thesis, may be partly accountable for the eruption states which the insect exhibits in a bi-annual cycle (Koi, unpublished). In the tables, Stage(x) refers the pooled number of individuals in each life stage wherein the analysis begins, stage (0) being the number of unhatched, newly laid eggs placed in each chamber, for example.

The next column “nx” refers to the number of individuals in that life stage. “Lx” is calculated by dividing the surviving number of individuals by the previous days’ survivors and indicates the proportion of individuals surviving at the start of interval “x”;

“dx” is the number of individuals dying from “x” to “x+1”. “Qx” shows the mortality

(dx/nx). “Bx” is the number of females per brood; males are not counted as females will determine the future brood size via fecundity/fertility and therefore the consequent growth of the colony. The sum of “lxbx” determines the net reproductive rate or number of daughters in the brood. The sum of “lxbxx” is the projected generation time.

The generation time (G), the length of time between the birth of the parents and the birth of offspring, is calculated by dividing the projected generation time (Σlxbxx) by the net reproductive rate (Σlxbx). In all chamber seasons, the calculated generation time was only 4, which quickly leads to exponential growth in the equation (r=ln(Ro)/G) to determine “r”, which is the rate of increase. Within one generation, a summer Atala colony with a generation time of 4 and growth rate of 94% has the potential to erupt into

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3,612 individuals. I have seen eruptions of this type, with several thousand adult individuals in wild colonies in South Florida.

Summer season has a 94% growth rate, fall shows a 98% growth rate, winter exhibits a 97% growth rate and spring displays a 95% growth rate. Generation time for all seasons was 4, which has the potential to quickly exhibit exponential growth without such mitigating factors as host plant depletion, drought, predation, etc. Life table graphs

(Figure 4-3) show high mortality in the immature stages, with increased survival in late larval and pupal stages, with little variation between seasons.

Survival plots of the ova stage (Figure 4-4) display the beginning of a significant seasonal difference between summer-fall and winter-spring development. Each step in the graph represents the stage at which the egg hatched in days. The intercept

(x,y=0,0) indicates that all the ova have hatched, and shows that summer ova hatched first, fall second, winter third and spring last.

Larval development (Figure 4-5) displays an even greater discrepancy between the warm seasons and cooler seasons. Each step represents the day after hatching at which the larvae became a pre-pupa (i.e, no longer a larva), not a true mortality. In this graph, the last summer and fall larvae pre-pupated on approximately day 13 after hatching, but the last winter larvae did not pre-pupate until about day 17 after hatching, while last spring larvae did not become pre-pupae until day about 19 after hatching. The intercept (x,y=0,0) indicates that all the larvae have become pre-pupa.

Pupal development (Figure 4-6) displays a significant difference between the development time of the summer-fall broods and that if the winter-spring broods. Each step in the graph represents the number of days after pre-pupa that the pupa eclosed

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as an adult and the intercept (x,y=0,0) indicates that all the pupae have become adults.

The last of the summer brood emerged first between13 days to 16 days after onset of pupation and the last of the fall brood followed closely behind, eclosing between 16 and

17 days after pupation. However, the last emergent in the winter brood did not eclose until approximately day 22 after pupation and the final spring emergent was on day 26 after pupation.

Pupae exhibit a seasonal polyphenism with summer and fall pupa developing a

“warm honey brown” case with soft black speckles (Figure 4-7). Winter and spring pupal cases displayed a heavy melanization of the integument and a darker brown casing

(Figure 4-7).

Males exhibit seasonal polyphenism (Figure 4-8), with cooler environmental conditions associated with green color and warmer conditions associated with blue colors. The polyphenism may be partially explained by genetics and partly as a seasonal response, as green and blue males eclosed consistently at a 50/50 ratio in the laboratory. Table 4-3 shows that more green males eclosed in cooler seasons in the chambers than did blue males.

Wing chord length was measured in a straight line from the basal sclerite on the left forewing from where it attaches to the thorax to the apex of the forewing. Females were the largest in spring. The wing chord length did not vary significantly between females and males in the summer season but did vary significantly in the other three seasons (p-values<0.000 to 0.001) (Table 4.3). There was no significant difference between the green males and blue males (p-value<0.855). There were no significant differences in intraclass correlations.

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Discussion

There were some discrepancies between the actual Homestead, Miami values in temperature, photoperiod and humidity and the simulated Chamber readings for fall and winter in particular:

1. The chambers could not be programed for temperatures as low or as high as Homestead values.

2. There may have been some mis-programming of the Percival chambers for a few days by the operator (SK) as we struggled to get the chambers correctly setting the programmed values.

3. Both the winter and fall programs were installed in the same environmental chamber (winter first and then fall after the last winter adult emerged. This could have affected the second program or may have been an artifact of the actual chamber itself.

4. The chambers were not programmed to vary at all during the season, so the early settings remained in effect for the entire time frame. In the natural environment, the photoperiod, humidity and temperatures would be fluctuating slightly (or sometimes a lot!) every day. This was not accounted for in the chambers.

5. The length of time that temperatures were actually cooler or warmer than the mean temperature values for the season that were used for the programming skewed the results. Baskerville-Emin sine wave curves were employed to compensate for that in these two seasons, but the differences were still greater than the method could compensate for in the end.

If doing this experiment again I would re-program the settings monthly, or at least weekly, to reflect previous temperature means for the week in another year. Ideally, a mean for the day for the entire three-month period could by programmed into the chambers and would give a much stronger data set. Nonetheless, the values recorded in the chambers are valuable in documenting the tremendous range in variability in

Atala development rates. I would also not be surprised if in looking through more archived weather patterns that we have had in South Florida that there is as much variability in weather patterns as the Atala has in life history strategies.

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Plasticity in life history traits in all life stages is an important adaptive response to changing ecological situations, especially in species that inhabit harsh environments and stochastic weather patterns (Abrams et al., 1996; Fischer & Fiedler, 2002). The atala has displayed a wide variety of adaptive traits that are advantageous for it reproductive fitness and colony survival.

Seasonal polyphenism is a combination of the effects of temperature, photoperiod and humidity. This experiment was not designed to tease out the separate factors. Polyphenism is seen in many species of butterflies (Daniels, 1999 and references within; Daniels et al., 2012) and acts in different roles. It may be a mating signal of the color corresponds to the species mating season, and may be particularly important for univoltine species recognition where the season is short and mating must be accomplished without hesitation. Darker colors, such as the melanization evident in the cooler season pupa, are strongly associated with increased absorption of sunlight in the shorter days of winter and spring which affects thermoregulation and development.

In addition, the larger size of females in cooler seasons is related to the longer development time spent as a larva and pupa. A larger size is a distinct advantage for females as the Atala is multivoltine displaying consecutive and overlapping generations.

A larger size would be able to lay larger and more robust eggs, resulting in larger, more robust larvae (Abrams et al., 1986; Daniels, 1999; Daniels et al., 2012). A larger size would allow for more feeding, development of larger fat bodies and general health of the female, contributing to her overall fitness.

Because these butterflies were reared in laboratory at the USDA/ARS Biocontrol in a quarantine facility, they were euthanized immediately after emerging (after

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necessary biological parameters were measured). It would have been helpful to follow the individuals to observe whether there were differences in the lifespan, what the mating success may have been between green or blue males (regarding male aggression and/or female preference.) All specimens have been deposited as vouchers in the collections at McGuire Center for Lepidoptera and Biodiversity.

Conclusion

Seasonal changes do not take into account the other environmental factors that indirectly influence development, plasticity, survival and fitness such as the urban heat sink (Dennis et al, 2004) that permeate most of south Florida, surrounding the islands of remnant pinelands, hammocks and wetlands. South Florida has traditionally hosted farming communities surrounded by remnant pinelands but the landscape is changing drastically and natural areas are being squeezed out of existence by planned communities and developments. This may help reduce agricultural use of chemicals

(fertilizers, pesticides and herbicides), but it is ultimately detrimental because of increased commercial use of the same chemicals (and often worse!)

It is thought that urbanized areas, however, with those very damaging increases in artificial fertilizers, may be beneficial in that there is an increase in host plants, and more prevalent sources of abundant nectar. In the early 1980’s it was thought that the pine rocklands in Everglades National Park did not support plant growth very well and that’s why Atalas have not persisted well there (Covell, pers. com.). Now that there are so many developments hosting a wide variety of host plants as ornamentals and many nectar plants, this may have a direct influence on Atala fitness regardless of seasonal fluctuations in native flower abundance or the usual winter state of the coontie plant.

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Ornamental and exotic cycad installations in botanical and private gardens have also allowed the butterfly to persist in areas where it may have died out in the winter, when native sources would be less than desirable for sustenance. Mechanistic factors such as these may act as drivers for the Atalas dispersal and persistence in urbanized areas rather than in historically occupied sites.

Dennis et al. (2004) states that what is apparent is that having access to multiple host plants, including secondary host plants, increases species’ exposure to a wider variety of survival strategies many of which may offer novel evolutionary paths and channels that could lead away from a specialist life style. “Loss of variability in host use with habitat loss implicates the reverse: vulnerability of species leads to extinction,” he concludes (Dennis et al., 2004). Season may play a less important role in the butterfly’s evolutionary tract than these other factors.

