How Can Allegheny Mound Ants (Formica Exsectoides) Provide an Optimal Environment for Karner Blue Butterflies (Lycaeides Melissa Samuelis)?
HOW CAN ALLEGHENY MOUND ANTS (FORMICA EXSECTOIDES) PROVIDE AN OPTIMAL ENVIRONMENT FOR KARNER BLUE BUTTERFLIES (LYCAEIDES MELISSA SAMUELIS)?
Preston M. Thompson
A Thesis
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
August 2019
Committee:
Shannon Pelini, Advisor
Karen Root
Ryan Walsh ii ABSTRACT
Shannon Pelini, Advisor
Understanding how the result of the mutualistic interaction between the Karner Blue
Butterfly (Lycaeides Melissa samuelis) (KBB) and the Allegheny mound ant (Formica esectoides) changes with temperature and how Allegheny mound ants modify potential KBB habitat is crucial to the persistence of the federally endangered Karner Blue Butterfly. A change in conservation techniques and an emphasis on new conservation plans will occur if there are advantages to maintaining a mutualistic relationship between these species. This research examined how the mutualistic interaction between KBB and the Allegheny mound ant buffers the negative effects of climatic changes by manipulating temperature and Allegheny ant presence. In an experimental study, I manipulated temperature and ant presence via infrared heating lamps and a connective tubing system to an ant mound on KBB larvae. I found that larvae distance from lupine soil decreases with increasing temperature and decreases with ant presence. KBB mortality, days to pupation, and days in the pupal stage did not change with changes in temperature and ant presence. I also examined how Allegheny ant mounds in the Oak
Openings region influenced abundance of invertebrates found near ant mounds and the community structure of the surrounding vegetation. I found a significant interaction between distance from ant mounds and spider abundance: the highest concentration of spiders was found the farthest sampling locations away from ant mounds. The abundance of other ground-dwelling invertebrates found near Allegheny ant mounds did not change with varying distance. Vegetation density and vegetation ground cover did not change with varying distance from ant mounds. The application of this research proposes the mutualistic interaction between KBB and Allegheny iii
mound ants—which provide reduction in predation as well as improved use of microclimate—to be implemented as a conservation tool, in hopes bolster KBB populations. iv ACKNOWLEDGEMENTS
This thesis has been directly and indirectly touched by so many people. My advisor Dr.
Shannon Pelini has been arguably one of the most influential people of my life. She showed me the path to become a successful and well-rounded scientist; Shannon always encouraged me that
I can do something rather than I can’t do something. Her emphasis on her students mental health is admirable and has influenced me to become the person I am today. My committee members,
Dr. Ryan Walsh and Dr. Dr. Root have attributed greatly to the setting of this experiment, structure of my experiment and what data I should collect and analyze, without them this project would not have been successfully completed. My lab-mates, Josephine Lindsey-Robbins and
Audrey Maran were always supportive and helpful with any questions I had or when I became so stressed I just needed a venting session. I was very fortunate to come into a lab where Josie and
Audrey always were detail oriented, this really changed my creativity and problem solving skills.
I am proud to say that the cohesiveness of our lab always felt like family (including Sal and
Michael Clawson). My family, friends and pets have supported me immensity through this process. My family: Bruce Thompson, Mary Thompson, Spencer Thompson, Pumpkin, Princy, and Silky gave me so much emotional and intellectual support I cannot thank you enough, one day I will help others the way you helped me. I am so happy to have met my best friend and significant other, Mellissa Seidel, through the journey of graduate school. You have always been supportive when stressful days would approach and showed patience when my love for music would sometimes…always blare in the apartment. Admittedly, I have more love for you then I will ever do for music. To my friends Jordan Barkey, Ben Bomlitz, Kevin Chiteri, Kevin
Connell, Mariah Dandar, Meigan Day, Domenic DiPetro, Sean Grady, Erin Johnson, Kenneth
Kohlhofer, Logan Moreno (future best man), Nabil Karnib, Shreyashi Pain, Rebecca Welker, v
Conner Willams, and John Woloschuk I am forever grateful to surround myself with friendly, funny, supportive, goofy people, I will remember and honor the memories we have made together. If you all ever read my thesis, this is me saying I owe you a beer. I would like to personally thank Bowling Green State University, Ohio Biological Survery, Barbara Long
Masters Award, and the Katzer Graduate Student Research Award for helping fund this study.
Lastly, I would like to thank Dr. Ryan Walsh and the Toledo Zoo for allowing me to conduct my experiment on zoo grounds, and Karen Menard and the Oak Openings metropark for allowing me to conduct my observational component of my thesis. vi
TABLE OF CONTENTS
Page
CHAPTER I ...... 1
Introduction ...... 1
Populations in Decline as a Result of Climate Change ...... 1
Association with Ants ...... 2
Methods...... 3
Research Facility ...... 3
Model Organism-Karner Blue Butterfly ...... 3
Model Organism-Blue Lupine ...... 4
Model Organism-Allegheny Mound Ant ...... 4
Experimental Setup ...... 5
Data Collection ...... 6
Statistical Analysis ...... 7
Results ...... 7
Discussion ...... 9
CHAPTER II ...... 11
Introduction ...... 11
Lycaenidae Association with Ants...... 11
Aphidoidea Association with Ants ...... 12
Vegetation Characteristics of Allegheny Ant Mound Surroundings ...... 12
Current Conservation Plan and Research Questions ...... 12
Methods...... 13 vii
Study Locations ...... 13
Data Collection ...... 13
Data Analysis ...... 14
Results ...... 15
Discussion ...... 17
REFERENCES ...... 20
APPENDIX A: TABLES ...... 25
APPENDIX B: FIGURES ...... 30 1 CHAPTER I
Introduction
Karner Blue Butterfly (Lycaeides Melissa samuelis) (KBB) populations are declining due to climatic changes. Mutualistic interactions with other organisms may be beneficial to the overall success of the Karner Blue Butterfly; however, promoting the habitat of blue lupine
(Lupinus perennis) is a major component of the conservation plan for KBB, whereas promoting
KBB-ant mutualisms are not. The majority of Lycaenidae species, including KBB, maintain larval associations with ants (Fiedler, 1991). KBB, a Lycaenidae species, and Alleghany mound ants (Formica exsectoides), belonging in the genus Formica, and with most Lycaenidae-Formica associations being mutualistic, a conservation effort must be considered to maintain these mutualistic relationships to buffer climate change. A recent experiment by Marquis et al., 2014 shows that mutualisms can buffer organisms from climate change and stabilize survivability rate.
