A Brief Natural History on the Ant Odontomachus Brunneus (Patton) Lauren M

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2009 A Brief Natural History on the Ant Odontomachus Brunneus (Patton) Lauren M. Hart

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A BRIEF NATURAL HISTORY ON THE ANT ODONTOMACHUS BRUNNEUS (PATTON)

By LAUREN M. HART

A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirement for the degree of Master of Science

Degree awarded: Fall Semester, 2009 The members of the committee approve the thesis of Lauren M. Hart defended on July 23, 2009.

______Walter R. Tschinkel Professor Directing Thesis

______Tony Stallins Committee Member

______Brian Inouye Committee Member

Approved: ______Bryant P. Chase, Chair, Department of Biological Science

The Graduate School has verified and approved the above-named committee members.

ACKNOWLEDGEMENTS

I would like to thank Dr. Joshua King for introducing me to Odontomachus brunneus and encouraging the study of this really interesting species. Many thanks to Dr. Walter Tschinkel for his help in project design and analysis, without whom I could not have completed this work. Also a heartfelt thank you to all of my friends, family and labmates that assisted in various aspects of field work, especially to my good friend Carli Seeba, who was always willing to dig big holes, provided she did not have to touch the ants.

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TABLE OF CONTENTS

List of Tables ………………………………………………………………. vi

List of Figures ……………………………………………………………… vii

Abstract …………………………………………………………………….. viii

1. GENERAL INTRODUCTION …………………………………………. 1

2. ANNUAL CYCLE OF REPRODUCTION

2.1 Introduction ……………………………………………………. 5

2.2 Materials and Methods ……………………………………….. 7

Description of Species ……………………………………. 7

Description of site …………………………………………. 8 Excavation and collection ………………………………… 8 Cocoon Dissections ……………………………………….. 9

2.3 Results ………………………………………………………….. 10

2.4 Discussion ……………………………………………………… 17

3. SEASONAL ENERGY ALLOCATION

3.1 Introduction …………………………………………………….. 19 3.2 Materials and Methods ……………………………………….. 21

Fat extraction and analysis ………………………………. 21 3.3 Results …………………………………………………………. 22 3.4 Discussion …………………………………………………….. 29 4. FORAGING BIOLOGY OF ODONTOMACHUS BRUNNEUS

4.1 Introduction …………………………………………………….. 30

4.2 Materials and Methods ………………………………………… 33

Trapping and marking ……………………………… 33

Forager population estimates ……………………… 34

Nest Excavations ……………………………………. 35 Results ……………………………………………………….. 37 Discussion …………………………………………………… 40 5. OVERALL DISCUSSION ………………………………………………... 43 Appendix A …………………………………………………………………... 47 Appendix B …………………………………………………………………… 48 Appendix C …………………………………………………………………... 50 References …………………………………………………………………... 51 Biographical Sketch ………………………………………………………… 55

LIST OF TABLES

Table 2.1: Results of cocoon dissections ………………………………. 12 Table 2.2: Seasonal variation in nest depth/architecture……………… 14 Table 4.1: Population Estimates ……………………………………...... 37 Table B.1: Colony composition throughout annual cycle ……………. 48 Table C.1: Mean worker weight by month throughout annual cycle… 50

LIST OF FIGURES

Figure 2.1: Excavation Materials ……………………………………….. 9 Figure 2.2: Example of excavation pit …………………………………. 9 Figure 2.3: Annual cycle of colony composition ……………………… . 10 Figure 2.4: Proportion of sexual pupae in relation to colony size …… 12 Figure 2.5: Correlation of sexual pupae production to total production of pupae …………………………………………………………… 13 Figure 2.6: Correlation of maximum nest depth to colony size ……… 14 Figure 2.7: Recently closed nest shaft …………………………………. 16 Figure 3.1: Mean dry weight of workers throughout a one-year cycle. 22 Figure 3.2: Comparison of worker dry weight, lean weight and fat content over annual cycle ………………………………………………… 24 Figure 3.3: Proportion of colony fat by life stage ……………………… 26 Figure 3.4: Monthly within-nest allocation of worker fat by depth……. 27 Figure 4.1: Marked forager ………………………………………………. 33 Figure 4.2: Location(s) of marked and unmarked worker ants by nest. 38

Figure A.1: Average Monthly Temperatures ………………………….. 47

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ABSTRACT

A population of Odontomachus brunneus, a primitive species of Ponerine ants, located in north Florida was studied for a one-year period. Through nest excavation and colony census, the annual cycle of reproduction and colony growth was determined with nests exiting a period of winter inactivity in late April and beginning brood production. Early brood is a combination of both sexuals and workers, with sexuals present in nests during June and July. After production of sexuals in May and June, all subsequent brood produced were found to be workers through dissection of pupal cocoons. Brood production ceased in October, with the final pupae eclosing in November at which time the colonies began a four month period of relative inactivity.

Within-nest seasonal energy allocation was determined by fat extraction. Seasonal energy stores coincided with the annual cycle of reproduction, with workers declining in energy stores (fat) during initial brood production and regaining these stores after production of brood in preparation for the winter season during which colonies are primarily inactive. Comparison of body fat provided the relative ages of worker ants, which suggested that O. brunneus nests display internal age stratification throughout the majority of the year with older, leaner workers being found in the upper chambers of most nests and younger, fatter workers in the lower chambers.

Using a combination of mark-recapture of foragers and nest excavation, the proportion of foragers per colony was shown to include a mean of 77% (S.D. 22) of the workforce. This proportion was not related to colony size. Female alates were also found to be a part of the foraging population of colonies.

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1 – GENERAL INTRODUCTION

Deciphering life history strategies is a key element in understanding a species and its interactions with the environment, these life history tactics are also referred to as a species‟ natural history. For social insects such as ants, the life history involves a description of the phenotype of the superorganism- the colony being considered the whole with each member acting as an individual part that helps make up the superorganism. There are numerous attributes that may be included in a natural history study including, but not limited to: (1) colony size, ant colonies are known to range in size from as small as twenty workers to several millions of worker; (2)queen number, many species of ants have multiple queens; (3) number of nests per colony, a colony can inhabit one or several nest structures; (4) worker size, workers may be of similar or highly varying size depending on their social caste and/or the age of the colony; (5) alate size and number, a element in reproductive studies; (6) nest location, some species require a very specific set of environmental variables for survival; and (7) nest architecture, which aids in understanding both the physical and social constructs of ant society (HÖlldobler and Wilson 1990, Gadgil and Bossert 1970, Stearns 1976, Tschinkel 1993, Laskis and Tschinkel 2007).

In 1991, Tschinkel introduced the notion of insect sociometry- the collection and analysis of the physical and numerical attributes of social insect colonies and their inhabitants. Tschinkel proposed that without such data, we may be “devising unrealistic schemes of social evolution” (Tschinkel 1991), suggesting that phylogenetic data and generalizations about ants may not be enough to paint an accurate picture of ant evolution, and that life history data should play a major role in determining relationships among the various taxa. The more the body of sociometric data grows, the more we will learn about the similarities and differences in this large taxon of animals.

The collection of such sociometric data can be broken down into three levels: (1) the compilation of descriptive data; (2) comparative studies; and (3) linkage among attributes (Tschinkel 1991). Compilation of descriptive data entails the collection and measurements of natural history traits as previously described. This stage of study is 1

time-consuming and tedious, thus not entirely appealing to many researchers. In North Florida, such work has been conducted on Pogonomymex badius, the Florida Harvester Ant, (Tschinkel 1998, Smith 2004), Trachymyrmex septentrionalis, the fungus-gardening ant (Seal and Tschinkel 2006, 2007) as well as Solenopsis invicta, the red imported fire ant (Tschinkel 1993). Additionally, several species endemic to the former USSR have been studied for specific sociometric attributes including their annual cycles and winter diapause (Kipyatkov 1993, 1995, 2001, Kipyatkov and Lopatina, 1997a, 1997b, 1997c).