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Table 4-1. Temperature, photoperiod and relative humidity were programmed in Percival Environmental Chambers to simulate South Florida seasonal variations. Summer Fall Winter Spring Temperature °C 26.9 28.9 23.6 23.0

Range 23.2-30.0 27.2-31.1 23.3-25.0 19.4-23.8 Photoperiod 16L:8D 12L:18D 9L:15D 10L:14D Humidity 76.3% 69.5% 71.9% 73.0%

Range 65-82% 59-79% 59-83% 58-97%

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Table 4-2. Development times of immature life stages of Eumaeus atala under environmental conditions simulating southeast Florida seasons. Total Ova Larval Pupal development development development Pre-pupae development (days)=Adult Season (days) (days) formation (days) emergence n=Females n=Males Summer n=125 n=114 n=84 n=84 n=84 43 41 Mean 6.55 11.15 2.17 12.23 31.87 St. Deviation 1.21 0.86 0.86 0.86 0.84 Range (days) 5-10 8-13 1-4 10-14 31-33

Fall n= 129 n=110 n=106 n=106 n=103 50 53 Mean 6.79 10.81 2.54 12.04 31.65 St. Deviation 1.46 1.04 0.99 0.713 2.21 Range (days) 6-12 7-12 1-3 11-15 30-35

Winter n=125 n=117 n=87 n=87 n=87 49 38 Mean 8.67 14.85 3.80 15.67 42.81 St. Deviation 1.07 0.73 0.68 1.00 2.14 Range (days) 7-11 13-17 3-5 14-18 40-48

Spring n=125 n=115 n=88 n=88 n=86 46 40 Mean 9.50 15.59 5.32 17.62 47.59 St. Deviation 1.32 1.63 1.63 1.45 1.65 Range (days) 8-13 13-19 2-8 14-20 44-53 Significantly different development times were between Fall-Spring (p-value<0.0001), Fall-Winter (p-value<0.0001), Winter-Spring (p-value<0.0001) and Summer-Spring (p-value<0.001). Summer-Fall showed no significant difference in development time (p-value<0.3895).

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Table 4-3. Wing chord length of E. atala female and male butterflies reared under environmental conditions simulating southeast Florida seasons. Sex ratio did not change seasonally, but male dorsal wing color varied. Wing chord length Wing chord length Wing chord length Wing chord length Season (cm)-Females (cm)-Males (cm)-Blue males (cm)-Green males Summer n=43 n=41 n=38 n=3 Mean 2.37 2.37 2.24 2.23 St. dev. 0.12 0.09 0.09 0.10 Range (cm) 2.1-2.6 2.0-2.4 2.0-2.4 2.1-2.3

Fall n=50 n=103 n=50 n=3 Mean 2.40 2.23 2.23 2.30 St. dev. 0.08 0.34 0.34 0.14 Range (cm) 2.2-2.5 2.0-2.4 2.2-2.5 2.2-2.4

Winter n=49 n=38 n=24 n=14 Mean 2.51 2.28 2.28 2.36 St. dev. 0.13 0.43 0.43 0.05 Range (cm) 2.0-2.7 2.2-2.5 2.2-2.5 2.3-2.5

Spring n=46 n=40 n=21 n=19 Mean 2.60 2.39 2.39 2.39 St. dev. 0.14 0.05 0.05 0.13 Range (cm) 2.23-2.7 1.9-2.5 2.1-2.5 1.9-2.5 Significantly different wing chord lengths in Atala butterflies were observed between fall females and males (p-value<0.000), winter females and males (p- value<0.001), and spring females and males (p-value<0.000). There was no significant difference in the wing chord length between blue males and green males regardless of season. There was no significant difference between summer females and males.

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Table 4-4. Development times for life stages in environmental chambers programmed for southeast Florida seasons. Ova development with 99% confidence interval (days) Summer 6.27 ≤ 6.55 ≤ 6.83 Fall 6.45 ≤ 6.79 ≤ 7.13 Winter 8.42 ≤ 8.67 ≤ 8.92 Spring 9.19 ≤ 9.50 ≤ 9.81

Larva development with 99% confidence interval (days) Summer 10.69 ≤ 11.2 ≤ 11.61 Fall 10.55 ≤ 10.8 ≤ 11.07 Winter 14.67 ≤ 14.9 ≤ 15.02 Spring 15.19 ≤ 15.6 ≤ 15.99

Pre -pupa development with 99% confidence interval (days) Summer 1.92 ≤ 2.17 ≤ 2.42 Fall 2.29 ≤ 2.54 ≤ 3.99 Winter 3.61 ≤ 3.80 ≤ 3.99 Spring 4.86 ≤ 5.32 ≤ 5.78

Pupa development with 99% confidence interval (days) Summer 31.63 ≤ 31.87 ≤ 32.11 Fall 11.86 ≤ 12.04 ≤ 12.22 Winter 15.39 ≤ 15.67 ≤ 15.95 Spring 17.31 ≤ 17.62 ≤ 18.03

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Table 4-5. Life table of brood reared in environmental chamber programmed to simulate Miami-Dade Summer conditions. Σlxbx Summer Stage (x) nx lx dx qx bx (Ro) Σlxbxx G=Σlxbxx/Σlxbx generation time 4 egg (0) 125 1 11 0.088 0 0 0 r=ln(Ro)/G growth rate 0.9403 larva (1) 114 0.912 30 0.263 0 0 0 pre-pupa (2) 84 0.737 0 0 0 0 0 pupa (3) 84 1 0 0 0 0 0 adult (4) 84 1 0 0 43 43 172

Table 4-6. Life table of brood reared in environmental chamber programmed to simulate Miami-Dade Fall conditions. Σlxbx Fall Stage (x) nx lx dx qx bx (Ro) Σlxbxx G=Σlxbxx/Σlxbx generation time 4 egg (0) 129 1 19 0.147 0 0 0 r=ln(Ro)/G growth rate 0.9830 larva (1) 110 0.853 4 0.036 0 0 0 pre-pupa (2) 108 0.982 0 0 0 0 0 pupa (3) 106 1 0 0 0 0 0 adult (4) 106 1 0 0 51 51 204

Table 4-7. Life table of brood reared in environmental chamber programmed to simulate Miami-Dade Winter conditions. Σlxbx Winter Stage (x) nx lx dx qx bx (Ro) Σlxbxx G=Σlxbxx/Σlxbx generation time 4 egg (0) 125 1 8 0.064 0 0 0 r=ln(Ro)/G growth rate 0.9780 larva (1) 117 0.936 30 0.256 0 0 0 pre-pupa (2) 87 0.744 0 0 0 0 0 pupa (3) 87 1 0 0 0 0 0 adult (4) 87 1 0 0 50 50 200

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Table 4-8. Life table of brood reared in environmental chamber programmed to simulate Miami-Dade Spring conditions. Σlxbx Spring Stage (x) nx lx dx qx bx (Ro) Σlxbxx G=Σlxbxx/Σlxbx generation time 4 egg (0) 125 1 10 0.08 0 0 0 r=ln(Ro)/G growth rate 0.9521 larva (1) 115 0.92 27 0.23 0 0 0 pre-pupa (2) 88 0.77 0 0 0 0 0 pupa (3) 88 1 0 0 0 0 0 adult (4) 86 0.98 2 0.02 46 45.08 180.3

A B

Figure 4-1. Percival environmental chambers. A) Interior view of fabric housing cages in the Percival chambers and B) plastic ‘deli-box’ larval cages inside with host plant material and young larvae. Photos courtesy of S. Koi

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30.0 3500 )

° 3000 25.0 Environmental 2500 20.0 Chambers, 2000 15.0 Homestead Baskerville-Emin mean Units Day 1500 adjustment 10.0 temperatures 1000 Homestead 5.0 500

Temperature (C Temperature Values

Chambers mean Degree 0.0 temperature 0 A B

Season Season

Figure 4-2. Development rates in different environmental chambers. A) Temperatures in the chambers were programmed to match Homestead, Miami temperatures as closely as possible (see discussion). B) Degree day units were analyzed using a Baskerville-Emin adjustment (see discussion).

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200 200 180 A 180 R² = 0.8996 B 160 160 140 R² = 0.9023 140 egg (0) egg (0) 120 120 100 larva (1) 100 larva (1) 80 pre-pupa (2) 80 pre-pupa (2) 60 60 pupa (3) pupa (3) 40 40 20 adult (4) 20 adult (4) 0 Poly. (adult (4)) 0 Poly. (adult (4))

200 200 180 R² = 0.9055 C 180 D 160 160 R² = 0.8994 140 140 120 egg (0) 120 egg (0) 100 larva (1) 100 larva (1) 80 pre-pupa (2) 80 pre-pupa (2) 60 pupa (3) 60 pupa (3) 40 40 adult (4) adult (4) 20 20 0 Poly. (adult (4)) 0 Poly. (adult (4))

Figure 4-3. Graphs of life table data showing high mortality in immature stages and decreasing mortality as larvae near pupal stage and adult emergence in different seasons. A) Summer B) Fall C) Winter D) Spring.

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Figure 4-4. Ova development time is indicated by the solid and dashed lines. Two distinct groups of seasonal difference are starting to be expressed between warm season (summer-fall) and cool season (winter-spring).

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Figure 4-5. Larval development time is indicated by the solid and dashed lines. Two groups of seasonal difference are expressing more strongly between warm season (summer-fall) and cool season (winter-spring).

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Figure 4-6. Pupal development time is indicated by the solid and dashed lines. The two groups of seasonal difference are fully expressed between warm season (summer-fall) and cool season (winter-spring).

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Summer Fall

Winter Spring

Figure 4-7. Polyphenism in Atala pupae. Increased melanization is associated with cooler temperatures. All pupae were ten days old when these photographs were taken. Photos courtesy of S. Koi.

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Figure 4-8. Seasonal polyphenism in the dorsal wing color of male Atala butterflies. Green is found more often in cooler temperatures and blue more often in warmer seasons. Photo courtesy of S. Koi.

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CHAPTER 5 IPM AND ATALA BUTTERFLY HOST PLANT CHOICE

Introduction

Cycads are so perilously endangered or threatened worldwide that botanists have recently established global networks to monitor the populations (Oberpreiler,

1995a, 1995b, 1995c, 2004; Chemnick et al., 2002; Donaldson, 2003). Causes of the severe declines in these beautiful ancient plants includes legal and illegal collection, removal of seed heads and root calyxes for food and/or “bush medicines,” and the two biggest on-going threats: unsustainable trade and habitat loss (Oberpreiler, 1995a,

1995b, 1995c, 2004; IUCN, 2003). The possibility that the specialist pollinator associated with an individual cycad species has been extirpated, or is extinct, has been cited as another possible reason for declining colonies (Oberpreiler, 1995a, 1995b,

1995c, 2004; Chemnick et al., 2002; IUCN, 2003; Gonzáles, 2004; Koptur, 2006). Both legal and illegal trade impact the colonies, many of which are isolated and highly vulnerable, regardless of legislation and laws designed to protect them (Oberpreiler,

1995a, 1995b, 1995c, 2004; Chemnick et al., 2002; Donaldson, 2003). González (2004) indicates that the cycad, the pollinator and the herbivores are all in danger in Colombia.