Due to these findings, it is imperative to study how KBB and Alleghany mound ants interact under climate change. This study focuses on the potential effectiveness of incorporating these associations into the KBB management plan.
Populations in Decline as a Result of Climate Change
The Karner blue butterfly (KBB) is a federally endangered bivoltine butterfly species and its distribution is largely dependent upon the larval host plant, wild lupine (Lupinus) (Dirig,
1994). Overwintering eggs, which are oviposited during the fall, hatch on wild lupine in April and May with larvae experiencing four instars before they begin pupation. The first brood of
KBB adults emerges May-June (Grundel et al.1998), while the second brood of eggs, oviposited by the first brood of females, hatches June- July. The second brood of adults emerges in July-
September and lays overwintering eggs around September (Grundel et al. 1998). 2 The year 2015 had the warmest global mean temperatures ever recorded and the years between 2011-2015 is 0.051°C warmer than the average temperatures of 2001-2010, and this change in climate will impact the survivorship of the KBB (Grundel et al. 2000; Grundel and
Pav-lovic 2007, Jones et al. 2015). Dry summers decrease the egg survival rate by reducing KBB host-plant (Lupine, Lupinus perennis) quality (Pollard 1998; Dennis & Shreeve 1991). In 2012, a severe drought combined with a false spring, a period of spring with unusual warming, occurred in the Great Lakes region, leading to a decline in the KBB population (Walsh, 2017).
Association with Ants
Lycaenidae butterfly larvae are known to have associations with fifty-three ant genera belonging to six subfamilies (Fiedler, 2001). Ants will protect Lycaenidae larvae, incuding KBB, from predators and parasitoids in exchange for nutritious secretions, and Lycaenids mediate this relationship through the use of a specialized organ that produces vibrations and secretions that attract tending ants (Pierce, 1989). Lycaenidae larvae have additional adaptations to associate with ants: a thick cuticle, appeasement pheromones, and nectar glands (Atsatt 1981, Wagner
1993). A study by Robert K. Robbins showed that the larvae of Arawacus lincoides (Lycaenidae) took 0.68 days longer to complete larval development without ants (Robbins, 1991). Lycaenidae significantly increase clutch size after contact with ants, and eggs laid in the presence of ants typically experience lower parasitism and predation rates, which results in an increase of survivorship (Atsatt, 1981). When engaged in these ant mutualisms, Lycaenidae species experience increased clutch size, decreased parasitism rates, decreased predation rates, decreased larval development time, and increased body mass (Atsatt 1981, Robbins 1991, Wagner 1993,
Pierce et al. 2002; Weeks 2003).
Understanding how the result of the mutualistic interaction between the Karner Blue 3 Butterfly (Lycaeides Melissa samuelis) (KBB) and the Allegheny mound ant (Formica esectoides) changes with temperature is crucial to the persistence of the federally endangered
Karner Blue Butterfly. This research examined how the mutualistic interaction between KBB and the Allegheny mound ant buffers the negative effects of climatic changes by manipulating temperature. I aimed to elucidate the role of ant-butterfly mutualistic relationships in bolstering
KBB populations against climate change. My research addresses two critical questions: 1) How does warming impact KBB larvae? and, 2) Does ant tending impact KBB larvae, and do these impacts change with temperature?
Methods
Research Facility
Research was conducted at the Toledo Zoo greenhouse (Toledo, OH). This facility allowed for the control of temperature and precipitation for rearing and experimental purposes. A
50% aluminet shadecloth covered the greenhouse, which maintained a steady temperature of 1.1-
1.7 °C above ambient temperature during summer months. Karner blue butterfly (Lycaeides
Melissa samuelis and its host plant Blue lupine (Lupinus perennis) were reared, with Ohio
Department of Natural Resources permission (Permit TE207180-4), at the Toledo Zoo for nearly a decade.
Model Organism-Karner Blue Butterfly
Dr. Ryan Walsh, Wild Toledo Biologist at the Toledo Zoo, provided the KBB for this study. In April 2018, Dr. Walsh and his team collected 1st brood 28 adult females and 2 adult males collected from Allegheny, Michigan. These adults were used for captive breeding purposes within the Toledo Zoo greenhouse. These KBB individuals produced 17 KBB larvae that I used for my experiment. Before KBB larvae were used for the experiment I collected width 4 (mm) and length (mm) measurements of each larva.
Model Organism-Blue Lupine
Blue lupine seeds purchased by the Toledo Zoo were from the Prairie Moon Nursery and
collected from the Toledo Zoo’s kind road library prairie if necessary. 17 Blue lupine plants
germinated in 128 cell packs and were transferred to 12.7 cm x 12.7 cm square pots, Lupine were
grown in a 3:2 Pro-Mix BRK (Riviere-du-Loup, Canada) and coarse perlite was supplemented
with Osmocote Plus professional 15-9-12 (Dublin, Ohio). Soil moisture underneath lupine plants
was monitored twice everyday by a Soil Moisture Meter (Grainger, Lake Forest, Illinois) and
controlled by adding the necessary amount of water by a watering can so each plant receives the
same moisture soil conditions. Enough water was added to moisten the lupine soil if mineral soil
moisture measurements were below 5 percent.