This body of knowledge can then be taken to the second level of sociometric analysis- comparative studies. These studies are also important because “comparisons between closely related species, of life history variation in one species in different parts of its range, or polymorphism in life history traits, provide the most rigorous analysis because we know that life history variation has developed from a common phylogenetic stock and divergence of traits has probably resulted from differential natural selection because of differing ecological opportunities and constraints on life history evolution” (Price, 1997 p.342). Several books have been published on the comparison and analysis of descriptive studies including The Ants (HÖlldobler and Wilson 1990) and Social Evolution in Ants (Bourke and Franks 1995). Both works include a vast amount of comparative information on aspects of the life histories of many species, however, there is still much to be learned about the biological and social processes that shape ant life histories. The goal of these comparative studies is to identify similarities and differences in attributes among species, which represents the third level of sociometric analysis. By relating these similarities and differences to the environment, we are better able to understand the selective pressure that shaped the life histories of these species and origins of various adaptive traits.

Living ants today are derived from primitive members of Formicinae and the poneromorphs (Wilson and HÖlldobler 2005). The earliest known ant fossil is from the Cretaceous period (approximately 110 mya), and phylogenetic reconstructions show my subfamily of interest, Ponerinae, emerging not too long after (approximately 90 million years ago) (Moreau et al. 2006). While we know some of the history of this subfamily

through the fossil record and phylogenetic analyses, little is known about the life history traits of the twenty-five genera within Ponerinae. The goal of this study is to provide one of the first detailed natural history studies for the subfamily.

My study focused on Odontomachus brunneus, a primitive Ponerine ant that inhabits a variety of terrestrial habitats throughout Florida, southern Georgia and Alabama as well as Cuba and the Dominican Republic (Deyrup and Cover 2004). Despite their great variation in physical appearance and colony structure, ponerines all share a primitive level of social organization (Bolton 2003, Peeters 1997, Wilson &

HÖlldobler 2005). Ponerine primitive social traits include (1) queens and workers are more similar in size than those of more derived ant subfamilies; (2) queens have relatively low fertility and generally produce about five eggs per day (Wilson and

HÖlldobler 1990); (3) colony sizes are small due to low fecundity; and (4) young ponerine queens generally begin new colonies independently but not claustrally, leaving their natal nest to mate then constructing a nest, and foraging in order to raise their first brood (Haskins and Haskins1955) ; and (5) they are generally solitary foragers and do not use odor trails or other pheromone signals to recruit nestmates to food sources

(Wilson & HÖlldobler 2005, HÖlldobler and Wilson 2009).

A large component of life history analysis is “understanding the diversity of reproductive allocation strategies” (Bourke & Franks 1995, p.301). All known ant species are perennial and iteroparous (HÖlldobler &Wilson 1990, p.143), however a detailed account of reproductive cycles is lacking for the majority of species. Such information would be valuable for understanding the evolution eusociality amongst ant species. Two important pieces of a reproductive allocation strategy are the timing of the annual cycle and the amount of energy invested in the creation of workers (colony growth) versus production of alates (reproduction) (Tschinkel 1993, 1998). These data can be obtained by collecting colonies of varying sizes throughout the year, the inhabitants of which are then censused and their individual weight and fat content determined (Tschinkel, 1993 and 1998). As the number of species for which this

information is known grows, much can be learned about comparative reproductive cycles as part of the life history tactics of various species.

2 - ANNUAL CYCLE OF REPRODUCTION

Introduction

The determination of colony growth and annual cycles of reproduction is an understudied area within the field of myrmecology, despite the fact that such data are essential to understanding the patterns of how colonies allocate resources between the essential tasks of seasonal and size-related colony growth (ergonomic phase), sexual reproduction and colony maintenance (Gadgil and Bossert 1970, Kipyatkov 1993, 1995, 2001, Oster and Wilson 1978, Tschinkel 1993, 1998). In social insects in particular, this information could potentially shed light on the over-arching questions about the evolution of eusociality that underlie all such studies.

The seasonal and life-cycle patterns of how colonies allocate resources are best seen in the rates at which the different units of production (i.e. workers, sexuals) and maintenance proceed (Tschinkel 1993) as well as when such production occurs seasonally. In 1993, Kipyatkov published a treatise on the reproductive cycles of ants in which he outlined different potential developmental modes for a colony‟s annual cycle and suggested an evolutionary rationale of the adaptations that led to the specific modes.

Each species of ant is characterized by a specific annual cycle, which should be organized so as to capitalize on the warmest period of the year for larval development (Kipyatkov 1993). In considering this, a generalized annual cycle should have brood production beginning as early in the spring as possible and continuing as long as possible, with only the stages capable of successful hibernation present at the beginning of the winter diapause (Kipyatkov 1993, 2001).

In his extensive discussion on known annual cycles in ants, Kipyatkov derived two main types of developmental modes that describe the majority of these cycles: homodynamous and heterodynamous (1993, 2001). In species termed to be homodynamous, all ontogenetic stages (from egg to pupa) are present year-round in the nest with no pronounced periods of dormancy. Heterodynamic species, on the 5

other hand, are characterized by their various states of dormancy which are species- specific. Such diapauses can be brought on by a combination of endogenous and exogenous factors, the regulation of which is still not fully understood (Denlinger, 1986, Kipyatkov 1993).

Yet another important component of the annual cycle is determining when a colony produces sexuals (alates) versus workers. In all of the temperate ant species that have been studied thus far, the rearing of sexual brood always occurs prior to worker production (Kipyatkov 1996, 2001). By this account, all of the colony‟s initial investment in brood should be towards the creation of alates for the sexual reproduction of the species followed by a secondary investment in the ergonomic phase in which the colony rears worker brood to increase or maintain the size of the colony as well as create a worker force that will survive the over-wintering diapause to emerge and begin the cycle all over again.

In addition to the reproductive cycle, cyclical variations in physical structure as a colony moves, grows or prepares for environmental change can be an important element to its yearly phenotype. Nest structures are known to be species-specific and may serve particular functions that are biologically important to the colony (Tschinkel 1998, 1999, Cerquera and Tschinkel 2009). Descriptive studies of the general structures are in their infancy, with little if any information on seasonal adaptations. One exception to this is the annual vertical movement of colonies of fungus gardens of fungus gardening ants in relation to temperature (Seal and Tschinkel 2006).

The annual cycles are known for very few ant species, with many taxa not represented. The aim of this section of the study is to provide the complete annual cycle of the Ponerine ant, Odontomachus brunneus in northern Florida, so that it may one day be used in comparative studies. Since each species has its own unique reproductive cycle, deciphering how these annual cycles have evolved can help us to gain a better understanding of the adaptations that have allowed ants to become so successful.

Materials and Methods

Description of the species

Paraphrased from Deyrup and Cover 2004: Odontomachus are large, quite conspicuous ants, averaging approximately 8 mm in length with large mandibles and a powerful sting. Despite these formidable weapons, these ants are not particularly fierce, and are generally found hunting slowly and solitarily about the ground. A 1985 review found that at least three known species inhabit the state of Florida, with O. brunneus being the primary occupant of the north Florida region.

O. brunneus appears to be a habitat generalist, occurring both in well-drained and poorly drained habitats spanning a variety of vegetation types, including flatwoods, mesic forest, swamp forest, upland scrub and sandhill (p. 142). Additionally, nests are found in a variety of microhabitats from the base of a tree to deep in leaf litter to sparsely covered sandy areas. Workers are often seen foraging on cloudy days, but are generally considered to be nocturnal foragers. Workers display an interesting hunting technique as described by Brown (1976) in which the foragers strike their prey, recoil and subsequently return to retrieve the prey item. It is thought that this is a method to avoid possible chemical defenses of the prey item.

Description of the site

This study was completed in compartment 219 of the Apalachicola National Forest in Leon County, Florida (30˚22‟ 11 N, 84˚ 19‟ 32 W). This area is designated as a sandhills ecotype, the vegetation of which is largely comprised of turkey oak and Smilax vines with interspersed long leaf pines, live oaks and the occasionally saw palmetto. Most of my study was carried out in a low-lying area with live oaks and other broadleaf trees mixed with longleaf pine. The ground in most areas was covered with a dense laver of decaying leaves. This study site was near several depression ponds with a fluctuating water table, so that the soil drainage in this location was poor, creating a moist substrate in which the ants may nest and hunt.