North America’s only native cycad, Zamia integrifolia L. (Zamiaceae: Cycadales), was found historically from southern Georgia to the Florida Keys but is still listed as

“Threatened” in wild natural areas (Coile & Gardner, 2003; IUCN, 2013). The starch industries of the last century depleted wild populations of the cycad, commonly called

“coontie,” almost to the point of extirpation, as the roots were harvested to make mildew-resistant flour (Small, 1913; Kirk, 1976, 1977; Knetsch, 1989, 1999; Blank,

1996; Coile, 2000; Coile & Gardner, 2003). The French explorer, Laudoníerre,

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documented the Florida Calusa Indian’s use of coontie for flour in his ships’ logs in the year 1564 (Laudoníerre ,1975). Cycad-originated flour is still a staple in many undeveloped and underdeveloped nations (Oberpreiler, 1995a, 1995b, 1995c, 2004;

Chemnick et al., 2002; IUCN, 2003) and is one cause of cycad declines, particularly because the reproductive cones are often removed as well (Oberpreiler, 2004).

However, native coontie has been making a strong recovery in the Florida landscaping industry during the past twenty years as nurseries and homeowners discovered how well-adapted the luxurious leafy plant is to Southeast Florida’s diverse ecosystems and stochastic weather cycles (Haynes, 2000; Culbert, 1995(2010);

Dehgan, 2002). With increasing urban development and world nursery trade, more exotic non-native cycads have been added to the repertoire available to landscape planners.

Utilization of these exotic non-native cycads has increased in south Florida’s urban developments because they are generally hardy and usually pest-resistant, as well as beautiful and highly sought-after (Haynes, 2000; Dehgan, 2002). However, cycads are susceptible to a few insect predators, including natives such as red scale

(Chrysomphalus aonidum), hemispherical scales (Saissetia coffeae) and longtailed mealy-bugs (Pseudococcus longispinus); these sucking insects may damage a plant to the level that encourages sooty mold (Cryptolaemus montrouzeri) to follow (Culbert,

1995 (2010)).

A more recent armored scale insect has been introduced into Florida from

Southeast Asia, Aulacapsis yasumatsui (Howard, et al., 1999). The Asian scale causes a more virulent infestation than our native insects and attacks many species of cycads

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as well as coontie. A root-attacking beetle larva from Mexico and Guatemala, Eubulus trigonalis, has recently been found in two botanical gardens in south Florida (Tang,

2001), but has not yet been found to domestic gardens.

Two species of native lepidopteran herbivores attack cycads in North America: the Echo Moth (Seirarctia echo Smith 1797) and the Atala butterfly (Eumaeus atala

Poey 1832). The range of the Echo moth covers Georgia south to the Florida Keys, and west to Mississippi (Wagner, 2005; Covell, 1984, 2005). The moth is widely polyphagous, utilizing Sabal Palmetto (Sabal palmetto), and various species of crotons, lupines, oaks and persimmons, as well as coontie (Wagner, 2005; Covell, 1984, 2005).

Wagner (2005) includes ‘other woody plants,’ indicating that the moth could show up on virtually any plant as it wanders. He mentions that the larvae may wander away from frass left at the feeding site to discourage predators or parasites (not identified) or to seek pupation sites and questions if the moth is seeking certain plants for the production of sex pheromones.

The Echo moth is found in many types of ecosystems, including urban areas, as evidenced from the many photography webpages now online. The moth was called

“destructive” in the early part of this century (Slosson, 1917) and lamented as being a possible competitor of food for the other lepidopteran cycad-herbivore, the Atala butterfly, at a time when the butterfly was thought to be nearly extinct (Baggett, 1982).

The Atala hairstreak butterfly historically inhabited southeast Florida’s now endangered pine rockland ecosystems and tropical hardwood hammocks in southeast

Florida only (MDC, undated; Myers, 1990; Snyder et al., 1986). Unlike the Echo moth’s eclectic choice of foods, the Atala is a specialist butterfly that used our only native

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cycad, Zamia integrifolia as its sole host plant in the past. The reduction of the native cycads, as previously noted, for the starch industry caused the drastic decline in the butterfly’s population by the 1930’s and by 1951, it was thought to be extinct (Klots,

1951). Therefore, when the Endangered Species Act was passed in 1973, the Atala butterfly was not listed. Unbeknownst to almost everyone, the butterfly had survived in isolated pockets (or had been re-inoculated via trade winds from the Bahamas. See

Chapter 1).

Beneficial Insect Associations with Cycads

There are some probable beneficial effects of insect-associated activity with cycads. Each cycad species has a mutualistic relationship with a specialist, sometimes obligate, pollinator weevil (Norstog & Fawcett, 1989; Tang, 1990; Vovides, 1991;

Oberpreiler, 1995a, 1995b, 1995c, 2004; Stevenson et al., 1998; Schneider et al., 2002;

Chaves & Genaro, 2005; Chemnick et al., 2002). Like butterflies, moths or other insects, including pests such as scale, they each play a part in the total ecology of the plants

(Norstog & Fawcett, 1989; Tang, 1990; Vovides, 1991; Oberpreiler, 1995a, 1995b,

1995c, 2004; Stevenson et al., 1998; Schneider et al., 2002; Chaves & Genaro, 2005;

Chemnick et al., 2002). The co-relationships between the specialized insect herbivores and pollinators may be more recent than previously thought, as new molecular phylogenies show the relationships have co-evolved since the late Miocene

(Nagalingum, et al., 2011).

Taylor (1999) also indicated that the butterfly larvae associated with the cycads may break down the germination-inhibiting sarcotesta on the female seed cones, helping the seeds sprout faster than they do otherwise, and are therefore helpful herbivores. The large volume of frass left behind by herbivores acts as a fertilizer as

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well. Nagalingum et al. (2011) notes that this may be one of the reasons for the low genetic diversity in the genera, which has made identification of the cycad species difficult (Vovides, 1983, 1991; Brenner & Twigg, 2003; Donaldson, 3002; Broom, 2004;

Nicolalde-Morejón et al., 2011; Meerow, et al., 2012; Calonje, et al., 2012). It is thought now that the diversification of the six cycad genera may have first been triggered when the current continents were moved into their present locations as the tectonic plated shifted during the Miocene (Nagalingum, et al., 2011), although there is still some controversy (Nicolade-Morejón et al., 2011; Taylor, et al., 2012). A recent paper indicates that the seeds may have been ingested by large birds, mammals and other megafauna, and dispersed in clusters along with large amounts of ‘fertilizer’ in places some distance from the mother plant (Hall & Gimme, 2013), explaining the growth habit of the plants, usually adapted to growing in high density. Hall & Gimme (2013) determined that opossums were primarily responsible for consuming the fleshy sarcotesta and dispersing the seeds of the Australian cycad Macrozamia miquelii

(Zamiaceae). They found that few seeds were dispersed more than a meter from the maternal plant and that most juvenile seedlings did not establish within a few meters of the parent, even though most seeds fell beside the maternal plant. In Florida, squirrels are known to ingest the sarcotesta, but opossums and other wildlife may be responsible for dispersal of the seeds as well. Because of this growth habit, Zamia plants may be locally abundant, but rare; this is true of Z. integrifolia in wild lands.

Chemecology of Cycads and Insect Associates

Each species of cycad contains various mixes of neurotoxins, such as cycasins and macrozamins in different concentrations and amounts (Teas, 1967; Bell, 1967;

Norstog & Fawcett, 1989; Tang, 1990; Vovides, 1991; Nash et al., 1992; Oberpreiler,

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1995a, 1995b, 1995c, 2004; Stevenson et al., 1998; Nishida, 2002; Schneider et al.,

2002; Chaves & Genaro, 2005; Chemnick et al., 2002). Teas (1967) was the first to record the metabolic pathways in Seirarctica echo by which the neurotoxic methylazoxymethanol (MAM) in the coontie host plant was transformed into less toxic cycasin via beta-glucosidase activity, thereby avoiding autotoxicty. It is thought that the same metabolic detoxification methods are used by other the cycad insect associates.

Beta-methylazoxymethanol (BMAA) is stored in idioblasts in the leaves of some cycads (Brenner et al. 2003); herbivory by insects has been shown to have beneficial effects regarding enhanced growth patterns as an agonist of glutamate receptors which induces increased hypocotyl growth, as evidenced in Arabidopsis. There is a question as to whether the BMAA neurotoxin may have evolved before it developed as an herbivore deterrent (Brenner et al., 2003). Brenner et al. (2003) questions if the idioblasts may have evolved to protect the other cells because of the high concentrations of the chemical. Azoxyglucosides are not released until leaf tissue injury occurs (Yagi, 2004). Methyl-azoxyglucoside-methanol (MAM) therefore released via herbivory or as enzymes in the digestive tract of herbivores (Schneider et al., 2002;

Yagi, 2004), but BMAA contents have not been analyzed in any of the herbivores yet

(Prado, 2011). Some cycasin and macrozamins have been analyzed in some insects and parts of some cycads, but there is a lot still unknown about the complex relationship between the insects and the plants.

Castillo-Guevara and Rico-Gray (2003) conjectured that macrozamins are the most primitive of azoxyglucosides, and may have evolved as a primeval method of storing nitrogen. The increased fitness allotted by the mechanism became an exaptation

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retained by the species as protection from herbivory. They mention that because the plants are very slow growing, this added protection would have proven a beneficial to prevent competition for space in crowded tropical regions where many cycads grow; it is known that the sarcotesta of the Z. integrifolia seeds contain a growth inhibitor (Taylor,

1999) and must break down over time usually germinating the next season unless the flesh is consumed by wildlife. Cycas circinalis inhibits germination of nearby plants through the root system (Castillo-Guevara & Rico-Gray, 2003).

Castillo-Guevara and Rico-Gray (2003) show a negative relationship between the types of herbivory and azoxyglucoside content of the plants, regardless of whether the herbivore was a lepidoptera, beetle, weevil, midge, mealy bug or scale insect. In other words, it did not matter what kind of herbivory occurred, it did not predict the neurotoxin concentration in the cycad.