Model Organism-Allegheny Mound Ant
I obtained one colony of Allegheny mound ants from the Oak Openings Metroparks with permission from the Toledo Metroparks (Toledo Metroparks Permit Number- 040618).
Allegheny ants were placed in a large holding container (43 cm x 33 cm) in the month of May where they were maintained. The ant colony acclimated for two weeks before the start of the experiment, during which the colony was held at Bowling Green State University. The
Allegheny ant colony was given three petri dishes of the Bhatkar Diet (Bhatkar, 1970) every three days, and the soil of the ant nest was moisturized regularly three times a week until the top layer of the ant mound became saturated. The methodology to properly rear an Allegheny ant mound was previously established in the Pelini Lab in the fall of 2018. A 2 cm layer of Insect-a-
Slip (BioQuip Products, Rancho Dominguez, California) was placed around the edges of the container to avoid any ant escape. The ant colony was moved to the Toledo Zoo’s greenhouse 5 while the experiment was active. At the end of the experiment the ant colony was properly disposed of.
Experimental Setup
I manipulated temperature and presence of Allegeny mound ant tending for 17 mesocosms placed in the Toledo Zoo greenhouse. Mesocoms were constructed out of 0.30m x
0.30m x 0.30m collapsible cages (BioQuip Products, Rancho Dominguez, California). The collapsible cages prevented predation from external organisms and organisms from freely moving between mesocosms. Each mesocosm contained one individual KBB larva on one lupine host plant (Figures 1 & 2). Mesocosms were randomly selected for each KBB larva as well as the original placement of larva on lupine plants. KBB larvae were transferred to mesocosms using a paintbrush to prevent harm to the larvae.
The Allegheny ant mound was placed in the center of 17 mesocosms and was approximately 0.1 meter away from each mesocosm (Figure 2). To prevent ants from escaping, binder clips were synched to mesh on the lid of the ant colony container. A total of nine mesocosms were accessible to the Allegheny ants via clear 0.16 cm inside diameter x 0.32 cm outside diameter tygon tubing (Home Depot, Bowling Green, Ohio), while the other eight mesocosms did not have access to the Allegheny ants. Accessibility was randomly assigned to each mesocosm (Figure 2). Thus, two-meter pieces of tygon tubing were connected between the ant colony and nine mesocosms, via drilled holes, into the Allegheny ant colony container. Each hole was 4-5 cm from each other and drilled approximately 2 cm above the ant colony soil. The connections between tygon tubing and mesocosms were synched with rubber bands and cloth to prevent model organisms from escaping and outside predation.
Mesocosms were subjected to ambient or elevated temperature—generated by infrared 6 heat lamps—treatments. Mesocosms were randomly assigned to one of the following treatments: four mesocosms were maintained at ambient temperature with absence of ants, five mesocosms were maintained at ambient temperature with presence of ants, four mesocosms were heated to ~
1°C-2°C above ambient temperature with absence of ants, four mesocosms were heated to ~
1°C-2°C above ambient temperature with presence of ants (Figure 2). Heating treatments were established using 250-watt infrared lamps suspended above mesocosms (Figure 1). Suspension of lamps was achieved by using PVC piping tied firmly together with string and clamps. Lamps were hung at varying heights above mesocosms to achieve these target warming. To furthermore achieve target warming temperature lamps were on throughout the experiment.
Data Collection
Data collection began when second brood KBB larvae were placed in mesocosms
(6/27/18) and lasted until adult KBBs emerged or larvae deceased (7/16/18). Data was collected daily until the completion of the experiment. KBB Measurements included survivorship, time to pupation, time from pupation to butterfly, vertical placement (cm)--measured by a straight-line distance from lupine soil surface of KBB larva—and larva location within mesocosm. While I observed KBB larvae I categorized them in the following categories: alive, dead, larvae stage, pupae stage or adult stage.
Environmental data included ambient temperature (°C) of the Toledo Zoo greenhouse, mean mesocosm temperature (°C) and lupine soil surface temperature (°C) by using an infrared thermometer (Jida HuaPu Instrument Co., ZhuHai, China). Soil moisture of lupine soil and ant mound soil was taken by using a SM150T Portable Soil Moisture Meter, (Dynamax, Fresno,
CA). To calculate mean mesocosm temperature I measured the surface temperature (°C) in each corner of each mesocosm and found the average temperature (°C). 7 Allegheny mound ant and Blue lupine measurements consisted of visual data on the number of individual ants within each mesocosm, ants tending KBB larvae/KBB pupae, lupine height (cm), and number of leaves (Table 1). Data was collected on lupine soil moisture, ant colony soil moisture, and ant colony temperature but these variables were not included in models.
Statistical Analysis
All statistical tests were conducted using R (version 3.5.3) and R studio (version 1.1.463).
The nlme package was used to fit linear mixed effects models (lme). For all lme models that consisted of repeated measures, mesocosm ID was considered the random variable. Some lme models assessed the main and interactive effects of temperature of lupine soil surface, presence of ants, number of lupine leaves, and height of lupine (cm) for the response variable of larvae vertical placement on lupine (cm). Other lme models examined how temperature of mesocosms and temperature of lupine soil changed the number of ants tending KBB larvae and number of ants in mesocosms. I used general linear models (glm) to examine the relationships between the average temperature of surface lupine soil and the presence of ants for the response variables of larvae days to pupation, larvae days from pupation to butterfly, and larvae mortality. In all circumstances, statistical significance was accepted at a p-value equal to or less than 0.05. The
Shapiro-Wilkes test was used to examine normality. Non-normal data were log transformed and a poisson regression model was used to normalize data. Least-squares trends from the lsmeans package were used for post-hoc analyses for statistically significant models.