Excavation and collection

Excavations were done by digging a pit directly adjacent to a known colony, with the edge of this pit no less than 15 cm from the nest entrance(s). This distance accommodates varying chamber sizes as well as the slight deviation of nest shafts from being entirely vertical. Chambers were exposed sequentially from the top and their contents removed in 20 cm increments down to 60 cm in total depth (initial nest depths did not exceed this). Inhabitants residing deeper than 60 cm were placed in a separate container and the maximum nest depth recorded. (Procedure modified from Tschinkel 1998)

Nest inhabitants were killed via rapid freezing. The ants were then dried at 50˚, sorted and counted to determine the number and vertical locations of the workers, brood, alates and the queen (location only). Additionally, nests were categorized by size class, with Class 1 nests having 50 works, Class 2 51-100 workers, Class 3 101- 150 workers, and Class 4 151-200 workers.

Fig. 2.1 Excavation materials: sharpened trowels, aspirator, collection jars (large shovels excluded).

Fig. 2.2 An example of an excavation pit, March 2009

Pupal Cocoon Dissections

To determine what type of brood early cocoons contained, dried cocoons from May through July were dissected under a dissection microscope using fine-tipped forceps to tear away the pupal sac. Developing brood were then determined to be sexual, worker or unknown pupae. Sexual pupae were recognized by the presence of various stages of wing development as well as lack of large mandibles in the males. Unknown pupae were too early in the developmental process for their caste to be determined.

Results

Annual Cycle

Fig. 2.3: The annual cycle of production in Odontomachus brunneus shown as a mean percentage of the colony population in each category. Nests were broodless throughout half of the year, followed by a period of production of both sexual and worker brood. Sexual brood were produced for only two months during the spring, followed by four months of worker production.

The monthly excavations and census of Odontomachus brunneus nests revealed the annual cycle of this species (Fig. 2.3). Brood production began with the arrival of the warmer temperatures in late spring and continued until temperatures were too cool for brood development in the mid-fall (see Appendix A for yearly temperatures). The first larvae appeared in small quantities in later April and increased to a peak in June. Pupal cocoons were present beginning in May and increased through October with only a few pupae left to eclose in November. Dissection of these cocoons (Tab. 2.1, Fig. 2.4) revealed the majority of recognizable pupae to be sexual in nature in the months of May and June; this was indicated by the presence of various stages of wing development as well as lack of large mandibles in male alates. Production of alate brood was recorded as late as the second week of June (6/9/08), after which point colonies switched to production of only worker brood. Interestingly, initial brood production in early spring was not focused solely on the production of sexuals, but rather a mixture of both worker and sexual brood, with sexual brood predominating. Both sexual and worker pupae were found in the same nests, without influence of size of the colony (Fig. 2.4, p= 0.137783) or the number of pupae present (Fig, 2.5 p= 0.12).

Alates appeared in mid-June (6/16-6/23), with a few female alates remaining as late as the third week of July (7/21/08). This suggests that this species‟ mating flights are likely to occur in mid to late June.

The lack of over-wintering brood shows this species to have a heterodynamous annual cycle of development, with all over-wintering individuals having eclosed prior to the winter diapause. Of note in this regard: several colonies moved brood to the surface of their nests in late October as temperatures were beginning to decrease. This was likely an adaptive behavior to take advantage of warmer near-surface temperatures in order to speed pupal development and achieve eclosion prior to the winter.

Cocoon Dissections

Table 2.1: Results of cocoon dissections

Percent Sexual to Percent Percent Month Unknown Total Pupae Worker Worker Pupae Sexual Pupae Pupae Pupae Ratio

May 12% 20% 68% 25 5:3

June 8.64% 16.05% 75.31% 81 13:7

Proportion of Sexual Pupae to Number of Workers 0.14 r2 = 0.1889; y = -0.0113151562 + 0.000631969528*x 0.12

0.10

0.08

0.06

0.04

Proportion of SexualProportion Pupae 0.02

0.00

-0.02 10 20 30 40 50 60 70 80 90 100 110 Number of Workers

Figure 2.4: Proportion of sexual pupae in relation to colony size for the months of May and June. This relationship was not significant (p= 0.11).

Correlation of Number of Sexual Pupae to Total Number of Pupae 14 Sexual Pupae = 0.43+0.09*x p = 0.12 12

6 Sexual PupaeSexual 4

0 0 10 20 30 40 50 60 Total Number of Pupae

Figure 2.5: No correlation between the production of sexual pupae to the total number of pupae in a nest was found during the months of May and June (p = 0.12)

Queen Presence

In very few colonies was a queen present. Of the 76 colonies excavated, only 26 were queen-right (34%), with one of these colonies actually containing two queens. It is unknown if both queens were reproductively active as they had been processed for fat extraction and therefore could not be dissected.

There was no relationship of the presence of a queen to size class (Chi square test: 11.2611, p= 0.421249).

Seasonal Variation on Nest Depth/Architecture

Table 2.2: Variation in nest depth by season.

Season Range of Depth (cm)

Fall: September-November 20 cm – 128 cm

Winter: December-February 60 cm – 170 cm

Spring: March-May 20 cm – 112 cm

Summer: June-August 15 cm – 60 cm

Correlation of Maximum Nest Depth to Number of Workers 200 Winter = 73.25 + 0.50*x 180 Fall = 35.25 + 0.47*x Summer = 43.01 - 0.001*x 160 Spring = 47.18 + 0.52*x

140

120

100

40 Maximum Nest Depth(cm) Nest Maximum 20 winter nests fall nests 0 -20 0 20 40 60 80 100 120 140 160 180 200 summer nests spring nests Number of Workers

Figure 2.6: Correlation of maximum nest depth to number of workers by season. Mean summer nest depth was 43 cm (S.D. = 16.5) with little variation, resulting in a near horizontal line. Winter nests were much greater in depth than summer, with spring and fall occurring in transitional locations between these extremes. Additionally, nests with a larger workforce were capable of digging to greater depths in the cooler seasons.

Unexpectedly, this study revealed changes in the nest depth and structure through the annual cycle, with nests as shallow as fifteen centimeters in the summer and as deep at 170 centimeters in the winter. Architecturally, the nests changed in structure in preparation for the winter, extending the final shaft down 60-100 cm from the maximum summer depth. Except on unseasonably warm days, all winter inhabitants of the nest were found in the final, nearly circular chamber at the end of this long shaft.

When the maximum depth of individual nests was regressed by the number of ants present, we see that, while the range of colony sizes is fairly consistent across seasons, the depth is not (Fig. 2.5). Winter nests are much deeper than summer nests of comparable size, with spring and fall nests displaying a transition between these two extremes.

Fig. 2.7: Recently closed shaft, filled with loosely packed soil. (horizontal view)

On March 23, 2009 an excavation showed evidence of a colony migrating upwards in its nest, filling the lower shafts/chambers with loosely packed soil, shown in Fig. 2.6. Around this time, all colonies decreased their depth as the ants moved from their period of winter inactivity into the reproductive period of their annual cycle. In this particular nest, the majority of ants were located in a chamber immediately above this shaft with only a few workers closing the shaft behind the colony.

Discussion

The overall picture of the annual cycle as a phenotypic property of Odontomachus brunneus is quite clear in both the reproductive pattern as well as the seasonal changes in nest architecture. As the spring temperatures increase in April, production of brood begins and nest depth gradually decreases. Minimum nest depths occur in the warmest months of the year, when the colony invests most of its energy in the production of brood. With cooler winter temperatures in the autumn, there is a decrease in the number of larvae, followed by a decrease in pupae, and an increase in nest depths in preparation for a winter period of relative inactivity. While temperature is often linked with brood production (Kipyatkov 1993, 1996, 2001, Kipyatkov and Lopatina 1997a, 1997b, 1997c), the consideration of changes in nest architecture to maximize productivity and minimize costs have yet to be considered. My observations and colony excavations showed differences in both the depth and structure of summer versus winter nests. In a recent study on the nest architecture of this ant, this variation in nest properties was recorded but not linked to seasonality (Cerquera and Tschinkel 2009), possibly because we knew so little about the biology of this species prior to this study.