The chemical concentrations and variations that occur between cycad species do not seem to affect the any of the insects’ interest in colonizing the plants or consuming the plants (Koi, unpublished), except for the weevils that are specialists and/or obligate pollinators of specific cycad species (Norstog & Fawcett, 1989; Tang, 1990; Vovides,

1991; Oberpreiler, 1995a, 1995b, 1995c, 2004; Stevenson et al., 1998; Schneider et al.,

2002; Chaves & Genaro, 2005; Chemnick et al., 2002). It is thought that the cycads from Central and South America utilized by the Atala butterflies are basal species recognized as ancestral host plants (Meerow, pers. comm.). The chemoecology is similar for most of the plants (DeLuca et al., 1980; Charlton et al., 1992; Nash et. Al.,

1992; Brenner & Twigg, 2003; Donaldson, 2002; Schneider et al., 2002; Castillo-

Guevara & Rico-Gray, 2003; Yagi, 2004; Nicolalde-Morejón et al., 2011), but many of

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the non-native cycads have not yet been chemically analyzed and the analyses of others have been performed using different methods or parts making comparisons difficult to interpret.

Integrated Pest Management

Integrated pest management (IPM) is a multifaceted methodology developed to prevent or control pest insect damage to economically valuable plants (Boyles &

Koehler, undated; Speight et al., 2008), including ornamentals that may be expensive, rare and/or aesthetically significant to botanical gardens and homeowners. IPM integrates various methods for detecting, identifying, controlling and managing insect pests by combining biological, cultural, physical and chemical means to keep insect damage at an acceptable level. It is the most economically balanced way to prevent potential health hazards to humans and animals, while controlling damages caused by insects (Boyles & Koehler, undated; Pimentel, 2009, 2011, 2013; Pimentel et al., 2013).

IPM is the safest approach for controlling infestations of unwanted insect pests while conserving beneficial insect species, as well as imperiled or endangered biota sharing the same habitat (Speight et al., 2008; Boyles & Koehler, undated).

There are four methods to IPM: cultural control, physical control, biological control and chemical control. The goal of IPM is to use the first three to prevent using the fourth. Cultural control is the basis for everything else and includes things such as understanding the biology of the plant and the pest insects’ life, maintaining a healthy garden through sanitation practices (such as removing sick plants as soon as they are identified) and timing are other cultural controls. Planting too early or too late in the season can be stressful to the plant making it more susceptible to pest insects. The timing and amount of watering practices may affect a plant’s ability to withstand

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seasonal changes, herbivory or strong sunlight. Another cultural control may be the use of trap crops, plants that attract insect pests away from desirable plants and may be planted slightly out of regular season. In that way, in proper season the pests have been eradicated because their life cycles will have been completed (Boyles & Koehler, undated; Speight et al., 2008).

Physical control denotes proper pruning methods, tilling and planting practices, mulching appropriately for the plant species, and pest exclusion. It can include mixed plantings that encourage beneficial insects and discourage pests. Biological controls may include releasing commercially propagated beneficial insects into a garden, such as Ladybird Beetles or Green Lacewings, or providing the plants and cultural practices that promoting natural beneficial insects. Biological controls avoid the use of potentially harmful chemicals that may affect beneficial insects and other biota, such as fish.

As a last resort for sever infestations of unwanted pest insects, chemical means may be used that target specific pest species and maximize effectiveness while minimizing hazards to humans and other animals (Speight et al., 2008; Pimentel, 2009,

2011, 2013; Pimentel et al., 2013).

IPM first entails monitoring plants for pests frequently to find them as soon as possible. Once an infestation has taken place, it if often too late to eradicate by means other than chemical, if not recognized and treated early (Boyles & Koehler, undated;

Speight et al., 2008). Unknown insects must be identified to ensure that beneficial insects are not destroyed inadvertently. It is indispensable to set “action thresholds,” the level of insect damage permissible or acceptable that determines when action needs to be taken to control insect damages.

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“Florida Friendly Landscaping” methods are designed to use integrated pest management practices and Florida native or Florida-compatible plants, to establish low maintenance, water-restricted, wildlife attractive yards and minimizing the use of pesticides, herbicides and fertilizers.

Host Plant Choice Tests with the Atala Butterfly

The Atala butterfly has adapted to utilize many of the introduced non-native and valuable cycads found in botanical and domestic gardens in south Florida and Zamia vazquezii L. (Zamiaceae: Cycadales) is one of the valuable non-native cycads being used by the Atala butterfly as an alternative host plant (Hammer, 1995; Koi, unpublished). Z. vazquezii is a critically endangered cycad in its native Mexico (Vovides,

2010) and is a highly utilized cycad in Miami-Dade botanical and domestic gardens, as well as in upscale urban developments (Haynes, 2000; Dehgan, 2002).

Herbivory of the cycad is a pest management concern of economic importance as it is one of the exotic cycad species produced in highly specialized south Miami-

Dade nurseries and is considered a valuable addition to a prestigious landscape (i.e., it is not generally utilized along highway median strips or shopping plaza parking lots).

However, Z. integrifolia, our native cycad, has shown itself to be hardy and resilient enough to be successfully utilized along mall parking lots and highway medians. Both are pest-resistant under optimal conditions, but show signs of stress, such as scale infestations (Figure 5-1) or sooty mold, when too crowded or poorly maintained.

Both cycads are readily used by the Atala butterfly in these ‘semi-natural’ urban environments, and may sustain considerable herbivory damage (Figure 5-1).

Pesticide use has been recorded repeatedly (Koi, unpublished) to control the insect,

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thereby triggering both an economic impact as well as a potentially detrimental environmental damages (Pimentel, 2009, 2011, 2013; Pimentel et al., 2013).

Larval and adult host plant choice tests were performed with comparisons between Z. integrifolia and non-native Z. vazquezii to determine larval survival, development rates and subsequent adult choice, as oviposition choice by offspring has been shown to be at least partially affected by the adults’ oviposition choices (Singer et al., 1988; Mousseau & Dingle, 1991; Paukku & Kotiaho, 2008; Meagher et al. 2011;

Mader et al., 2012; Henry & Schultz, 2013). Understanding host preference (Singer,

1983; Thompson & Pellmyr, 1991; Meagher et al., 2011; Lindman et al., 2013), egg distributions (Floater & Zalucki, 2000), heritability of oviposition sites (Singer et al.,

1988; Mousseau & Dingle, 1991; Floater & Zalucki, 2000; Paukku & Kotiaho, 2008;

Meagher et al., 2011; Mader et al., 2012), colonization of non-native host plants (Mader et al., 2012; Forister et al., 2013), larval feeding strategies and choices (Floater &

Zalucki, 2000; Dolek et al., 2013) and oviposition site selection (Thompson & Pellmyr,

1991; Paukku & Kotiaho, 2008; Meagher et al., 2011; Mader et al., 2012; Dolek et al.,

2013; Henry & Schultz, 2013) for the Atala butterfly and larvae will help lepidopterists develop management protocol (Henry & Schultz, 2013) focusing on cultural practices and reducing pesticide use to control herbivory.

Results indicated that both adults and larvae chose native more often than non- native, but that survival and development decreased when utilizing Z. vazquezii.

Survival to successful mating and fecundity as adults occurs with either choice. Non- chemical mechanical control methods were also explored for control of larval herbivory on Z. vazquezii.

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Materials and Methods

All tests were completed under the same environmental conditions in a laboratory at the University of Florida with approximately 8:16h photoperiod and stable ambient temperatures between 24°C and 26°C. Cages were sprayed twice daily with water misters to maintain normal humidity.

Adult Choice Test I

Adult butterflies of both sexes were freely housed together in a large walk-in adult flight cage (LiveMonarch, 70” x 70” x 70”, Greenhouse Cage, Boca Raton, FL) to approximate natural environments. Cages were equipped with nectar and water feeders

(see Chapter 2) as well as host plants as described here. For the adult oviposition choice tests, one frond each from both Z. integrifolia and Z. vazquezii, closely matched in size, were installed in the flight cage. The host plants were each encased in a water pic to keep the leaves fresh, and were hung approximately 15” from the ceiling to make them more apparent to the adults. The leaves were installed about two feet apart on the same side of the cage so that they were not touching. Fresh leaves were installed daily, and the positions of the fronds alternated daily to avoid position preference by the females.

Leaves were collected from the flight cage daily and ova counted to assess oviposition preferences by the females. Trials were executed for forty-two uninterrupted days. Because of the nature of the free-flying adults in the flight cage, multiple parents were most likely represented by the ova in this test. Ova were counted and the fronds stored in water pics that were refreshed daily with water to keep the leaves fresh.

On hatch, the larvae were reared with their brood mates on the host plant of the adult choice or reared singly if only one egg hatched. The larval boxes containing adult-

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choice hatched larvae were maintained as described above until pupal emergence. The eclosed adults became First Generation Adult Test.

First Generation Adult Choice Test I

Larvae that successfully pupated and emerged as adults from “Adult Choice Test

I” were housed together as they emerged in smaller fabric cages (LiveMonarch Jumbo

Cage, Boca Raton). Clutches were housed together as much as possible but because of the variable hatch rate and deaths, the sample sizes are small for this test. The cages were supplied with nectar and water feeders (see Chapter 2) as well as two fronds of host plant, one each Z. integrifolia and Z. vazquezii. Fronds were hung approximately ten inches from the ceiling and installed about six inches apart but not touching. The position of the fronds was not alternated and adults were set free in the large flight cage as soon as the females chose an oviposition site. (Only one cage had more than one female.)

First Generation Adult Choice Test II

Larvae that successfully pupated and emerged as adults from “Larval Choice

Test II” were housed together as they emerged in smaller fabric cages (LiveMonarch

Jumbo Cage, Boca Raton) as previously described. Clutches were housed together as much as possible but because of the variable hatch rate and deaths, the sample sizes are small for this test as well. The cages were supplied as described above and adults released to the big flight cage when females made ovipositing choices.

Larval Choice Test I

Ova were collected from any non-plant substrate, such as the wall, floor, nectar feeders or roof, from inside the flight cage described above. Cages were equipped with nectar and water feeders (see Chapter 2) as well as host plants as described below.