Results
All larvae subjected to ant presence treatments were tended. 8 KBB Number of Days to Pupation- Ant Presence (GLM: χ2=0.000, p = 0.995, Table 1,
Figure 3A) and lupine soil temperature (°C) (χ2=0.117, p = 0.733, Table 1, Figure 4A) was not
significant. KBB Number of Days from Pupation to Butterfly- Ant presence (GLM: χ2=0.048, p =
0.827, Table 1, Figure 3B) and temperature of lupine soil (°C) (χ2=0.058, p = 0.809, Table 1,
Figure 4B) was not significant. KBB Mortality- Temperature of lupine soil (°C) (GLM: χ2=0.158,
p = 0.695, Table 2, Figure 5) and ant presence (χ2=0.118, p = 0.731, Table 2) was not significant.
Number of Ants Tending Larvae- The interaction between temperature of lupine soil (°C) and the
number of ants tending larvae was not significant (LME: χ2=0.706, p = 0.401, Table 2, Figure
5A). Number of Ants in Mesocosms- The interaction between temperature of lupine soil (°C) and
the number of ants in each mesocosm was not significant (LME: χ2 =0.863, p = 0.294, Table 2,
Figure 6B). KBB Vertical Placement (cm)- Within the model my measurements decreased significantly with increasing temperature (LME: χ2=9.144, p = 0.003, Table 2). An lstrends post-
hoc analysis test was performed to detect if there was a statistically association between
temperature of mesocosms (°C) influences KBB vertical placement (cm) (trend=-0.33, df=426,
Std. Error=0.065, lower CI=-0.456, upper CI=-0.203, Figure 7B). Also, lstrends post-hoc
analysis test was performed on how the presence of ants affects KBB vertical placement (cm)
(trend=-4.4,df=15,Std. Error=1.58, lower CI=-7.76, upper CI=-1.04, Figure 7A). Number of
Lupine Leaves- The interaction between lupine soil temperature (°C) and ant presence was not
significant on the number of lupine leaves (LME: χ2=0.608, p = 0.457, Table 2). Height of
Lupine (cm)- The interaction between lupine soil temperature (°C) and ant presence was not
significant on the height of lupine plants (LME: χ2=0.143, p = 0.718, Table 2).
9 Discussion
Through my experiment it appears that the Karner Blue Butterfly (Lycaeides Melissa samuelis) (KBB) and the Allegheny mound ant (Formica exsectoides) does improve the performance of KBB under warmer temperatures. KBBs were generally found at similar vertical positions when lower temperatures occurred in mesocosms; however, at higher temperatures
KBBs that were subjected to ant presence had higher vertical positions (Figure 7), suggesting that ant presence influenced KBB larvae thermoregulatory behavior
Microhabitat selection has been shown to have an effect on ectotherm’s, including juvenile Lepidopterans’, body temperature and activities (Stevenson, 1985). A study showed that
Eastern tent larvae (Malacosoma americanum) growth was positively correlated with an increase of body temperature, due to behaviorioral thermoregulation increasing rates of food processing and a total increase in herbivory (Casey et al., 1998). In other cases larvae will seek refuge for cooler temperatures (Nielsen and Papaj, 2015). Larvae of the pipevine swallowtail butterfly
(Battus philenor), have been observed to change microhabitats by moving on vegetation to avoid excessive heat (Nice & Fordyce, 2005). Larvae of the range caterpillar moth (Hemileuca oliviae) has been observed to cease feeding, assuming a resting position, in a response to high temperatures (Capinera et al., 1980).
Lycaenidae-ant interactions can be induced when environmental stressors occur. The communication between lycaenidae larvae and ants is a result of transmission between chemical and audible signals; stressed signals of lycaenidae larvae can result in ants offering security or shelter (Devries, 1990). Large-blue butterfly larvae (Maculinea rebeli) have been documented to be carried by ants directly to their nests greatly influencing microhabitat characteristics (Elmes et al. 1991). In this study I observed KBB larvae at lower vertical positions when Allegheny ants 10 were present I did not detect subsequent effects on the growth or mortality of KBB in this study,
but a higher sample size may produce a pattern of increased growth and development as ants
facilitatetheir escape from lethal environmental stressors.
Understanding how the result of the mutualistic interaction between the KBB and the
Allegheny mound ant changes with temperature is crucial to the persistence of the federally endangered KBB. A change in conservation techniques and an emphasis on new conservation plans will occur if there are advantages to maintaining a mutualistic relationship between these species. This research provides a deeper understanding to how the mutualistic interaction between KBB and the Allegheny mound ant buffers the negative effects of climatic changes.
Larvae vertical placement (cm) was significantly lower when lupine soil temperature (°C) increased, while ant presence also decreased vertical placement. A change in vertical placement
However, larvae development and KBB mortality was not impacted by lupine soil temperature
(°C) or ant presence, which suggest a facultative relationship between the KBB and Allegheny mound ants. These findings may contribute to conservation efforts for the federally endangered
KBB species and permits future research to be explored into the explanations of this complex system, in hopes to increase the odds of the federally endangered species to recover in the Oak
Openings region.