O. brunneus does not appear to adhere to the reproductive cycles described by Kipyatkov (1993, 2001). In all of his studies of Myrmica, he showed that, upon resuming production after the winter diapause, the ants produced only sexual brood, soon followed by worker brood. I expected similar results; however, because these are quite different species that live in very different climates, it is not surprising to see that the pattern is not the same. While sexuals appeared in the nests early in the reproductive cycle, they did not appear in the relatively high numbers that one would expect, comprising eleven percent or less of the June colonies (Fig. 2.1). To clarify the reproductive pattern, pupal cocoons were dissected to see what fraction of the brood present in each nest would have eclosed as either alates or workers. While the majority of the young pupae were indecipherable, in the early months of brood production, sexuals were produced nearly twice as often as workers (Table 2.1). Why these ants do not completely separate sexual production from the worker production is unclear.

Perhaps their life history does not require a very high production of sexuals, or perhaps it is important for them to grow or maintain their workforce alongside production of sexuals, replacing older workers that die during this period.

A second question raised by these data relates to the presence or absence of a queen. In early collections, I had attributed the lack to a novice researcher‟s error and thus collected additional colonies. However, many of those colonies were also lacking a queen. From a combination of literature searches as well as observational data, I have arrived at two possible answers: (1) O. brunneus is polydomous and/or (2) queens continue foraging even after their colony has been successfully established. Only one other species of Odontomachus, O. mayi from South America, has thus far been determined to be polydomous (Debout et al. 2007). Since there has been very little work done on the genus Odontomachus, we do not yet know if this taxon is monodomous as generally assumed, or if polydomy occurs with some frequency. The observed spatial clumping of colonies often seen in my study site also suggested polydomy (personal observation). A test of polydomy in the early spring of 2009 failed because of a long rainy period, during which many of the clumped colonies were drowned before they assumed their summer depths.

The second possibility for a lack of queens is based on what is known, in general, about colony founding in Ponerine ants and supported by an observation made in the early stages of this study. After mating, a Ponerine queen finds a suitable location to dig her initial burrow. In this burrow she lays her eggs and then, to feed her young, forages until she has a sufficient workforce to take over this task (HÖlldobler and Wilson 1990, 2005, 2009). In July of 2008, while testing foraging traps, a queen was found alongside several workers in these test traps. As this was well after the mating period, it is possible that she was foraging. Although risky, it is possible that the queens continue to forage for some time after founding the colony. Further study on the reproductive biology of this species may shed light on this possibility.

3 - SEASONAL ENERGY ALLOCATION

Introduction

Annual cycles of energy allocation have been determined for several other ant species found in the Apalachicola National Forest including the Florida harvester ant, Pogonomymex badius (Tschinkel 1998), and the fire ant Solenopsis invicta (Tschinkel 1993), both of which are found in overlapping territories with Odontomachus brunneus (personal observation). Energy allocation patterns are determined by measuring fat, the primary energy stores, in the various life stages throughout the annual cycle, revealing colony energy investment patterns. Additionally, in several ant species, the proportion of fat in a worker‟s body has been shown to be age-related and can be used to estimate relative age. In general, younger workers have the largest proportional fat stores, which decrease as the workers age (Wilson 1985, Tschinkel 1993 and 1998).

Previous studies on seasonal energy allocation have shown strong correlations among the investment rates in worker production, fat storage, production of alates (sexuals) and colony maintenance with season (Tschinkel 1993 and 1998). In most species, following overwintering, production rates of alates increase for a late spring/early summer mating season. During this time very little energy (often none) is allotted towards the production of workers, as the main focus of the colony is colony reproduction. After the reproductive period, energy is shifted towards colony growth (worker production) and maintenance and the colony expends its energy towards building up the work force, as well as storing energy for the next overwintering period so that the colony is ready to produce sexual alates once again in the spring (Passera and Keller 1987, Rissing 1987, Ricks and Vinson 1972, Tschinkel 1993 and 1998, Yang 2006). This cyclical colony growth has also been shown to create a seasonal age structure with young workers predominantly in the fall and older workers in the spring (Rissing 1987, Tschinkel 1998).

In addition to determining the annual cycle of reproduction and energy allocation, it is interesting to consider the colony-level investment in the various intra-colony tasks.

Despite their status as a primitive species of ant, O. brunneus colonies generate a division of labor within their ranks (Powell and Tschinkel, 1999) through “interaction- based task allocation.” Dominant individuals (dominance status is determined via antennation ritual) achieve a location closer to the brood within a nest, participating in brood care while subordinate individuals are forced into the risky task of foraging. However, it is unclear if spatial age stratification is associated with the division of labor in O. brunneus. In many species of ants the youngest workers, after eclosing on the brood pile, remain and provide brood-care, thus displacing older workers into other jobs more distant from the brood (Beshers and Fewell 2001, Tschinkel 1998). This demographic shift in roles has been termed „temporal polyethism‟ (Franks et al. 1997). By determining the relative age(s) of workers in relation to their location in the nest, we should be able to determine if age truly does influence what role(s) a worker may play in the life of the colony (Tschinkel 1993 and 1998).

Materials and Methods

Excavation and collection

See chapter 2.

Determination of seasonal energy allocation throughout one year

After ants were dried and counted as in Chapter 2, each ant was weighed (mg), assigned an identification number and placed in a labeled, perforated gelatin capsule. Capsules were threaded onto a wire skewer and placed in a Soxhlet extractor, extracted with diethyl ether for 48 hours, dried and reweighed. The difference between the pre- and post-extracted dry weights represents the extracted fat, and together with the dry weight, allow the determination of the percentage of fat stored in each individual ant (modified from Tschinkel 1993, Seal and Tschinkel 2006 and 2007, Soxhlet 1879), and by summation, in the colony as a whole. From this calculation we can determine energy (fat) allocation by life stage and throughout the yearly cycle as well as the relative age(s) of workers at each level within the colony

Results

Dry weights of workers throughout a one year cycle

Dry Weight of Workers By Month 5.0 Dry Weight + S.E.M. (mg) Dry Weight + S.D. (mg) 4.5

4.0

3.5

3.0 Dry Weight (mg) Weight Dry

2.5

2.0 April June August October December February May July September November January March Month

Fig. 3.1 Mean dry weight of workers, April 2008-April 2009 (April data are combined). Error bars denote the standard deviation and standard error of the means. Mean dry weight decreases to an annual low in August and increases to an annual high in January.

Figure 3.1 displays the monthly means and ranges of worker dry weight. In April, ants emerging from their winter diapauses ranged in weight from approximately 2.14 mg to 5.27 mg, a range of 3.13 mg with a mean dry weight of 3.75 mg (COV= 0.15). While the range of dry weights remained similar throughout most of the year, the mean weights show a much clearer trend. From initial emergence after winter inactivity in April and early May, the mean worker dry weight decreased substantially through August then increased again up until the winter period. These fluctuations tie in with the annual cycle (Fig. 2.3) and the production of alates in May and June, during which time energy is diverted towards reproduction, thus decreasing the amount of energy 22

allocated towards production of new workers. Energy (nutritional resources) is probably also transferred from the workers to the developing brood. After this period, an increase in worker production occurred until the beginning of November and included an increase in worker weight. Also at this time, there were several presumably older, less fat individuals in each nest as seen in the low end of the dry weight distributions (Fig. 3.1, Appendix C). From September through December, there was in general an overall increase in fat stores within each colony in preparation for over wintering, when the colony must be able to survive off its fat stores until it may begin foraging again in the late spring.

Comparison of worker dry weight, lean weight and fat content over the annual cycle

Comparison of Monthly Worker Dry Weight, Lean Weight and Fat Content 7 28 Dry w eight (mg) Lean Weight (mg) 6 Percent Fat/4 24

5 20

4 16 Fat Content (%)

3 12

2 8 Dry Weight/LeanDry Weight (mg)

1 April June August October December February May July September November January March Month

Figure 3.2: Lean weight, dry weights and percent fat of workers by month. Error bars denote 95% confidence intervals. By decomposing the dry weight into its components, it is apparent that the majority of the fluctuation in mean dry weight of workers is due to a change in the fat content.