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Because of the nature of the free-flying adults in the flight cage, multiple parents were most likely represented by the ova in this test. Sanitized plastic petri dishes (9 cm) were prepared as choice test arenas by trimming Whatman filter papers to fit inside the petri dishes. The filter papers were lightly folded in half to find the center and a line was drawn with a graphite pencil to delineate the middle. The top of each half of the arena was labeled either “N” (designating native Zamia integrifolia) or “NN” (designating non- native Z. vazquezii). The labels were alternated in each arena to reduce possible side- preference effects by the larvae (i.e., 1=“N-NN”, 2=“NN-N”, 3=“N-NN” etc.). Just before ova were placed into the arena, the filter paper was dampened with deionized water and the dates of collection recorded so that expected hatch dates were known. Hatches were monitored immediately after eclosure was noted; larval eclosure, behavior and choice were photographed.

A single ovum from the day’s collected eggs was placed in the center of each prepared petri dish and lightly sealed with string to prevent larvae loss, if eclosure occurred earlier than expected. The arena and egg was allowed to rest undisturbed until the larvae hatched. On the expected day of larval hatch, the lid and string were removed from the petri dish, and single leaflets were pinched off at the rachides of each of the two host plants. The individual leaflets were installed on opposite sides of the center line approximately 1 cm from the center (Figure 5-2). The leaflets were changed daily if the larvae did not hatch as expected until it did emerge.

The larvae were monitored to record behavior and choice immediately during and after eclosure. Choice was recorded when the larvae climbed onto the plant it chose and commenced feeding, i.e., settling in” (Figure 5-3A) If the larvae did not remain on

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the leaf to continue feeding after an initial “test-bite,” it was not recorded as a choice

(Figure 5-3B). The time from eclosure to choosing its host plant was recorded. Larvae were reared singly on the host plant of their choice in standard square plastic boxes

(19.05 cm x 19.05 cm x 10.16 cm). The box tops were covered with 1 mm tulle netting to prevent larvae escape; the boxes were cleaned daily and food of the larvae’s choice was replaced. The rachides of all food fronds were housed in water pic tubes to maintain freshness for the larvae.

Larval Choice Test II

The same basic protocol was followed as above, with two changes: instead of one egg per arena, an entire brood of eggs from the same location was deployed in the arena. These small clusters were likely from the same female. Ova were lined up on the center line in haphazard order. This test explored how and if larvae influenced each other in their choice of host plant (Figure 5-4).

The second change in this test involved trimming the both ends of the leaves with scissors (rather than pinched off at the petiole), to both simulate herbivory and release volatile plant chemical clues (Figure 5-4). This change was made based on observed behavior exhibited by the larvae in Larval Choice Test I that included larval attempts to garner these volatiles from the atmosphere in the arena by rearing up and swaying with their “nose in the air” and returning repeatedly to the ova, circling it and touching it.

Larvae were reared with their brood mates on the host plant of their choice in square plastic boxes (19.05 cm x 19.05 cm x 10.16 cm); individuals that chose differently than brood mates were reared singly or with their congeners, depending on their choices. The box tops were covered with 1 mm tulle netting to prevent larval escape; the boxes were cleaned daily and food of the larvae’s choice was replaced. The

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rachides of all food fronds were housed in water pic tubes to maintain freshness for the larvae. The larval boxes containing larval-choice larvae were maintained as described above until pupal emergence. The eclosed adults became First Generation Adult Test II.

Figure 5-5 is a graphical representation of the experiment design.

Results

Adult Choice Test I

Ova collected from the flight cage daily were counted and a total of 9055 ova were laid during the forty-two day trial. There was a significant difference in female host plant choice, with 8094 ova laid on Z. integrifolia and 961 ova laid on Z. vazquezii (p- value <0.000). Native Z. integrifolia (Zi) received 89% of the ova and 11% were laid on

Z. vazquezii (Zv) (Figure 5-6). Although it is possible for all life stages to be completed successfully while feeding on the non-native plant Zv, the survival rate was very low and many larvae feeding on both plants did not complete the life cycle (Figure 5-7).

Differences in development times was not significantly different for any life stage between the two groups (Adult or Larval Choice) or between the two plants (p>0.05), although there were pronounced differences (Table 5-1).

For example, average number of days from oviposition to adult emergence (Adult

Choice I) was 46.80 days on non-native Zv and 38.69 days on native Zi (Table 5-1).

Only 2% of eggs laid on non-native Zv hatched and only seven out of fifteen adult butterflies reared on Zv successfully emerged from the pupa (66%). Only 15% of eggs laid on native Zi hatched, but 254 adults successfully emerged from 347 pupae (73%).

Ova to adult survival was 0.021 for larvae consuming Zv and 0.153 for larvae feeding on native Zi. Pupa to adult eclosion survival was 0.666 for larvae that had

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consumed Zv, and 0.732 for larvae that had consumed Zi. Survival was markedly lower for larvae reared on Zv, but not significantly so (Figure 5-9).

Immature development time and duration of life stage not significantly difference for larvae reared on either plant, but there was a significant difference between the number of ova laid on Zv and Zi (휒2=df14, 0.05, 169.00) (Figure 5-10).

Longevity of adults did not vary significantly between individuals reared on either plant, but adults reared on Zv (Adult Choice) lived longer than other adults in the trials

(Figures 5-11 and 5-12).

First Generation Adults from “Adult Choice” Test I

First generation adults that eclosed from “Adult Host Plant Choice Test I” pupae chose to oviposit on native Zi 100% of the trials (n=32, 11 females, 4 cages).

Larval Host Plant Choice Test I

Most larvae chose native Zi (77%), but many nibbled the non-native plant first.

One neonate eclosed from egg that had been laid on a non-host plant, Pithecellobium bahamense, a southeast Florida native that occurs in coastal areas where there are extant Atala colonies. The plant had been installed in the flight cage as a roosting tree, and was not intended to be used for this experiment. When the larvae eclosed, it moved toward the petri-dish sides and circled consistently, wandered to the native leaflet but leaving it again without feeding. It would wander to the non-native leaflet in the same manner but did not commence feeding. After nearly four hours, I counted it as “no choice” and it was the only “no choice” in all of the trials.

Most of the larvae found their way to the native leaflet within 6 minutes (±14.10)

(Figure 5-13). A third short trial within this test used 112 ova which developed into 42 larvae, 93% of which chose native.

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Larval Host Plant Choice test II

Ova collected from broods appeared to make independent choices, as not all of the larvae chose the same host plant (Figures 5-4 and 5-14). A higher percentage chose native over non-native (83%) than in the first trial, and 17% chose Zv non-native host plant. Total development time from egg to adult (Larval Choice II) showed 42.75 days for larvae reared on Zv, and 39.16 days for larvae reared on Zi (Table 5-1). The sample size was small (n=23) as individual mortality was high reared on Zv.

First Generation Adults from “Larval Host Plant Choice” II

First generation adults that had been reared on both native and non-native plants in “Larval Host Plant Choice Test II” chose native Z. integrifolia 100% of the time.

Discussion

All cycads contain variations of the same basic chemicals (cycasins, macrozamins, etc.) and are most likely recognized by the Atala butterfly as basal host plants. However, both larvae and adults preferred native Z. integrifolia over non-native

Z. vazquezii. Growth and survival were arrested to some extent on the non-native cycad, and development was influenced as well, although there were no significant difference between the growth rates. There was a significant difference in which plant received the most eggs, indicating that female adults choose the native plant preferably for the offspring. It was interesting that the first generation adults from larvae that chose non-native as a larval food chose native for oviposition.

In general, insects reared on Z. vazquezii lived longer than those reared on native host, although not significantly so. It could be that insects that are tough enough to survive on the non-native plant simply have a more robust constitution. The surface epidermis of Z. vazquezii is much thinner than that of native Z. integrifolia, requiring

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larvae to feed longer and grow more slowly to acquire the nutrients needed to successfully complete their life cycle to adult emergence.

If I were to redesign the tests, I would isolate single females to better control female age and brood effects. Although this is not entirely under human control, significantly more larvae should be reared on Z. vazquezii for the tests, as many died during the trials.

There were two attempts to rear individuals from eggs that had been laid on native and non-native plants by removing the eggs from their substrate and switching them to the other plant. However, in the first trial, only one of eight ova hatched that had been laid on Z. integrifolia and the larva finally chose to consume Z. integrifolia after 1.5 hours of wandering around the petri-dish. In the second trial, again only one larva hatched from a brood of seventeen ova. It also chose native Z. integrifolia within a few minutes. These trials were not used in any analyses. The other sixteen ova developed fungal bodies as well.

Conservation Concerns: Encounters between Wildlife and Humans

One of the possible reasons that the Atala butterfly has also made a dramatic recovery from near-extinction is because of the increased use of Zamia plants in landscaping, as well as increased conservation and restoration projects by biologists, botanists, park managers, scientists and concerned citizens. In 2008, Everglades

National Park, for instance, grew additional coontie for the park from seed harvested four years prior in the park by volunteers, to prevent possible dilution of genetic material

(Perry, pers. comm.). Most restoration endeavors include replanting native vegetation as well as removing non-native invasive plants.

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Although it is generally accepted as good that the Atala has increased its range and distribution because of increased use of cycads for landscaping (Koi, unpublished), the adult butterflies have expanded their oviposition choices during the past twenty years as well. That expansion includes many of the non-native introduced ornamental cycads found in south Florida’s botanical and domestic gardens (Hubbuch, 1991;

Hammer, 1985; Koi, unpublished). The larvae are able to successfully complete their life cycle on many, if not most, of these non-native plants (Hubbuch, 1991; Hammer, 1995;

Koi, unpublished).

This expansion into exotic Cycadales, many of which are extremely rare and valuable, has in turn increased potential struggles between home-owners, botanists, property managers and city planners with park managers, biologists, entomologists and conservationists as the Atala butterfly larvae attack these urban cycads (Hubbuch,

1991; Culbert, 1994;Oberpreiler, 1995a, 1995b, 1995c; Culbert, 1995,2012; Koi unpublished). Fortunately there have been no records of the Echo Moth using any of these non-native cycads as host plants at this time, although they do find their way to urban garden occasionally.