11 CHAPTER II
Introduction
Karner Blue Butterfly (Lycaeides Melissa samuelis) (KBB) populations are declining due to climate change events. Mutualistic interactions with other organisms may be beneficial to the overall success of the Karner Blue Butterfly; however, mutualisms are not in the current conservation plan for KBB. A recent experiment by Marquis et al. (2014) showed that ant- herbivore mutualisms could buffer organisms from the physiological stresses of and population size responses to climate change. The majority of Lycaenidae species, including KBB, maintain larval associations with Formica ant species (Fiedler, 1991). KBB, a Lycaenidae species, and
Alleghany mound ants (Formica exsectoides), belonging in the genus Formica, and with most
Lycaenidae-Formica associations being mutualistic, a conservation effort must be considered to maintain these mutualistic relationships to buffer climate change. Due to these findings, it is imperative to understand the Allegany mound ant habitat characteristics and maintain their populations in hopes to better conserve the KBB. This study aimed to maintain KBB-Allegheny mound ant mutualism in order to aid in the conservation efforts of KBB’s.
Lycaenidae Association with Ants
Lycaenidae butterfly larvae are known to have associations with fifty-three ant genera belonging to six subfamilies (Fiedler, 2001). Ants will protect Lycaenidae larvae, incuding KBB, from predators and parasitoids in exchange for nutritious secretions, and Lycaenids mediate this relationship through the use of a specialized organ that produces vibrations and secretions that attract tending ants (Pierce, 1989). When engaged in these ant mutualisms, KBB experience increased clutch size, decreased parasitism rates, decreased larval development time, and increased body mass (Atsatt 1981, Robbins 1991, Wagner 1993 Pierce et al. 2002; Weeks 2003). 12 Aphidoidea Association with Ants
The Allegheny mound ant is known to associate themselves with multiple species of
Homopterans including aphids (Cory and Haviland 1938). Ant-aphid mutualism is very similar to the ant-lycaenidae mutualism in that ants will protect aphids from predation in turn to remove honeydew (Strickland 1947, Majer 1982). In particular, Allegheny mound ants have been found to create enemy-free space for aphids (Bishop & Bristow, 2003). With the protection of ants, aphid populations may increase due to the removal of other insects that can harm aphids’ host- plants (Holldobler and Wilson 1990, pp. 522-526). Thus, this mutualism could influence local vegetation characteristics and insect diversity.
Vegetation Characteristics of Allegheny Ant Mound Surroundings
The Allegheny mound ant (Formica exsectoides) is a common mound-building ant, which can be found in dense populations often found in oak savannah habitats (Creighton, 1950).
Mound building ants are known to influence the surrounding vegetation either through seed dispersal or injecting formic acid into plant tissues which may result in killing vegetation surrounding the ant mound (Beattie & Culver 1977). Allegheny mound ants, by removing competitors of Chaitophorus aphids, reduce herbivory on and therefor increase local abundance of cottonwood trees (Populus) (Bishop & Bistow, 2003). Interestingly, Allegheny mound ants do not recognize ants from different colonies, allowing multiple mounds to inhabit the same area may result in intensified vegetative changes (Holldobler and Wilson 1990).
Current Conservation Plan and Research Questions
Current conservation efforts for the KBB include restoring and maintaining habitat for mating, oviposition and nectar plant selection (Wiklund, 1977). These techniques include planting lupine to expand the density and range of the KBB host-plant and conducting controlled 13 burns to reduce canopy cover. Reduction of canopy cover can result in KBB females ovipositing on lupine when there are differences in canopy cover, specifically when moderate shade is present (Grundel et al. 1998). With my research, I studied how Allegheny mound ants, which interact mutualistically with KBB (Chapter 1) modify their (and KBB) surrounding habitat. My research addresses the question: How do Allegheny mound ants influence the vegetation density and arthropod communities in the Oak Openings Region?
Methods
Study Locations
To investigate how the Allegheny mount ant presence influences vegetation and insect composition I collected data within the Oak Openings region (Toledo Metroparks Permit
Number-040618). For the duration of the study I collected data from two locations in Toledo
Metroparks properties within the Oak Openings Region: Site 1 is a butterfly meadow
(N41.56340 W083.84610) comprised of low laying vegetation and tree saplings and Site 2 is a forest off of Girdham Road (N41.56332 W083.84659), which is a secondary forest mostly comprised of maple and oak trees. These two sites were chosen due to their high frequency of
Allegheny ant mounds. Data were collected once per week for each location, and then was pooled among sites. The data collection period for butterfly meadow site were 8/8/18-10/1/18 and the forest off of Girdham Road was 8/24/18-9/30/18.
Data Collection
For each location, 10 Allegheny ant mounds were haphazardly selected for a total of 20 ant mounds. Each ant mound was comprised of a total of 16 sample locations (Figure 8). One week before data collection occurred, flags were placed and labeled at each data sample location for reference. 14 For each ant mound the following data was collected: Temperature (C°), temperature of the ant mound soil (C°), soil moisture (mV), litter depth (cm), soil type, o-horizon length (cm), a- horizon length (cm), b-horizon length (cm), height of ant mound (cm), and area of ant mound
(cm2), the only variable that was analyzed further was the area of ant mounds (cm2) In addition, visual obstruction reading of vegetation was measured along with the composition of vegetation, presence of cottonwood trees, number of cottonwood trees, presence of Allegheny mound ants, number of Allegheny mound ants, behavior of Allegheny mound ants.
Visual obstruction readings were taken by using a modified Robel Pole (Smith, 2008).
The pole consisted of twelve bands with each band being 10 cm long. To measure visual obstruction, I stood 1 meter away at eyelevel with the top of the pole. Then, I observed the first visible band from the ground and counted the number of bands that were not visible. The number
I recorded was the number of bands not visible and the first band visible from the ground. To record the vegetation composition I used a 20 x 50 cm Daubenmire frame (Daubenmire, 1959). I haphazardly placed the frame on the vegetation and estimated the percentage of shrub, forb, warm season grass, cool season grass, detritus, bare ground, and moss. I used ranges of percentages to record a number value in my data. The ranges and the corresponding number values to the ranges were as follows: 0-20% = 1, 21-40% = 2, 41-60% = 3, 61-80% = 4, 81-
100% = 5.