Dry weight (mg) and lean weight (mg) displayed similar trends throughout the cycle, with fat content varying the most. After winter inactivity, ants emerged from their nests in late April and early May of 2008 with just over 17% fat composition. The mean dry weight then decreased through the loss of both lean weight and fat, reaching the yearly minimum in August (Fig. 3.2; Appendix C), mostly as a result of the loss of fat. Between August and November, an increase in both fat and lean weight caused the dry weight to increase, but after November, most of the dry weight increase resulted from increased fat. The overall decrease in both dry and lean weights through August parallels the annual cycle of colony composition (Fig 2.3) as young workers replace old

ones that die. This demographic shift may also explain the dramatic changes seen in the fat content of workers.

Fat content decreased as the workers aged and as nutritional resources were provisioned towards other life stages- alates and brood (Fig. 2.3). The largest decrease of worker body fat coincided with the production of alates in May and June (Fig. 2.3). Female alates had a mean of 26% body fat, suggesting that they sequestered a large amount of available resources. Fat content slowly increased post-alate production from the addition of younger, fatter workers and an overall fattening of all workers. This gradual increase continued through October, with the addition of young workers ending in early November (Fig. 2.3). In October through January, there was a constant, large increase of worker fat stores, which decreased during the remainder of the winter while the colonies were primarily inactive.

Fat allocation by life stage per month

Figure 3.3: Proportion of colony fat stores in all life stages by month.

The proportion of the colony‟s total fat found in each life stage per month was analyzed to show where colonies were allocating energy stores. As shown previously (Fig. 3.2), fat stores of workers decrease immediately after a period of winter inactivity. At the same time, fat stores of brood (pupae and larva) increased as brood production began in late April and decreased with the cessation of brood production in late October/early November. Concurrent with the early decrease of worker fat in May and June, alates were produced; female alates acquired a large amount of fat, as is 26

necessary for them to prepare for mating and colony founding. Post-alate production, the proportion of fat in workers increased as young, fatter workers eclosed and joined the population and as older workers began replacing their fat stores.

Monthly fat allocation within the worker class by nest depth

Proportion Fat of Workers by Location and Month 0.35

0.30

0.25

0.20

0.15

0.10 Proportion Fat Proportion 0.05 Location in Nest Surface/ Top 20cm 0.00 Location in Nest 20-40 cm Location in Nest 40-60 cm Location in Nest > 60cm -0.05 April June August October December February May July September November January March Month

Figure 3.4: Monthly within-nest allocation of worker fat by depth. Error bars denote 95% confidence intervals. Throughout most of the year, leaner (older) workers were found in the upper chambers of all nest with fatter (younger) workers in the lower regions.

The proportion of fat in workers was analyzed by level within each nest by month. Significant differences were found for the proportion of fat by location within nests for 9 out of 12 months. Additionally, in the months of January and February, there appears to have been an increase of fatter workers in the upper regions of the nests. This was

probably caused by a small sample size of fatty workers emerging on warmer days to forage.

It has been shown in other species of ants, such as Pogonomyrmex badius (Tschinkel 1993), that as an ant ages, it loses fat and gains lean weight. So by analyzing the fat content by location within nests, we can see if Odontomachus brunneus is stratified within the nest by age. With the assumption that the proportion of fat decreases with age, there appears to be stratification within the nests of O. brunneus for the majority of the year, with the exception of January. The January similarities in proportion of fat by level can be tied to the life cycle in with all workers having gained fat content for survival over a long period of minimal activity, thus all workers have a comparable fat content.

Discussion

Figures 3.1 and 3.2 depict the seasonal cycling of fat stores throughout the worker class in Odontomachus brunneus. This information, combined with the annual cycle (Fig. 2.3) describes the general structure, and to some extent, activities of the worker class in respect to the overall colony. In the late spring through summer, the mean starting weight of the worker class decreases as the colony allocates the majority of its energy stores into the production of alates (Fig. 3.3), which are present in nests from mid-June through early July. While colonies do produce new workers during this time (Fig. 2.3), the majority of workers are aging and thus lose fat stores and decrease the mean worker weight in the late summer months (Fig. 3.1,Appendix C, Fig. 3.4). During the fall, the mean worker dry weight increases as young ants eclose and the colony stores fat in preparation for the winter period, similar to several species of Myrmica (Kipyatkov 1997c) and Pheidole morrisi (Yang 2006).

Within-nest fat allocation to workers by depth was analyzed to show potential age stratification. It is commonly accepted that ants organize themselves in an age-specific pattern in which the youngest (fattest) workers should eclose near the brood pile and remain within the nest as they have more future value to the colony as a whole, whereas older, less valuable workers should engage in foraging (Beshers and Fewell 2001, Powell and Tschinkel 1998). The data presented here suggest that O. brunneus also display similar stratification with the exceptions of January (Fig. 3.4).

It is unclear if the data presented here are common to primitive ants in general, as the majority of studies of this nature have been completed on more derived species. This study serves as a jumping off point to question just how different „primitive‟ ants are in their colony structure from the more „derived‟ species; more studies must be completed on other ants known to be primitive to come to a more complete understanding.

4 - FORAGING BIOLOGY OF ODONTOMACHUS BRUNNEUS

Introduction

Ant colonies are often referred to as „superorganisms‟, with each individual ant being both an individual as well as a part of a larger entity: the colony. Within a colony, the parts can be broken down quite simply into a reproductive caste comprised of the queen and alates, and a worker caste, which is always more or less sterile. This worker caste can be further divided by the roles of the workers: brood-care, nest maintenance and foraging. Looking at the members of the worker caste raises several questions: How is the labor divided amongst individuals? Are workers capable of switching roles or are they faithful to their roles throughout life? How much of the colony is actively engaged in the various tasks?

Within the field of ant biology, there exist three main ideas on how the division of labor among workers is determined: (1) age-based polyethism, (2) foraging-for-work, and (3) evolutionary origination. Age-based polyethism is part of adaptive demography, according to which the schedule of birth, worker body size, aging and death are the product of natural selection, and thus an adaptive, colony-level trait. Under adaptive demography, age-related changes and changes of physical position are associated with worker roles, and are a function of age and size of the individual (Wilson 1985, Oster and Wilson 1978, HÖlldobler and Wilson 1990). Under the foraging-for-work hypothesis, task specialization is a product of idle individuals randomly foraging for work within the colony and performing any task that needs to be completed (Bourke and Franks 1995). And finally, according to the evolutionary origins concept, individuals compete for preferential tasks. The losers of these encounters are then prevented from direct reproductive tasks and thus must find work at locations more distal to the center of the nest (Powell and Tschinkel 1999, Franks et al. 1997, Robson and Beshers 1997, Bourke and Franks 1995, West-Eberhard 1981).

Powell and Tschinkel (1999) examined task distribution within laboratory colonies of Odontomachus brunneus. Workers of O. brunneus use a method aptly named

„interaction-based task allocation‟, whereby workers engage in pair-wise antennal dueling, the winner of which becomes the dominant individual and assumes a role nearer the brood pile while the subordinate is driven further from this location into the roles of nest maintenance and, ultimately, foraging. This dominance interaction, coupled with the well-documented phenomenon of age-based polyethism in most other ant species was used to decipher the internal social structure in the laboratory O. brunneus colonies. What is unclear is how faithful workers are to their designated roles; in laboratory observations, marked workers were capable of moving among the foraging arena, brood-less and brood zones frequently (personal observation).

Though occurring less frequently, a lack of age-based polyethism has been shown in both Leptothorax unifasciatus (Sendova-Franks and Franks 1993) as well as Odontomachus troglodytes (Dejean and Lachaud 1991). In both of these studies, workers that eclosed at the same time could develop separate specializations which did not always change with age. Additionally, in L. unifasciatus older workers were able to move from the role of forager back to working inside the nest (Sendova-Franks and Franks 1993, 1994). While these species represent a minority of species with respect to age correlation of tasks, such a lack of age-based polyethism shows that a worker can shift from a role usually associated with older individuals (foraging) to that of younger ones (brood care).

