PERROTTI-DISSERTATION-2018.Pdf (2.953Mb)

PALYNOLOGICAL EVIDENCE FOR TERMINAL PLEISTOCENE

PALEOENVIRONMENTAL CHANGE AT TWO SITES IN THE SOUTHEASTERN

UNITED STATES

A Dissertation

ANGELINA GIOVANNA PERROTTI

Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Chair of Committee, Vaughn M. Bryant Co-chair of Committee, Michael Waters Committee Members, Kelly Graf Fred Smeins Head of Department, Cynthia Werner

May 2018

Major Subject: Anthropology

ABSTRACT

Researchers interested in late-Pleistocene extinctions continue to debate the potential drivers of this event and, at times, do not even agree on the timing of extinction for various species in different regions. This dissertation presents two studies of the timing of megaherbivore disappearance and the effects of this process on local vegetation at two sites in the southeastern United States. This research uses two relatively novel methods that required extensive exploration and testing. Chapter II highlights an effective method for the removal of tiny, unwanted organic material from pollen samples. This method, sonication-assisted sieving, proved useful for eliminating such debris from organic-rich sediments. Chapter III describes the opportunities and limitations of using dung fungal spores as a proxy for herbivore abundance and extinction. Sustained interest in the timing of megaherbivore extinction in regions with poor organic preservation has led to a rapid increase in the use of dung fungi as a proxy for large herbivore abundance in archaeology and paleoecology. This chapter synthesizes modern mycological literature and suggests that there are many factors involved in the formation of a dung fungal spore record.

Chapter IV presents the results of a palynological study of terminal Pleistocene sediments in two cores from the Page-Ladson archaeological site, Florida. The disappearance of

Sporormiella, a proxy for megaherbivore abundance, by ~12,700 cal BP is consistent with the timing of terminal Pleistocene megafaunal extinction elsewhere in North America.

Pollen evidence from the site also reflects dramatic vegetation changes, which are likely a response to both changing climate and fluctuating herbivore populations. Chapter V demonstrates the utility of fungal spores as paleoenvironmental proxies at White Pond,

South Carolina. White Pond is a small pond located at the intersection of the Coastal Plain and the Piedmont in the southeastern United States. The data from White Pond also has implications for understanding the best application of dung fungal spore data as a proxy for large herbivore abundance. Overall, this dissertation research reflects the utility of applying innovative methods to palynology and archaeology.

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DEDICATION

To my Mother, whose endless sacrifice enabled me to pursue my dreams.

ACKNOWLEDGEMENTS

I extend my deepest gratitude to my advisor, mentor, and friend, Vaughn M.

Bryant. Dr. Bryant: thank you for always taking the time to provide me with a lifelong example of what it means to be a good mentor. I hope I can even begin to reflect your commitment to dedicated mentorship and superb scholarship.

Thank you to my dissertation co-chair, Michael Waters, for presenting me with the opportunity to learn about Sporormiella at Page-Ladson and for all of the guidance along the way. I’d also like to thank my other committee members, Kelly Graf and Fred Smeins.

Without them and their expertise on vegetation and environmental archaeology, this dissertation research would not have been possible.

Obtaining a Ph.D. in Anthropology from Texas A&M University was about so much more than just scholarship. I treasure the lifelong friendships I have cultivated here.

Thank you to all of my friends I have made along the way, both those mentioned here and those who remain unmentioned. Specifically, I would not have been a functional human without my dearest friends, Katie Bailey, Crystal Dozier and Elanor Sonderman. Thank you for the endless emotional support, laughs, and the occasional lunch beer. Thank you also to my lab mates, Chase Beck, Tony Taylor, Katelyn McDonough, Rossana Parades,

Mary Katherine Bryant, and Taylor Siskind. To have such a positive and caring working environment is truly special. In particular, I appreciate the opportunity I was given to mentor Taylor Siskind. One of my proudest achievements was the research we did together over the past three years. Taylor, I am honored to be your friend and couldn’t have wished for a better undergraduate mentee.

I am endlessly indebted to the Department of Anthropology and the people who keep it going. I deeply appreciate our department head, Cynthia Werner, for constantly demonstrating her care for maintaining a positive environment for graduate students.

Thank you to Rebekah and Clint, for all you do to keep the department functioning. And to

Nicole, thank you for your commitment to all of your administrative duties, but more, for your friendship. My time spent in your office was always my favorite part of my day.