Data Analysis
All statistical tests were conducted using R (version 3.5.3) and R studio (version 1.1.463).
The nlme package was used to fit linear mixed effects models (lme). Lm models were used to examine the relationship between distance from ant mounds and the response variable of number of cottonwoods and invertebrate diversity. For all lme models that were created, ant mound ID 15 was considered the random variable. Lme models were created to examine the relationships
between the area of ant mounds, distance, presence of aphids, presence of cottonwoods on the
response variable of VOR average and the number of ants recorded. In all circumstances,
statistical significance was accepted at a p-value of less than 0.05. The Shapiro-Wilkes test was
used to examine normality. Non-normal data was log transformed and a poisson regression
model was used. Least-squares trends from the lstrends package were used for post-hoc analyses
for statistically significant models.
Results
Number of Cottonwoods. The number of cottonwoods present near Allegheny ant mounds
did not significantly change with increasing distance away from ant mounds (LM: p = 0.734,
Table 3, Figure 9A). Number of Ants- The number of Allegheny ants present near Allegheny ant
mounds did not vary with increasing distance away from (LME: p = 0.325, Table 4, Figure 9C)
or the size of (p = <0.001, Table 3) Allegheny ant mounds. The interactions between the area of
ant mounds, distance (LME: χ2=9.379, p = 0.002, Table 4) presence of cottonwoods (LME:
χ2=17.334, p = <0.001, Table 6) and the presence of aphids (LME: χ2=9.184, p = 0.003, Table 4) relating to the number of ants found in a sampling location was significant. An lstrends post-hoc analysis test was performed on how the interaction between the area of Allegheny ant mounds and the number of ants in a sampling location (trend=-<0.001, df=1891, Std. Error=<0.001, lower CI=<0.001, upper CI=<0.001) revealing that area of the ant mounds did not change the number of ants observed. Another ls trends post-hoc analysis test was performed on how the interaction between the distance away from ant mounds and the number of Allegheny mound ants in a sampling location (trend=-0.062, df=1891, Std. Error=0.036, lower CI=-0.132, upper
CI=0.008), showing that interaction between distance and the number of Allegheny mound ants 16 in a sampling location was not significant. It was found that presence of cottonwoods increased
the number of Allegheny mound ants in a sampling location in a lstrends post-hoc analysis test
(trend=3.28, df=1732, Std. Error=<0.001, lower CI=2.88, upper CI=3.68), and the presence of
aphids increased the number of Allegheny mound ants in a sampling location (trend=8.13,
df=1890, Std. Error=0.226, lower CI=7.69, upper CI=8.57). Invertebrate Diversity vs. Distance
(m) from Ant Mounds- The interaction between number of spiders present near Allegheny ant
mounds and distance away from ant mounds was significant (LM: p = 0.024, Table 3, Figure
9B). The interaction between distance away from ant mounds and number of crickets (LM: p =
0.737, Table 3), caterpillars (LM: p = 0.056, Table 3), grasshoppers (LM: p = 0.562, Table 3),
moths (LM: p = 0.528, Table 3), leafhoppers (LM: p = 0.228, Table 3), beetles (LM: p = 0.802,
Table 3) and the total number of invertebrates (LM: p = 0.113, Table 3) was not significant.
Vegetation Density- The interaction between the predictor variables: area of ant mounds and
distance from ant mounds, and the predictor variable: vegetation density, was not significant
(LME: χ2=0.68, p = 0.413, Table 4). However, the interaction between vegetation density and the area of the ant mounds were significant (LME: χ2=4.949, p = 0.027, Table 4). An ls trends post- hoc analysis test was performed on the interaction between the area of ant mounds and vegetation density (trend=<0.001, df=466, Std. Error=<0.001, lower CI=<0.001, upper
CI=<0.001), with a minimal this post hoc-analysis showed that area did not change vegetation density. Deeper analysis suggests that my random variable within my model, ant mound location, significantly interacted with vegetation density (χ2=0.68, p = <0.001). Vegetation Composition-
The vegetation composition around the ant mounds in the Oak Openings region are mostly
dominated with shrubbery (21-40%), with the rest of the vegetation categories (0-20%) (Table
5). 17 Discussion
In my study, my goal was to see how Allegheny ant mounds influenced the surrounding landscape, including vegetation characteristics and ground-dwelling invertebrate abundance characteristics. Overall, I found that invertebrate diversity, vegetation density, vegetation composition, and number of cottonwoods were similar for all distances away from ant mounds. I did find that spider abundance increased 7 meters away from ant mounds, compared to 1, 3 and 5 meters way.
I found that the number of spiders significantly increased with increasing distance from ant mounds (Figure 5B). However, distance from Allegheny ant mounds was not significantly associated with the abundances of other invertebrates) crickets, caterpillars, grasshoppers, moths leafhoppers, and beetles; Table 4).