Yet another aspect of foraging biology that has received limited study is the determination of what proportion of a colony is actively involved in the risky task of foraging. Previous studies of forager populations using Formica polyctena (Kruk-De Bruin et al. 1977) and Pogonomymex badius (Porter and Jorgensen 1981) suggest that (1) forager populations function almost as a separate entity from the rest of the nest and (2) that the foraging population should be comprised of primarily older workers who have already served as within nest workers, performing brood care and general nest maintenance work (Golley and Gentry 1964, Kruk-De Bruin et al. 1977, Porter and Jorgensen 1981). Despite such studies, it is unknown if the proportion of foragers in a colony is related to the size of the colony for all species such as it is in Solenopsis

invicta (Tschinkel, per. Comm..), or if it is a species-specific trait or if this proportion is an evolutionarily derived trait such that primitive ants (i.e. Odontomachus species) and more derived ants (i.e. Pogonomyrmex species) would have differing proportions of their workforce participate in foraging.

Materials and Methods

Forager collection and marking

Ten nests were chosen for forager monitoring. A foraging area with a diameter of 60 cm was cleared around the nest entrance(s). Foragers were collected using an aspirator (shown in Fig. 2.1) for two 20- minute periods separated by forty eight hours, allowing time for the marked workers to thoroughly mix within the population (Ryti and Case 1986). To ensure collection of only foragers, ants were collected upon returning to their nest or exiting past ten centimeters to prevent collection of nest maintenance workers. Each ant was then individually marked on the posterior of its head using Testors enamel (Fig. 4.1) and replaced near the entrance to its nest.

Fig. 4.1: Marked forager held with a Bioquip® soft touch tweezer.

Estimates of forager populations based on mark-recapture data

The number of foragers in a colony was estimated by using the Lincoln Index Method (Chew 1959, Kruk-De Bruin et al. 1977, Southwood 1978), which provides an estimate of the total foraging population of a colony based on mark-recapture data. This is calculated using the formula:

Eqn. 1: Forager population estimate

N represents the forager estimate equal to the product of the initial number of ants marked (a) and the number of ants marked in the recapture sample (n) divided by the number of marked ants recaptured (r) (Southwood 1978). This method makes several assumptions: (1) all individuals in the forager population have an equal chance of being caught and marked, (2) the marked ants mix thoroughly with the unmarked ants before resampling, and (3) marking is permanent for the duration of the sampling period and does not affect the behaviors or survival of the marked individuals (Chew 1959, Southwood 1978).

The variance (Eqn. 2), standard deviation (Eqn. 3) and standard error of the means (Eqn. 4) can then be determined as follows:

Eqn. 2: Variance

Eqn. 3: Standard deviation

The standard deviation is derived from the square root of the variance of N, the estimate of the foraging population, divided by the number of observations minus one.

For this equation, s is equal to the total number of observations in sample one and sample two of foraging ants.

Eqn. 4: Standard error of the means

Standard error of the mean is found by dividing the standard deviation by the square root of s, the total number of observations in sample one and sample two of foraging ants. This provides a standard deviation of the sampling distribution, with a larger sample size having a smaller distribution and with this a more precise measure of the mean.

Nest excavations

Nests were hand-excavated as described in chapter 2 to determine how workers were vertically distributed throughout the nest. Workers were collected as each chamber was exposed, and the depth recorded. Collection was done in the morning at similar times to marking events so that workers would be engaged in similar daily tasks at the time of excavation.

Using the number of marked foragers (m) from the excavation and the proportion of recaptured marked foragers (p) from the mark-recapture data, the number of foragers (both marked and unmarked) within the nest during the excavation was determined (F):

Eqn.5 Foraging population in nest

Once the foraging population within the nest (F) was determined, the number of foragers actively foraging was found by subtracting the foraging population within the

nest (F) from the forager population estimate (N). The total colony size was then determined by adding this value to the number of ants collected during excavation.

Results

Table 4.1: Population estimates

F) - Nest ID ID Nest & marked Number ( released (n) Sample Recapture marked Number of (r) recovered foragers of Proportion ants recaptured (p) marked (N) estimate Forager Standard deviation the of error Standard mean population Total nest (excavation) marked ants Total (excavation) Total foraging (F) nest in population nest of out Foragers (N total Estimated size colony colony of Percent foraging in involved

F1 31 21 14 0.67 47 24 3.33 71 18 27 20 91 51

F4 10 11 3 0.27 37 29 6.33 35 8 29 7 42 87

F5 21 20 6 0.30 70 58 9.06 76 20 67 3 79 88

F6 18 14 5 0.36 50 45 7.95 41 7 20 31 72 70

F7 20 11 2 0.18 110 316 56.76 62 5 28 83 145 76

F8 27 28 12 0.43 63 38 5.12 95 22 51 12 107 59

F9 11 9 1 0.11 99 179 40.03 11 3 27 72 83 119

F10 11 8 4 0.50 22 16 3.67 25 3 6 16 41 54

F11 6 4 1 0.25 24 40 12.65 4 1 4 20 24 100

F12 10 10 2 0.20 50 53 11.85 52 6 30 20 72 69

By using the mark-recapture data and excavation censuses, colony sizes and the proportion of foragers per colony can be determined. Female alates were included in the population census of workers as they were observed foraging in the majority of the nests. According to these calculations, the ten colonies used in this study ranged in

size from 24 to 145 workers with 51% to over 100% of the workforce participating in foraging, with no effect of colony size. In one colony, F9, this percentage was found to be over 100%, but confidence in this estimate is low because it was based on a very low proportion of recapture (0.11).

Excavation: Location of marked and unmarked workers

Figure 4.1: Location(s) of marked and unmarked ants by nest. Marked foragers were found distributed throughout nests; this distribution was unequal, with the majority located in the upper region of the nests.

Figure 4.1 shows that ants that were previously marked as foragers were distributed throughout the nest upon excavation, but possibly unequally among the

levels. A Chi-square test was performed assuming equal distribution of marked foragers within each nest; this test showed the distribution of marked foragers not to be equal between nest levels (Chi-square = 63.78831, p= 0.000204), with more marked foragers located in the upper levels and a smaller, but varying number distributed through the middle and bottom levels. In the all nests, brood was found in the bottom and middle chambers along with both marked and unmarked ants; brood was also found in the top chambers of nests F10 and F12. Within-nest locations were combined at what appeared to be natural breaks; i.e. ants located in the topmost chamber were observed to frequently exit nests and thus were combined with the returning surface foragers.

It should be noted that the total number of ants collected is not the total individuals per colony as excavations were performed while colonies were actively foraging. An estimate of colony sizes is provided in Table 4.1.

Discussion

The estimates of the foraging populations (Tab. 4.1) suggest that a large proportion of workers engage in foraging in Odontomachus brunneus. Due to the high proportion of recapture ( 20% in eight out of ten nests), a high degree of confidence can be placed in the estimates for all nests except F7 and F9 (<20% recapture). A large proportion of recapture yields a more precise estimate. The short interval between marking and recapture assured that the assumptions of no births and deaths within each closed population (colony) were met; i.e. the population was constant throughout the study.

The percentage of each colony that was involved in foraging in this study ranged from 51% to over 100% of each colony‟s total worker population, which is far more than the foraging population of a similarly primitive Ponerine and, Neoponera apicalis, which was found to have 1/3 of its colony involved in foraging (Fresenau 1985). These data suggest that there may be differing levels of eusociality within Ponerinae. Since this taxon is known to have been in existence for over 90 million years (Moreau et al. 2006), it is not surprising to see a variety of evolutionary trends within the subfamily. It would be particularly interesting to look at proportion of foragers as a comparative trait, and to correlate this proportion to the ecological situation and habits of the ants, as it would provide insight into the factors that influence division of labor within this subfamily.

One peculiar finding of this study is that female alates forage alongside their worker sisters. Perhaps the female alates of such primitive Ponerine ants should be counted amongst the worker caste, as was done for this study, to the extent that they participate in activities (i.e. foraging) commonly associated with workers. This behavior has also been noted in N. apicalis (Fresneau and Dupuy 1988), in which female gynes (alates) were noted to function as workers. Fresneau and Dupuy (1988) suggested that this trait could be a primitive characteristic typical of the division of labor in more primitive species of ants. Based on my laboratory observations, this is a plausible description as unmated female alates were often seen performing tasks generally

associated with the workers such as foraging and brood care, working alongside their nestmates despite their reproductive capabilities (personal observation).