Conservation methods are complicated because the often severe herbivory caused by the Atala larvae may damage the plant, but control of the larvae may impact the fragmented populations of the butterfly. The butterfly is currently listed as imperiled by the State of Florida and the natural ecosystems of coontie, the pine rocklands and tropical hardwood hammocks, are both now recognized as endangered (MDC, undated;

Myers,1990; Snyder et al., 1986). The question of how to address one endangered species’ natural but possibly destructive interactions with another endangered species

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is a dilemma faced by conservationists worldwide (Castner 1986; Hubbuch, 1991;

Culbert, 1994;Oberpreiler, 1995a, 1995b, 1995c; Culbert, 1995,2012).

Examples of human-endangered-wildlife conflicts include the endangered Rock

Hyrax (Procavia capensis), a small mostly herbivorous mammal, that has been invading domestic gardens in new developments in Northern Israel, where its’ native vegetation may not be as succulent as the irrigated vegetables growing in people’s gardens

(Kershenbaum et al., 2011). Because rock piles from building home sites often remain near the developments, the hyrax has utilized these ‘better’ nest sites rather than the natural rock crevices found in the desert outcrops. The natural crevices usually have one entrance split into the rock and little shade during certain times of the day; the rock piles, however, have several passageways, shade throughout the structure, and offer more secure nests from predators (Kershenbaum et al., 2011). This is also a human health issue because of the possibility of cutaneous leishmaniasis carried by the hyrax

(Kershenbaum et al., 2011).

The conflict between human safety and threatened animal populations is pronounced and obvious with predators such as wolves and black bear, but may include species usually thought of as common and harmless, such as white-tailed deer

(Odocoileus virginianus). Vehicular deaths for instance, are increasingly caused by roaming wildlife such as deer, as they are further pushed out of decreasing wild areas into urban roadways and back yards (Rondeau, 2001). In addition, deer carry the tick that causes Lyme disease, another major public health concern (Rondeau, 2001).

In Eastern Oregon and Washington, forestry managers face similar conflicts between balancing pest insect species, such as mosquitoes, while protecting scores of

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threatened insects that live in the tracts of forested area (LaBonte, et al., 2001). The paper points out that many insect species are ephemeral, and/or diminutive; that they are therefore difficult to monitor, challenging to find, may live in scattered fragmented habitats, but are nonetheless vital to overall health of the forest. Areas bordering urban localities are especially vulnerable; LaBonte et al. (2001) point out the need for mosquito control spraying in recreational areas, controlled burns to maintain forest ecology, road building for access to locations within the forest, changes in soil chemistry from compaction and erosion, herbicide use to control unwanted invasive plant species; the possible extirpation of the Polites mardon butterfly and a flightless beetle, Agonum belleri, may have been caused because of these challenges.

Urban interface areas such as this become classic fields of controversy when conflicts between human wishes and endangered species are involved, in any country, in any state (Hubbuch, 1991; Culbert, 1994; Culbert, 1995(2012); LaBonte, et al., 2001;

Rondeau, 2001; Kershenbaum et al., 2011). The environmental impacts of pesticide use, herbicides and other chemical means of controlling pests while managing wildlife is extremely complex to work with (Pimentel, 2009, 2011, 2013; Pimentel et al., 2013).

Deer, bear and wolves, as well as the little Atala butterflies, that were once thought to be nearly extinct, have all made impressive recoveries thanks to the vigorous conservation efforts of many people and legislations. However, there is a down side to the recovery when human health, or preferences, come into conflict with the species

(Rondeau, 2001; Kershenbaum et al., 2011). And unfortunately, management policies of increased wildlife populations come under fire as conservationists juggle human needs against wildlife survival (Rondeau, 2001 and references within).

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Insects under conservation concerns have matters such as non-target pesticides to battle as well as habitat loss and declining host plants. (Oberpreiler, 1995a, 1995b

1995c; Hardy & Dennis, 1999; Pimentel, 1995, 2009, 2011; Pimentel & Hart, 2001;

Salvato, 2001, 2002; Salvato & Henssey, 2003; Oberpreiler, 2004; Minno, 2010, 2012;

Bargar, 2012; Hoang et al., 2011).

Conclusion: Hand-Management and IPM Practices for Atala Butterflies

Hand management practices must evolve as we learn more about the requirements of the atala butterfly, including landscape architecture, nectar sources and host plants. Recommendations involve a number of protocols, some of which have not yet been defined for this species. Pesticides to control unwanted insect pests such as mealy bugs should be used sparingly in butterfly gardens as non-target insects, such as

Lepidoptera, are often impacted adversely by their use (Salvato, 2001; Hoang et al.,

2011; Bargar, 2012) (see Chapter 5).

Some practices, such as hurricane preparedness, are intensive and may be difficult to apply in large areas such as Everglades National Park. However, for small domestic gardens, natural areas under twenty areas, botanical gardens and domestic gardens, practices to ensure optimal environments for the butterfly are essential.

Individuals who are lucky enough to live in southeast Florida and have a butterfly garden, or park managers who have had coontie planted as an ornamental on the property or that have areas of naturally occurring but sparse coontie in the field, may find it necessary to make decisions about their colony.

Planting native and non-native together may be another possible management protocol for protecting valuable cycads, as intercropping has been shown to be an effective pest management for conservation (Floater & Zalucki, 2000). Because the

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Atala prefers native to non-native cycads, utilizing more native plants would be beneficial. Of course, one of the tricks to any successful butterfly garden is to move highly attractive host plants to areas where they will not be scrutinized by the public; public complaints from people who do not understand the value of the host plants is one of the biggest reasons the host plants get torn out. It is a shame that rather than using the herbivory to teach the public about the insects, the plants are removed. I have seen it happen over and over again, especially as coontie becomes more popular because of its hardiness and beauty, but also as the Atala is utilizing these stepping stones from one area to another.

A cultural management method could be used to prevent herbivory such as screening the most valuable plants while adult butterflies are active. A tomato cage covered with tulle mesh and buried into the ground an inch or so will prevent female ovipositioning on the plants. The adult females will flatten their wings ‘airplane style’ to crawling into tight places, so the mesh must be securely closed on all sides and along the bottom.

Hand-removal of ova on either Z. integrifolia or Z. vazquezii is easy with a thumb nail as they are not strongly adhered. The eggs can be frozen to humanely dispatch the embryos or simply left in the substrate where the ants and other insects will enjoy them.

Although it can be time-consuming, hand-removal is certainly less expensive than buying a new cycad, especially since some valuable plants can cost thousands of dollars! If larvae hatch, they will most likely be unable to find food before dying as the larvae must find food within a few hours after eclosure or they will die.

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Because it requires more time to develop on non-native Z. vazquezii, there is additional time for owners to remove the larvae from the plant. If possible, I suggest contacting the local chapter of the North American Butterfly Association, or Extension office Master Gardeners program to find new ‘foster homes’ for unwanted or excess

Atala larvae or pupae. Contacting the parks and natural areas management staff may welcome hosting this beautiful butterfly and be willing to take excess Atala.

And lastly, a thought from the world-renowned cycad botanist, Rolf Oberpreiler

(1995c):

The natural insect fauna of cycads should not be brazenly dismissed as ‘pests.’ These insects are a natural and mostly vital component of the environment of the cycads, and their destruction can have severe impacts on the survival of the plants. The most obvious examples in this regard are the pollinators, but other insects may also play important roles in e.g., the disintegration of the cones and release of the seeds, the decomposition of old cones, leaves and stems, the recycling of nutrients, etc. . . .their survival is so inextricably attached to that of the plants that extinction of their host plants will inevitably lead to the extinction of these insect species.

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Table 5-1. Development time varied between groups depending on the plant consumed, and whether it was Adult Choice or Larval Choice, but there was no significant difference in number of days.

Ova Larval Pupal Total Adult development development development Development Lifespan (days) (days) (days) (days) (days) n=

rval choice: Z. integrifolia 7.47 16.47 15.21 39.16 27.68 19 St. dev. ±0.70 ±1.74 ±1.18 ±1.30 ±13.47

Larval choice: Z. vazquezii 7.00 14.00 19.00 42.75 25.75 4 St. dev. ±1.15 ±2.00 ±2.06 ±3.69 ±16.32

Adult choice: Z. integrifolia 8.44 14.25 16.00 38.69 33.44 16 St. dev. ±0.51 ±1.18 ±1.21 ±1.96 ±12.28

Adult choice: Z. vazquezii 8.87 22.20 15.73 46.80 22.13 16 St. dev. ±0.35 ±2.76 ±3.65 ±4.83 ±15.32

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Figure 5-1. Zamia encephalartoides is one of twenty non-native cycads found in upscale gardens in southeast Florida. When infested with hundreds of Atala larvae, the plant is in danger of being damaged so severely that it may be unable to recover. The arrow points to two other cycad herbivores, Saissetia coffeae and Chrysomphalus aonidum. Photo courtesy of S. Koi.

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Figure 5-2. Larval Host Choice I arena and newly eclosed single larva. Photo courtesy of S. Koi.

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A B Figure 5-3. Neonate feeding behavior. A) Choice was recorded when the larva climbed onto the plant and settled on a feeding site; this is the native leaflet. B) Larvae sometimes took tentative ‘test-bites”, but did not stay to feed; this is the non-native leaflet. Photos courtesy of S. Koi.

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A B

C D

Figure 5-4. Arena for Larval Test II trials utilized the entire brood. A) Five larvae of five chose native Z. integrifolia. B) The first eclosed larva chose non-native Z. vazquezii, but the six eggs did not hatch.C) Five larvae in the brood chose non-native and one chose native. D) All five larvae chose native, but there was evidence of frass and test bites (nibbles) on the non-native plant. Photos courtesy of S. Koi.

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Figure 5-5. Graphical representation of test trials. Photos courtesy of S. Koi.

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11%

Z. integrifolia 89%

Z. vazquezii 11%

89% N=9055 ova

Figure 5-6. The “Adult Host Choice Test I” originated with 9055 ova laid on two host plant choices. The First Generation Adult Test I reared from these eggs all chose Z. integrifolia (100%).

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Figure 5-7. Many of the larvae feeding on non-native Z. vazquezii died before reaching pupation. This larva developed a fissure in the midgut from unknown causes and died. Photo courtesy of S. Koi.