I observed that Allegheny mound ants have a close association with aphids (Chitophorus populicola) and their host plant Swamp cottonwood (Populus heterphyya) in the Oak Openings region. Previous studies have shown that Chitophorus, aphids and tending-ants have close interaction; Chitophorus aphids attract tending-ants, which may result in an overall reduction in herbivory for Chitophorus (Floate & Whitham, 1993). With this interaction observed in the Oak
Openings region, one can predict tending-ants will remove predatory invertebrates and positively affect Populus heterophylla. In a recent study, Allegheny mound ants provided an enemy-free space for populations of aphids, in the presence of Allegheny mound ants, aphid populations increased while predator populations decreased (Bishop & Bristow, 2003). This is a possible explanation for why spider abundance increased from the farthest observed distance from
Allegheny ant mounds. In another study, Formica altipetens, was observed to decrease predatory spiders while mutualistic nymphs were present (Cushman & Whitham, 1989). As predator 18 abundance decreases near ant mounds then there is a higher chance of mutualistic interactions due to decreases in predator-prey interactions. A study by Nahas et al.,(2012) evaluated how spiders and ants would influence herbivory in Qualea multiflora, and they found that the presence of ants reduced the number and species richness of spiders. As predator abundance decreases near ant mounds then there is a higher chance of mutualistic interactions due to decreases in predator-prey interactions. Overall, this study raises more questions about the complex mutualistic and predator-prey interactions between that of the Allegheny mound ant, spiders, swamp cottonwoods and aphids in the Oak Openings region.
This study provides a deeper understanding on how Allegheny mounds ants interact in mutualistic relations and how they influence the surrounding vegetation in the Oak Openings region. Future efforts in the conservation of the federally endangered Karner Blue Butterfly
(Lycaeides Melissa samuelis) (KBB) and how they interact with ants that provide mutualistic benefits should be considered. In our study, we found that spider abundance significantly increased 7 meters away from Allegheny ant mounds compared to distances closer to the ant mounds. With spider abundance decreasing near ant mounds this could provide more of an enemy-free space for the KBB.
I recognize that my study couldn’t definitively answer how vegetation characteristics differ when Allegheny mound ants are present. I recommend that future studies compare the vegetation characteristics between oak savannah habitats occupied by Allegheny mound ants with those not occupied by Allegheny mound ants. More specifically, attention to canopy cover and the associations with ant mounds is warranted. We know, for example, that KBB females oviposit on lupine when there is a difference in canopy cover, specifically when moderate shade is present (Grundel et al. 1998). During the study Grundel et al. found that larval growth was 19 significantly faster on shade-growth lupine in comparison to sun-growth lupine. With canopy cover being an important factor in lupine quality and KBB larval growth rates, it is important to characterize how the potential Allegheny mound ant-KBB interaction can affect KBB habitat.
Future research has the potential to quantify the trade-offs between the direct interactions between the Allegheny mound ant and KBB, as well as the indirect relationships that may exist between the Allegheny mound ant, KBB, and the surrounding landscape.
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25 APPENDIX A: TABLES
Table 1. Results displaying general linear models (glm) in chapter 1, for each model, AIC,
Chisq, Df, Std. Error, and p-value will be displayed.
Std. GLM Models χ2 Df Error P KBB Number of Days to Pupation ~ Lupine Soil Temperature (°C) 0.117 1 ±0.094 0.733
~ Ant Presence 0.000 1 ±0.251 0.995
KBB Number of Days from Pupation to Butterfly
~ Lupine Soil Temperature (°C) 0.058 1 ±0.083 0.809 ~ Ant Presence 0.048 1 ±0.211 0.827 KBB Mortality ~ Lupine Soil Temperature (°C) 0.158 1 ±0.218 0.695 ~ Ant Presence 0.118 1 ±0.587 0.731
26
Table 2. Results displaying linear mixed effects models (lme) in chapter 1, for each model,
Chisq, Df, Std. Error, and p-value will be displayed. P-values in bold are significant (p<0.05).
LME Models χ2 Df Std. Error P Number of Ants Tending Larvae ~ Lupine Soil Temperature (°C) 0.706 1 ±0.025 0.401 Number of Ants in Mesocosms ~ Lupine Soil Temperature (°C) 0.863 1 ±0.259 0.294 KBB Vertical Placement (cm) ~ Lupine Soil Temperature (°C) 38.746 1 ± 0.09 <0.001 ~ Ant Presence 14.245 1 ±3.888 0.002 ~ Lupine Soil Temperature (°C):Ant Presence 9.144 1 ±0.117 0.003 KBB Vertical Placement (cm) ~ Lupine Temperature (°C) 27.551 1 ±0.134 <0.001 ~ Ant Presence 0.333 1 ±2.544 0.594 ~ Number of Lupine Leaves 1.898 1 ±0.025 0.205 ~ Lupine Soil Temperature (°C):Ant Presence:Number of Lupine Leaves 0.422 1 ±0.001 0.545 KBB Vertical Placement (cm) ~ Lupine Soil Temperature (°C) 13.515 1 ±0.139 0.002 ~ Ant Presence 2.039 1 ±4.009 0.198 ~ Height of Lupine (cm) 10.076 1 ±0.247 0.006 ~ Lupine Soil Temperature (°C):Ant Presence:Height of Lupine (cm) 4.423 1 ±0.009 0.058 Number of Leaves ~ Lupine Soil Temperature (°C) 0.48 1 ±1.018 0.508 ~ Ant Presence 0.393 1 ±51.895 0.554 ~ Lupine Soil Temperature (°C):Ant Presence 0.608 1 ±1.343 0.457 Height of Lupine (cm) ~ Lupine Soil Temperature (°C) 8.35 1 ±0.117 0.008 ~ Ant Presence 0.780 1 ±6.009 0.408 ~ Lupine Soil Temperature (°C):Ant Presence 0.143 1 ±0.154 0.718
27
Table 3. Results displaying linear models (lm) in chapter 2, for each model, Std. Error, t-value and p-value will be displayed. P-values in bold are significant (p<0.05).