The nest excavation census (Figure 4.1) shows that not only were marked foragers found foraging during their colony‟s excavation, they were also found distributed throughout all levels of the nests, though in higher numbers in the upper chambers than in the middle and lower chambers. In 70% of nests, marked foragers were found in the bottom chamber of their nests, a location generally occupied by brood-care workers in other species. These data suggest that not only do a high proportion of workers in O. brunneus colonies forage, but that they might also be capable of switching roles of performing multiple intra-colony tasks as is seen in Amblyopone pallipes, a ponerine species known to have one of the most primitive caste systems in ants (Traniello 1978). This large proportion of foragers may be due to a high demand for food as all colonies excavated had brood present, which suggests that the success of individual hunters is low enough that many workers must hunt to provide enough food for the colony. This would be compatible with the finding of Powell and Tschinkel (1999) that, under conditions of limited food supply, or in this case, high nutritional demand due to brood presence, a greater number of individuals are forced into foraging.

With such a high percentage of nestmates participating in foraging, it is interesting to consider why several of the individuals remaining in the nest upon excavation would be ants that recently fulfilled a foraging role, some of whom bore markings of both the initial capture and recapture. It is possible that these foragers are carrying food items directly to the larvae as observed in several other species of primitive ponerine ants (Traniello 1978, Fresneau and Dupuy 1988). This finding is of particular interest as division of labor studies have clearly shown a both a spatial stratification of workers by age and role within nests (Beshers and Fewell 2001, Tschinkel 2006, Tschinkel 1998). While O. brunneus were shown to stratify within nests by age (Chapter 3), the presence of foragers throughout all nest locations shows that most workers, regardless of age, participate in foraging. With the majority of workers

both foraging and bringing food items to the brood, there is no chain of transport within nests as is seen in more derived species (Tschinkel 2006). The data presented in this chapter raise more questions including: How and when did the division of labor that is commonly associated with ants arise? Are there varying degrees of labor division within the primitive ant species?

5 - OVERALL DISCUSSION

From the data collected throughout this study, much has been learned with regard to the life cycle of Odontomachus brunneus colonies as well as the individual ants that make up the colony. At the colony level, the annual cycle of growth and reproduction was determined (Fig. 2.3); the reproductive portion of the colony life cycle occurring in late April and early May with the appearance of larvae in nests, some of which developed into alates, which were found in nests at their highest numbers in mid to late June. O. brunneus display what may be a novel trait, with worker brood produced at the same time as that of sexual (Tab. 2.1), while it has been shown in several other species that colonies produce sexual and worker brood at distinctly different time (Kipyatkov 1996, 2001). Perhaps this separation is more common to derived species and/or species with larger colony sizes as the research on this topic has largely been done on species of Myrmica (Kipyatkov 1996, 2001).

Given the small colony sizes of O. brunneus (Appendix B), simultaneous production of sexuals and workers may be an evolutionary adaptation to enhance colony survival; a way in which to maintain/grow the colony‟s workforce during the production of sexuals. Because no other studies of this nature have been performed on primitive species of ants, it is as of now unknown if this is a species-specific trait or an adaptation common to primitive ants in general. It is assumed that, while worker life span remains unknown, it is likely that workers live for at least a year if not longer so that the colony is capable of yearly growth. Worker longevity generally increases with body size, and because O. brunneus is a large ant, this implies a fairly great longevity (Walter Tschinkel, pers. comm.) Additionally, with Ponerine queens known to lay approximately 5 eggs per day (HÖlldobler and Wilson 1990) though few make it to the larval stage (personal observation); longevity of workers is necessary in order for the colony to grow in size. Post alate production, the colonies begin their growth phase (Oster and Wilson 1978), during which colonies grow in size by the production of new workers (Fig. 2.3) and retention of old through allocation of nutritional resources in the form of fat to workers of all ages (Fig. 3.3).

The colony life cycle depicted through the colony composition data (chapter 2) parallels the pattern of allocation of fat among life stages (Fig. 3.3). During the mid- spring production of brood, some of which will become sexuals, there was an overall decrease in the proportion of fat found within the worker caste (Fig.3.3), meanwhile the proportion of fat increased within the brood (pupae and larvae) through a peak in production in the late summer and early fall (Fig. 2.3 and 3.3). In June, when female alates were present, their high nutritional demand was apparent in their high proportion of body fat (Fig. 3.3). Storing energy in the form of body fat is particularly important for female alates so that, once mated, they will have sufficient energy stores to found a colony. Odontomachus species, like most primitive Ponerine ants, are known to be semi-claustral, meaning that these energy stores are not sufficient to rear her first brood, so that the queen must forage in order to provide nutritional resources for her brood (Wilson and HÖlldobler 2005).

These colony-level attributes are essential to understanding colonies as a unit, or superorganism. It is also important to consider the life cycle of the individual ants that make up the colony. It has been shown in more derived species such as Pogonomymex badius (Tschinkel 1998) that individual ants follow a particular route throughout their lives: they eclose on the brood pile where they remain as brood care workers, as they age and new ants eclose, the older ants move away from the brood to perform nest maintenance tasks, and ultimately end their lives as foragers (Tschinkel 1998). This process, termed adaptive demography (Wilson 1985), is commonly accepted as the core of division of labor of most species. This clear age-related task distribution appears to be more flexible in O. brunneus (Fig. 3.4, Fig. 4.1): while there are distinct separations throughout most of the annual cycle as per the data presented in chapter 3, the locations of active foragers (Fig. 4.1) show that workers can and do migrate within their nests. It is possible that an age-related division of labor is less apparent for this species in natural settings. The finding of foraging female alates in both O. brunneus and Neoponera apicalis (Fresneau and Dupuy 1988) lend support to either a lack of division of labor or a more primitive version of this distribution. Because nests produce very few sexuals per season, it seems maladaptive to allow these alates, 44

who represent a large colony energy investment (Fig. 3.3) to engage in this risky endeavor. Why colonies do not retain these female alates under safer conditions until such time that they mate as is seen in derived species such as Solenopsis invicata (Tschinkel 1993) is not yet understood. It is possible that the colonies require the alates to forage in order to maintain sufficient resources to not only nourish the brood but also maintain the fat stores of the alates until it is time for them to leave the nest. Also, because Ponerine queens found colonies in a semi-claustral fashion, it may just be in the very nature of the female alate to forage during the early portion of her life.

Overall, this study has provided information on basic biological attributes of Odontomachus brunneus including the annual cycle of nest composition and energy (fat) allocation as well as the foraging biology of this species. The data provided here provide a basis for further exploration on both O. brunneus as a species in general, as well as for comparative studies of basic attributes of primitive ants and comparison with more derived species. While few studies of this nature have been performed, from those that do exist, there is a similarity in the foraging of O. brunneus with Neoponera apicalis, a fellow Ponerine species that also engages a large proportion of its workforce, including female alates, in the task of foraging (Fresneau 1985, Fresneau and Dupuy 1988). Additionally, there are similarities between O. brunneus and Pogonomyrmex badius (Tschinkel 1998) in regards to within-nest age structure. Similar to this more derived species, O. brunneus display a spatial partitioning of workers by age, with the youngest residing primarily in the lower regions of nests and the older in the upper region, likely performing primarily as foragers.

Comparative studies of ants will aid in our understanding of the evolution of eusociality in social organisms by providing insight into the composition and social structure of this rather large taxon. Fresneau and Dupuy (1988) make mention of the subfamily of Ponerinae as a potential model for studying the evolution of social organization due to varying combinations of both primitive and derived traits, and after spending nearly two years in the company of Odontomachus brunneus, I whole- heartedly agree; these ants are quite the enigma, displaying both traits common of

derived ants in the form of spatial division of ants by relative age within the nest while at the same time having a loosely organized division of labor, more similar to their primitive sister species. A thorough study (or compilation of studies) across Ponerinae could yield powerful insight into how and when the various traits of eusociality were derived within the diverse family of ants.

APPENDIX A – AVERAGE MONTHLY TEMPERATURES

Table A.1 Average Monthly Temperatures, April 2008-March 2009

Yearly Temperatures 100 Mean Temp 90 Min Temp Max Temp

Temperature (F) Temperature 50

30 April June August October December February May July September November January March Month

* Tropical Storm Fay influenced a decrease in temperatures in mid and late August.