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Figure 5-8. Comparison of six-day old larvae reared on non-native Z. vazquezii (arrow) and native Z. integrifolia. Note the surface scraping on the non-native, which has a much thinner epidermis than native plants. Photo courtesy of S. Koi.

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1 Z. vazquezii Z. integrifolia 0.75 Expon. (Z. vazquezii) Expon. (Z. integrifolia) 0.5

0.25 y = 1.2375e-0.571x R² = 0.7875 Proportion Surviving Proportion -1.315x 0 y = 2.7234e R² = 0.9388 Ova Larvae Pupae Adults

Figure 5-9. Proportion of Atala surviving to the next life stage was lower on non-native Z. vazquezii host plant than on the native Z. integrifolia (Adult Choice Test I).

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20 Zi n= 25

15 Zv n= 15 Z. vazquezii 10 Z. integrifolia

5 Development Time (days) Time Development

0 Larvae Pupae Imagoes

Figure 5-10. Immature development time and duration was not significantly different for individuals reared on native or on- native host plant (Adult Choice Test I), but was pronounced.

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50 Larval choice: 40 Z. integrifolia

Larval choice: 30 Z. vazquezii

20 Adult choice: Z. Number ofDays Number vazquezii

10 Adult choice: Z. integrifolia

0 Ova Larval Pupal Adult Adult Lifespan

Figure 5-11. Comparisons of development time per life stage and adult lifespan between larval and adult choice trials.

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Adult choice: Z. vazquezii Larval choice: Z. integrifolia Adult choice: Z. integrifolia Larval choice: Z. vazquezii Larval choice: Z. vazquezii Adult choice: Z. integrifolia Larval choice: Z. Adult choice: Z. integrifolia vazquezii 0 20 40 60 Lifespan (days)

Figure 5-12. Adult lifespan in larvae and adult choice trials.

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11% 2% Larvae chose native

Larvae chose non-native

10% Larvae made no choice

77% Nibbled non-native

n=126 ovae, 47 larvae

Figure 5-13. In Larval Choice Test I, one of the neonate larvae did not make a choice, many nibbles the non-native Z. vazquezii, but most chose native Z. integrifolia.

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17%

Larval Choice: Z. integrifolia

Larval Choice: Z. vazquezii

83% n= 23

Figure 5-14. Larval Host Plant Choice Test II exhibited a similar pattern to Larval Host Plant Test I, but there were no “nibbles” on the non-native Z. vazquezii plant and all of the larvae made a choice.

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APPENDIX A CONSUMPTION DATA FOR EUMAEUS ATALA LARVAE IN A CAPTIVE COLONY

One of the difficult and challenging aspects to maintaining a natural wild land or habitat is assuring that it provides the necessary ingredients for a healthy population of wildlife, including assessing the potential food content available for the wildlife. This may entail multiple and overlapping avenues for maintaining the ecosystem, including removing invasive plants and animals, replanting native vegetation and restoring derelict remnants of land, providing connectivity between fragmented lands, planning fire regimes to simulate natural ecology, anticipating changes in seasonal hydration and many other husbandry factors. Maintaining a landscape is even more challenging when there is not enough information about the species for which land managers are trying to provide. The Atala butterfly (Eumaeus atala Poey 1832) is one such species. Until this research, much of its basic life biology was unknown and published reports were incomplete or in disagreement.

Pine rocklands in Miami-Dade County, southeast Florida, are a critically endangered ecosystem (Land & Cooley, undated; Avery & Loope, 1980; Baggett, 1982;

Snyder, 1986; Snyder et al., 1990; Hubbuch, 1991; Hammer, 1995; U.S. Fish & Wildlife,

1999; González, 2004; Schultz et al., 2008; Henry & Schultz, 2013; Lindman et al.,

2013), and harbor a myriad of state and federally listed plants and animals, including the Atala butterfly. Federally listed animals include Key deer (Odocoileus virginianus clavium), eastern indigo snakes (Drymarchon corais couperi), bald eagles (Haliaeetus leucophalus), gopher tortoises (Gopherus polyphemus) and the Florida panther (Puma concolor coryi). Approximately 30 species of plants are endemic to the pine rocklands and some, such as deltoid spurge (Chamaesyce deltoidea) are listed as endangered by

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the State of Florida. Small’s milkwort (Polygala smallii), an endemic to the pine rocklands, is listed as endangered both by the state of Florida and the United States at the federal level.

Another important pine rockland herb is Croton linearis, which while not listed federally or state-wide, is the host plant for Bartram’s Scrub Hairstreak (Strymon acis bartrami) and the Florida Leafwing (Anaea troglodyte floridalis), both new candidates for listing as federally endangered at the time of this writing (August 2013). The Atala butterfly’s host cycad plant, coontie (Zamia integrifolia), is Florida-listed as threatened

(Coile, 2000; Coile & Garland, 2003) in the remnant pine rocklands, due to over- harvesting of the plant during the early part of this century as a source of starch by the early settlers (see Chapter 1). Because the cycad is extremely slow-growing, and the pine rocklands are an exceedingly harsh and nutrient poor ecosystem (Snyder, 1986;

Snyder et al., 1990), the plant has been unable to fully recover from the wanton destruction that took place over 100 years ago.

The Atala butterfly was thought to be extinct during the 1950’s, and when rediscovered in the barrier islands off the shore of Miami, efforts were intense to ensure its survival. New colonies seemed to be continually wiped out by hurricanes or other unknown causes. One of the biologists involved was Dave Baggett (1982) who suggested that perhaps a lack of adequate host in the pine rocklands was a cause for the Atala butterfly’s seeming inability to re-colonize in Everglades National Park (ENP).

It had been documented in the 1930’s there.

When I assisted park biologists at ENP with a third Atala butterfly reintroduction attempt in 2004-2008, we arrived at a similar conclusion as Baggett. Because of the

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plants’ protected status and the fact that we did not want to change the genetic make-up of the plants indigenous to the park, we harvested seed from mature coontie plants on site and grew them from seed.

Naturally occurring seedlings establish themselves in the substrate very slowly over time, usually starting in a solution hole that has filled with whatever detritus has fallen into it from the surrounding scattered understory vegetation, as the substrate is mostly exposed limestone (Snyder et al., 1990). The sarcotesta of the ripe seed is broken down over a years’ time by various rodents and opossums (which are not susceptible to the toxins in the seed flesh) and by microbes and detritus-associated insects, such as Oniscidea species (commonly called sowbugs and their kin). When the summer rains arrive in May or early June, the seed will be ready to sprout.

The plant sets roots slowly over many years, weaving into the open cracks and forcing channels into the limestone. Four years were required for the seedlings at ENP to reach a survivable size because the pine rocklands are so rugged an environment; the new plants were installed into holes that were first drilled into the limestone substrate which were filled with natural detritus from on-site. To remove a coontie plant from the limestone substrate requires a chisel and hammer---and muscle! (Colleagues and I have removed plants from construction zones, not natural areas, in south Miami-

Dade and replanted them in safe habitats within the county.)

The coontie tap root will extend deep into the limestone as the plant grows. This is likely the reason there is any coontie left in wild locations, because the wild coontie was heavily harvested in the early part of this century. However, if enough of the tap root remained embedded in the limestone, the plant could recover over time. I have

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documented four sites in southeast Florida which were razed of invasive plants and shortly thereafter coontie seedlings emerged that had been covered by the invasive non-native plants--for up to fifty years! One site, John U. Lloyd State Park in Broward

County, removed the Casuarina plants along the shoreline and months later, over twenty coontie plants emerged from under the wood chips.

Another site, privately owned in south Miami-Dade, had been leveled of native vegetation in the 1950’s and rock-plowed for vegetable growing. When the current owner bought the derelict property in the 1990’s it was covered with Schinus terebinthifolius and other invasive plants. He had the property razed again to destroy the non-native vegetation and began restoring the original pinelands, discovering unplanted Coontie emerging in places throughout the broken-up limestone substrate.

Ironically, coontie has made a much stronger and easier recovery in ornamental urban landscapes, where it receives watering and fertilizers while rooted in rich topsoil substrates. The Atala butterfly has likewise followed its host plant to these urban areas, where it is often unwelcome because of the severe herbivory its larvae cause to the hosts. However, restoring wild populations of coontie in natural areas is a still a major concern for park managers and naturalists.

One of the most common questions asked is “How many plants are required to sustain an Atala colony?” The real question is how much plant resource will allow a colony to persist for an extended period of time (being two or more years)(Dempster,

1997; Smith, 2000; Hanski & Heino, 2003; Awmack & Leather, 2002; Baguette &

Schtickzelle, 2003; Kraus et al. 2003; Dennis et al. 2004; Hanski et al., 2006; Schultz &

Dlugosch, 2008). Based on prior field work, I could suggest a minimum of twenty

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healthy, well-established plants; however, I have documented successful colonies

(persistent for two or more years) in remnant pine rockland sites which have small plants and/or small numbers of plants on the site (Figure A-1), most of which are also located in heavily trafficked, urban locations. In answer to the question of how much food sustains a colony, I analyzed leaf consumption by captive larvae and compared that total to the potential food content of typical pine rockland coontie plants (Figure A-

3). Additional photos of forty-nine typical coontie plants with approximate food content are available for download at the University of Florida Digital Collections (Object A-1).

Materials and Methods

Eumaeus atala larvae were maintained in a captive colony housed in a standard laboratory at the University of Florida, Gainesville, in a 8:16h light:dark photoperiod.

Temperatures in the laboratory were a constant 24-26°C. Larvae were lightly misted twice daily with deionized water from a standard sprayer to help maintain humidity and provide water. Larvae were reared in plastic “shoebox” cages; the cages were cleaned of frass daily, a fresh paper-towel substrate was installed and new host plant food was distributed to the cages ad libitum twice daily. Larvae were counted every morning to monitor changes in the population.

Leaf material from living coontie plants (Zamia integrifolia) was cut daily and kept fresh in buckets of deionized water (to prevent possible reactions from tap water chemicals) until being distributed to the larval cages for consumption. Individual fronds were weighed fresh (wet weight) and inserted into a water pic to maintain freshness for the larvae. Fronds were replaced as often as necessary during the work day and larvae

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were given sufficient food to last until the next morning before the laboratory was closed for the night, although occasionally it was barely enough.