Model X2 Std. Error P Number of Cottonwoods ~ Distance (m) -0.011 ±0.117 0.734 Number of Spiders ~ Distance (m) 0.052 ±0.045 0.024 Number of Ants ~ Distance (m) <0.001 ±1.709 0.325 Number of Crickets
~ Distance (m) -0.011 ±0.052 0.737 Number of Caterpillars
~ Distance (m) 0.034 ±0.008 0.056
Number of Grasshoppers ~ Distance (m) -0.008 ±0.026 0.562 Number of Moths ~ Distance (m) -0.008 ±0.008 0.528 Number of Leafhoppers ~ Distance (m) 0.006 ±0.019 0.228 Number of Beetles ~ Distance (m) -0.012 ±0.040 0.802 Number of Invertebrates ~ Distance (m) 0.019 ±0.078 0.113 Number of Ants ~ Number of Cottonwoods 0.489 ±1.180 <0.001 Number of Ants ~ Area -0.012 ±<0.001 0.805
28
Table 4. Results displaying linear mixed effects models (lme) in chapter 2, for each model,
Chisq, Df, Std. Error, and p-value will be displayed. P-values in bold are significant (p<0.05).
Model χ2 Df Std. Error P Vegetation Density
~ Area 4.949 466 ± <0.001 0.027 ~ Distance 1.445 466 ±0.019 0.232 ~ Area:Distance 0.680 466 ±<0.001 0.413 Number of Ants ~ Area 9.716 1891 ±<0.001 0.002 ~ Distance 11.274 1891 ±0.049 0.001 ~ Area:Distance 9.379 1891 ±<0.001 0.002
Number of Ants ~ Area 0.823 1732 ±<0.001 0.365 ~ Distance 112.970 1732 ±0.034 0.004 ~ Presence of Cottonwoods 8.603 1732 ±0.247 <0.001 ~ Area:Distance:Presence of Cottonwoods 17.334 1732 ±<0.001 <0.001 Number of Ants ~ Area 0.716 1890 ±<0.001 0.398 ~ Distance 820.506 1890 ±0.028 0.198
~ Presence of Aphids 1.665 1890 ±0.268 <0.001 0.003 ~ Area:Distance:Presence of Aphids 9.184 1890 ±<0.001
29
Table 5. Table is displaying average percentages of vegetation types around all ant mounds.
Vegetation Type Distance from ant mound Percentage
Shrub 1m 21-40% 3m 21-40% 5 m 21-40% 7 m 21-40% Average 21-40% Forb 1m 0-20% 3m 0-20% 5m 0-20% 7m 0-20% Average 0-20% Warm Season Grass 1m 0-20% 3m 0-20% 5m 0-20% 7m 0-20% Average 0-20% Cool Season Grass 1m 0-20% 3m 0-20% 5m 0-20% 7m 0-20% Average 0-20% Detritus 1m 0-20% 3m 0-20% 5m 0-20% 7m 0-20% Detritus 0-20% Bare Ground 1m 0-20% 3m 0-20% 5m 0-20% 7m 0-20% Average 0-20% Moss 1m 0-20% 3m 0-20% 5m 0-20% 7m 0-20% Average 0-20% 30
APPENDIX B: FIGURES
Warming Chamber
0.30 Meter
= 250-Wa< Infrared Light Bulb
= Blue Lupine 0.30 Meter
= KBB Caterpillar
= Tygon Tubing Tubing (2 m) Tygon
Allegheny Ants
Figure 1. Mesocosms contained one Blue lupine plant and one KBB larvae. Each mescosm randomly selected for warming will be warmed by a 250-Watt infrared light bulb hanging directly above the plant. Mesocosms were and randomly selected for Allegheny Mound Ant presence. 31
Allegheny Ant Mound
Mesocosm treatments:
• 0°C (ambient) • Heated • Ants present
Figure 2. The Allegheny ant mound was placed in the middle of 17 mesocosms. Four mesocosms were maintained at ambient temperature with absence of ants, five mesocosms were maintained at ambient temperature with presence of ants, four mesocosms were heated to ~ 1°C-
2°C above ambient temperature with absence of ants, four mesocosms were heated to ~ 1°C-2°C above ambient temperature with presence of ants. 32 A B
C
Figure 3. Relationship between ant presence and number of days KBB Lavaeachieved pupation
(A). Relationship between ant presence and number of days KBB larvae were pupating (B).
Relationship between ant presence and the vertical height (cm) of KBB larvae (C). All error bars represent standard error. 33
A B
(°C) (°C)
Figure 4. Relationship between temperature of lupine soil (°C) and number of days KBB larvae achieved pupation (A). The relationship between temperature of lupine soil (°C) and number of days KBB larvae were pupating (B). 34
Figure 5. The relationship mortality and the average temperature of lupine soil (°C). All error bars represent standard error. 35
A B
(°C) (°C)
Figure 6. Temperature of lupine soil (°C) and number of ants tending larvae (A) and number of ants in each mesocosm (B). 36
A
(°C) B
(°C)
Figure 7. The relationship between larvae vertical placement (cm) and temperature of lupine soil
(°C) with ant presence (A). The relationship between larvae vertical placement (cm) and temperature of lupine soil (°C) without ant presence (B). Grey shadowed region indicates the
95% confidence interval. 37
N: 7 M
N: 5 M = Ant Mound
= 1 Meter N: 3 M
= Data Sampling Location N: 1 M E: 1 M E: 3 M E: 5 M E: 7 M W: 7 M W: 3 M W: 5 M W: 1 M
S: 1 M
S: 3 M
S: 5 M
S: 7 M
Figure 8. Visible representation of the 16 sampling locations at each Allegheny ant mound. 38 A B
C D
Figure 9. The interaction between distance (m) away from each mound and the total number of cottonwoods observed (A). The interaction between distance (m) away from each mound and the total number of spiders (B). The interaction between distance (m) away from each mound and the total number of ants observed (C). The interaction between distance (m) away from each mound and the visual obstruction reading observed (D). All error bars represent standard error.