** Mid-December had several warm days in the high 70s and low 80s.

APPENDIX B – COLONY COMPOSITION

Table B.1 Colony Composition throughout the Annual Cycle (raw data)

# Collection Collection # # # Male # # Total Colony ID Female Month Date Queens Workers Alates Pupae Larvae Individuals Alates

1 April 6-Apr-08 1 63 0 0 0 0 63 2 May 13-May-08 1 106 0 0 0 4 110 3 May 15-May-08 0 57 0 0 9 15 81 4 May 19-May-08 1 58 0 0 9 4 71 5 May 19-May-08 0 27 0 0 0 0 27 6 May 20-May-08 0 39 0 0 1 0 40 7 May 20-May-08 0 45 0 0 23 28 96 8 May 26-May-08 0 33 0 0 0 0 33 9 June 5-Jun-08 0 18 0 0 0 3 21 10 June 5-Jun-08 0 21 0 0 0 3 24 11 June 9-Jun-08 0 98 0 0 39 15 152 12 June 16-Jun-08 0 75 0 0 36 67 178 13 June 16-Jun-08 0 41 3 2 0 1 47 14 June 23-Jun-08 1 68 7 0 0 0 75 17 July 7-Jul-08 1 55 0 0 49 19 123 18 July 14-Jul-08 0 107 0 1 0 0 108 19 July 21-Jul-08 1 69 0 0 0 0 69 20 July 21-Jul-08 2 41 0 5 54 8 108 21 July 27-Jul-08 0 27 0 0 28 0 55 22 July 18-Jul-08 0 51 0 0 1 7 59 23 July 29-Jul-08 1 37 0 0 0 0 37 24 July 31-Jul-08 1 45 0 0 34 16 95 25 August 4-Aug-08 0 102 0 0 2 2 106 26 August 6-Aug-08 1 85 0 0 77 5 167 27 August 21-Aug-08 0 81 0 0 21 24 126 28 August 29-Aug-08 1 13 0 0 13 0 26 29 August 29-Aug-08 1 38 0 0 10 6 54 30 August 30-Aug-08 1 55 0 0 10 2 67 31 September 5-Sep-08 0 20 0 0 0 0 20 32 September 5-Sep-08 0 17 0 0 0 0 17 33 September 8-Sep-08 1 36 0 0 53 13 102 34 September 15-Sep-08 0 32 0 0 61 0 93 35 September 21-Sep-08 0 88 0 0 17 4 109 36 September 21-Sep-08 0 61 0 0 64 14 139 48

Table B.1 Continued # Collection Collection # # # Male # # Total Colony ID Female Month Date Queens Workers Alates Pupae Larvae Individuals Alates 37 September 28-Sep-08 1 120 0 0 24 15 159 38 September 28-Sep-08 0 81 0 0 18 1 100 39 October 5-Oct-08 0 37 0 0 93 0 130 40 October 5-Oct-08 1 21 0 0 0 0 21 41 October 5-Oct-08 1 31 0 0 0 0 31 42 October 17-Oct-08 1 53 0 0 26 0 79 43 October 17-Oct-08 1 4 0 0 25 0 29 44 November 2-Nov-08 1 74 0 0 2 0 76 45 November 23-Nov-08 0 59 0 0 0 0 59 46 November 24-Nov-08 0 70 0 0 1 0 71 47 November 26-Nov-08 0 19 0 0 0 0 19 48 November 27-Nov-08 1 126 0 0 0 0 126 49 December 12-Dec-08 1 190 0 0 0 0 190 50 December 14-Dec-08 0 28 0 0 0 0 28 51 December 16-Dec-08 0 65 0 0 0 0 65 52 December 16-Dec-08 0 25 0 0 0 0 25 53 December 17-Dec-08 0 77 0 0 0 0 77 54 December 17-Dec-08 0 18 0 0 0 0 18 55 January 9-Jan-09 0 35 0 0 0 0 35 56 January 12-Jan-09 0 19 0 0 0 0 19 57 January 16-Jan-09 0 67 0 0 0 0 67 58 January 23-Jan-09 1 145 0 0 0 0 145 59 January 27-Jan-09 1 119 0 0 0 0 119 60 January 31-Jan-09 0 48 0 0 0 0 48 61 February 18-Feb-09 0 26 0 0 0 0 26 62 February 17-Feb-09 0 60 0 0 0 0 60 63 February 21-Feb-09 0 35 0 0 0 0 35 64 February 23-Feb-09 0 16 0 0 0 0 16 65 February 27-Feb-09 0 12 0 0 0 0 12 66 February 28-Feb-09 0 17 0 0 0 0 17 67 March 21-Mar-09 0 18 0 0 0 0 18 68 March 22-Mar-09 0 4 0 0 0 0 4 69 March 22-Mar-09 1 100 0 0 0 0 100 70 March 23-Mar-09 0 28 0 0 0 0 28 72 April 10-Apr-09 0 145 0 0 0 0 145 73 April 10-Apr-09 0 62 0 0 0 0 62 74 April 21-Apr-09 0 95 0 0 0 0 95

Table B.1 Continued # Collection Collection # # # Male # # Total Colony ID Female Month Date Queens Workers Alates Pupae Larvae Individuals Alates 75 April 30-Apr-09 1 125 0 0 0 3 128 76 April 30-Apr-09 0 54 0 0 0 1 55

APPENDIX C – MEAN WORKER WEIGHTS BY MONTH

Table C.1 Mean Worker Weights by Month throughout the Annual Cycle

Std. Month N Mean Min. Max. Dev. April 235 3.75 2.14 5.27 0.58 May 194 3.64 2.03 5.51 0.62 June 181 3.52 1.73 5.00 0.51 July 171 2.99 0.91 4.63 0.81 August 106 2.86 1.24 4.19 0.63 September 185 3.05 0.98 4.48 0.63 October 104 3.33 1.63 4.18 0.41 November 141 3.30 1.23 5.91 0.82 December 132 3.97 1.83 5.77 0.78 January 124 3.91 2.51 5.92 0.66 February 109 3.81 2.31 5.62 0.61 March 85 3.53 2.23 5.57 0.63

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BIOGRAPHICAL SKETCH

Lauren Hart was born on July 17, 1980 in St. Petersburg, Florida. The first thirteen years of her life were spent reading books and rummaging through the nearby woods where she fell in love with all manner of living creatures- both for what they were and for the joy of terrorizing her older sister. During the Midwest floods of 1993, her family uprooted for a new adventure to be found in St. Louis, Missouri, where Lauren would fall deeper in love with biology and pick up new musical skills earning a role as a true band geek.

A love of biology and the Euphonium brought her to the University of Missouri- Columbia where Lauren would earn her degree in Biology while also performing in multiple musical ensembles including Marching Mizzou, Symphonic Band and the Tuba/Euphonium Ensemble. In addition to the musical mayhem that ruled her college years, she was introduced to the wonders of biological research in the laboratory of Dr. Donald L. Riddle, studying Caenorhabditis elegans with an interesting ensemble of researchers.

Upon graduation, Lauren headed east for new adventures while earning a Master’s degree in Science Education at Syracuse University. After two winters, she readily accepted a job teaching high school science in St. Louis, Missouri and said good-bye to the terrible phenomenon known as lake-effects snow. Teaching high school was an interesting experience, one that would shape her future greatly; while Lauren loved teaching others about biology, it just wasn’t enough. She tried to fill her time by coaching lacrosse, sponsoring several science clubs, but it still did not fulfill her and she decided that perhaps teaching college students and being able to participate in research would make her happy.

By some crazy luck, Lauren was offered an opportunity to study Ecology at Florida State University and jumped at the offer. But studying ants? That was entirely new. Within the first few months at FSU, she became entranced by the crazy behaviors of several species and was fortunate to happen upon one to which she has devoted the

past two years of her life. This experience was beneficial in helping Lauren decide exactly what path she wants her career to follow: after finishing a second Master’s degree at FSU, she will be continuing the study of Entomology working towards a PhD at her alma mater, the University of Missouri-Columbia.