Any leaf material or stems remaining after consumption were collected and oven- dried to less than 1% of original weight for dry-weight comparison sensu Su &

Tamashiro (1986) and Su & LaFage (1984). The three-dimensional quality of the coontie rachides, and thickness of the leaflets, made standard “leaf area meters” and other two-dimensional measuring methods, too imprecise for calculating consumption.

Although consumption data for 153 cages was collected, only 29 cages had complete data in that these cohorts had few or no changes in larval numbers (missing, death from cannibalism or other causes) and there were no gaps in the daily records. Some of the other cage data may be used in a future analysis, but because they exhibited many changes in larval density, the analyses will be time-consuming and may not lend anything more valuable to the research.

Coontie cut fronds cut from live naturally growing plants were weighed (wet weight) before being presented to the larvae for food; any material remaining after consumption was dried and re-weighed for dry weight.

Fresh fronds from potted plants, simulating field plants from wild colony locations, were maintained in a screenhouse behind the Entomology Department. The fronds were cut and weighed in the same manner as the fronds prepared for larval consumption (i.e., fresh weight, dry weight). These non-consumption fronds from the potted plants were dried to less than 1% of the original wet weight and re-weighed from dry weight in order to compare total leaf content from potted plants to consumption data.

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In this manner, plants could be evaluated for potential food quantity based on weight and number of fronds.

The fresh weights of fronds from potted coontie plants (Zamia integrifolia) were used to determine approximate food content of similarly-sized plants located in natural areas or gardens, based on the quantity of foliage consumed by larvae in a captive colony. Each frond rachis was cut from live plant material 4” above the soil line, weighed fresh (wet) and dried in the green-house. The fronds were dried in open brown paper bags in the greenhouse and stirred monthly to encourage even drying. The material was considered fully dry when the foliage contained less than 1% of its original wet weight.

The dry weight was compared to the dry weight of plant material consumed by a captive

E. atala colony.

Each photographic record of the potted plants was taken against a backdrop of squares measuring 30 cm x 30 cm (approximately 1’ x 1’) and fronds were counted on individual plants. This will assist land managers in judging the size and potential food content of plants on their properties.

Height of the plants was measured from the ground (bottom of pot) to the top of the tallest frond. Width was measured from the furthest tips of the leaves on either side.

Plants were allowed to rest naturally (fronds were not artificially extended in any photograph).

All frond length measurements are approximate. Leaf width is variable between plants, which alters the food content of the plant. The accompanying weight can be used to assess the width or rachis radius of the plants.

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The nutritional quality of the plant material is highly variable depending on soil chemistry, age and potential pathogens, such as scale or fungus. The chemical constituents will vary between plants according to the plant genetics, soil elements, age and environmental factors (such as drought, flooding, fertilizers, pesticides or herbicides, sunlight, seasonal photoperiod, and other factors) (Oberpreiler 1995a;

1995b; 1995c).

Not all leaf material may be appropriate for all larval consumption; for example, younger larvae require fresher new plant growth for consumption. Larvae may ingest more food than experimentally documented in the laboratory; although the larvae were fed ad libitum, there was a routine feeding schedule that may have affected the larval feeding rates.

Results

Twenty-nine cohorts containing a total number of 396 larvae consumed 2338.76 g. wet weight coontie plant material, a mean of 5.91 g per caterpillar. Larvae in the laboratory captive colony consumed between 5 and 14 grams (0.2-0.5 oz.) of plant material during their life span, varying according to brood size and age. The data for these plants will allow potential food resources of similarly sized plants to be estimated for wild colonies of E. atala.

Individuals were capable of ingesting as much as 22.31 grams (in a cage containing one larva, which also cannibalized the other six larvae in its original brood).

Plants were often consumed to virtually nothing, leaving remnants of stem fibers and little else within an 8-hour period, so as many twenty full fronds (~12-18” tall) were

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placed in cages for the night. The number of larvae in a brood per cage varied from one to over a hundred, but most large broods were divided to reduce larval competition.

Larvae consumed between 99.62% and 99.96% of all fresh leaf material offered.

Number of larvae per cage and mean brood consumption per larvae per cohort are shown in Figure A-2 and Table A-1 shows the mean larval consumption volume (g) for broods containing different numbers of larvae. Table A-2 shows how many broods consisted of how many larvae. In most cases, larval development occurred over a period of 14-28 days (see Chapter 2).

It is likely, based on data indicating a single larva is capable of consuming an average of 22.31g, that they would have eaten more had more been offered to them, even though they were fed as close to ad libitum as possible twice daily (or more often if they were out of food). This Appendix will allow wild plants in situ to be judged in assessment of how many larvae it could potentially support.

Conclusion

The task of assuring adequate resources for wildlife is especially formidable when assessing potential requirements for imperiled, vulnerable, endangered, threatened or otherwise compromised biota, whether for mammals (LaBonte et al.,

2001; Rondeau, 2001; Kershenbaum et al., 2011) or for insects (Land & Cooley, undated; Weems, 1977; Baggett, 1982; Castner, 1986; Snyder, 1986; Snyder et al.,

1990; Hubbuch, 1991; Kremen, 1992a; Cushman & Murphy, 1993; Hammer, 1995;

Schultz & Dlugosch, 1999; Smith, 2002: González, 2004; Schultz et al., 2008; Speight et al., 2008; Schweitzer et al., 2011; Thomas, 2011; Henry & Schultz, 2013; Lindman et al., 2013). When both the animal and its food resource are jeopardized in some way,

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sometimes by each other it is nearly impossible to balance the needs of one with the needs of the other (Killer Whales and their favorite food, Chinook salmon, are both endangered, for example).

This research will help directors, managers and biologists in charge of establishing management plans for property holding colonies of the imperiled Atala butterfly, Eumaeus atala. City, county, state and federal parks, natural areas and botanical gardens that either have persistent Atala butterfly colonies or wish to re- establish or introduce the butterfly into suitable habitat will find it a valuable resource for determining how many Zamia integrifolia plants are sufficient to maintain a strong healthy plant and butterfly population.

The Atala’s host plant is a cycad, one of the most ancient plant genera on the planet, and one of the most endangered plants worldwide. Cycads have a fascinating and complex co-relationship with highly specialized insects, including obligate pollinators and herbivores capable of metabolizing neurotoxins. Worldwide efforts to preserve this beautiful and fascinating plant must include the insects associated with it.

The Atala butterfly, a.k.a. the ‘coontie hairstreak’, is one of the most charismatic and beautiful lepidopteran species, given its relative ‘tameness.’ This approachability makes it a perfect insect for teaching stewardship values in natural areas and parks.

Together, this endangered plant and imperiled butterfly form a strong magnet to promote eco-tourism and stewardship for the natural treasures we have in southeast

Florida. Additional photographs of coontie plants with resource data on each plant are available for free download in the Digital Collection

Object A-1. Zamia integrifolia host plant rubric

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Table A-1. Number of Atala larvae in broods and mean coontie plant consumption (g) per larvae for 29 broods. The number of broods with the same number of larvae varied in this analysis. Number of larvae in broods 1 2 3 4 5 6 8 10 11 12 15 16 19 21 22 35 39 44 Mean wet-weight plant consumption (g) per 13. 11. 10. 7. 7. 11. 8. 6. 10. 6. 8. 5. 6. 3. 5. 4. 4. 4. larva 8 1 3 9 1 5 9 7 3 5 4 0 2 8 4 8 9 6

Table A-2. Number of broods containing designated number of larvae in the 29 broods used for consumption data analysis. Number of larvae in brood 1 2 3 4 5 6 8 10 12 15 16 19 21 22 35 41 44 Number of broods 3 3 1 1 3 2 2 2 3 1 1 1 1 1 3 1 1

Total larvae 3 6 3 4 15 12 16 20 36 15 16 19 21 22 105 41 44 398

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Figure A-1. This 10-acre remnant pine rockland natural area in south Miami-Dade County has had a persistent wild Atala colony for more than ten years that ‘blinks in and out’ periodically but consistently reappears. The coontie plants on site are small, narrow-leaved and hidden under dense Saw Palmettoes (Serenoa repens) and the shed needles (duff) from slash pine (Pinus elliottii var. densa). The coontie plants are circled. Photo courtesy of S. Koi.

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Figure A-2. Mean wet-weight plant consumption (g) per Atala butterfly larvae per brood density. Consumption was recorded in 29 stable broods exhibiting no cannibalism or other unexplained deaths.

Figure A-3.Typical native coontie, Z. integrifolia. Data on this plant: Height: ~65 cm (2.133 ft.); Width: ~120cm (3.937 ft.); Fresh Wet fronds: 286.5 g. (~10.1 oz.); Dry weight fronds: 94.7 g. (~3.3 oz.); Number of rachides: 25; Sex: Female; Cones: None. Photo courtesy of S. Koi.

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BIOGRAPHICAL SKETCH

Sandy Koi earned an MSc in entomology in 2013 from the University of Florida under the guidance of Dr. Jaret Daniels and is pursuing a doctoral in Lepidopteran studies. She has a BSc in environmental science from Nova Southeastern University; her published scientific research revolves around the object of her thesis, the Atala butterfly (Eumaeus atala), an imperiled South Florida species that she has been studying since 2001.

Sandy has been spearheading an assisted relocation project for the Atala butterfly from eruption sites into suitable new sites within its historical range in

Southeast Florida since 2003. She coordinated butterfly surveys for Florida Natural

Areas Inventory (FNAI) and is an active member of the North American Butterfly

Association (NABA) and the Xerces Foundation. Sandy is a contributing member of the

Imperiled Butterfly Working Group (IBWG), under the authority of the Florida Fish and

Wildlife Conservation Commission (http://share2.myfwc.com/IBWG/default.aspx).

Many of her photographs and plant information about South Florida butterfly host and nectar plants grace the national NABA website (www.naba.org) and she is a frequent speaker at local organizations in Florida, including Butterfly Days at Fairchild

Tropical Botanic Garden, the University of Florida’s McGuire Center for Lepidoptera and

Biodiversity and the Southern Lepidopterists’ Society. She maintains a blog that highlights her research, eco-tours and children’s nature programs (http://e- atala.blogspot.com).

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