I greatly appreciate the following people and who provided invaluable guidance and advice on pedagogy, research design and execution, and manuscript preparation:

Catharina Laporte, Christopher R. Moore, David Carlson, Cortland Eble, Jen O’keefe,

Jessi Halligan, Morgan Smith and Angela Gore. Specifically regarding the Page-Ladson project, I’d like to thank J. and B. Ladson and the Ladson family for site access and assistance with the project; E. Green, T. and B. Pertierra, J. Simpson, and S. Ellison for fieldwork, equipment, and logistics support; LacCore for core storage, analysis, and assistance; Capital Rubber, University of Wisconsin–La Crosse, Wakulla Dive Center, the

Center for Maritime Archaeology and Conservation at Texas A&M University, and Florida

Bureau of Archaeological Research Underwater Archaeology Division for field equipment and technical support. Thank you to Michael Waters, John Albertson, Shawn Joy, Morgan

Smith, Dave Thulman, Grayal Farr, Neil Puckett, Brendan Fenerty and Jessi Halligan for collecting the cores.

Thank you to everyone who organized conferences and workshops at which I networked and learned from the best in the field. I also extend my gratitude toward the journals that let me use Chapters III and IV in this dissertation, which are both published papers in Palynology and Quaternary International, respectively.

I would like to extend my endless gratitude to my family. From financial to moral support, I would not have been able to do it without them. In particular, to my mom: what can I say to a woman who worked three jobs in hopes that her children could achieve more and who never missed an opportunity to support me? You have shown me what it means to be a strong woman. You have been my longest running and most dedicated cheerleader.

You have shared in all of my sorrows and triumphs, and I can never thank you enough.

And finally, thank you to my husband, Ben: I am the luckiest woman in the world to have a husband who supports my dreams, takes interest in my research, and accompanies me to academic social events and “talks shop” with my colleagues- even when it ends up being about the types of fungi that grow on various animal turds. Last but not least, thank you for following me wherever my career might take me. I look forward to a lifetime of adventure with you.

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CONTRIBUTORS AND FUNDING SOURCES

Contributors

This work was supervised by a dissertation committee consisting of Professor

Vaughn Bryant (co-advisor), Professor Michael Waters (co-advisor), and Professor Kelly

Graf of the Anthropology Department, and Professor Fred Smeins of the Ecosystem

Science and Management Department.

Funding Sources

The research in Chapter II was financially supported by the Palynology Lab at

Texas A&M University.

Chapter IV was financially supported by Elfrieda Frank Foundation; the North Star

Archaeological Research Program and the Chair in First Americans Studies at Texas A&M

University; the University of Wisconsin La Crosse; National Geographic Waitt Foundation grant W224-12; Geological Society of America (GSA) graduate research grant 10445-14; the Society for American Archaeology Geoarchaeology Interest Group MA/MS Research

Award; the Texas A&M Department of Anthropology; and the Claude C. Albritton Jr.

Award of the Archaeological Geology division of the GSA.

Chapter V was financially supported by the South Carolina Department of Natural

Resources, the University of South Carolina, and the Texas A&M Department of

Anthropology.

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TABLE OF CONTENTS

Page

ABSTRACT ...... ii

DEDICATION ...... iv

ACKNOWLEDGEMENTS ...... v

CONTRIBUTORS AND FUNDING SOURCES ...... viii

TABLE OF CONTENTS ...... ix

LIST OF FIGURES ...... xi

LIST OF TABLES ...... xv

CHAPTER I INTRODUCTION AND LITERATURE REVIEW ...... 1

I.1 Overview ...... 1 I.2 Background ...... 2 I.3 Research Objectives ...... 6 I.4 Study Site Descriptions ...... 9 I.5 References ...... 14

CHAPTER II EFFICACY OF SONICATION-ASSISTED SIEVING ON QUATERNARY POLLEN SAMPLES ...... 23

II.1 Introduction ...... 23 II.2 Methods ...... 28 II.5 Conclusions ...... 40 II.6 References ...... 42

CHAPTER III DUNG FUNGI AS A PROXY FOR LARGER HEBIVORE ABUNDANCE: CONSIDERATIONS AND LIMITATIONS FOR ARCHAEOLOGICAL APPLICATION ...... 44

III.1 Introduction ...... 44 III.2 Applications for Archaeologists ...... 45 III.3 Opportunities and Limitations ...... 48 III.4 Future Directions and Conclusions ...... 63 III.5 References ...... 65

CHAPTER IV POLLEN AND SPORORMIELLA EVIDENCE FOR TERMINAL PLEISTOCENE VEGETATION CHANGE AND MEGAFAUNAL EXTINCTION AT PAGE-LADSON, FLORIDA ...... 77

IV.1 Introduction ...... 77 IV.2 Study Site ...... 84 IV.3 Methods ...... 87 IV.5 Discussion ...... 98 IV.6 Conclusion ...... 107 IV.7 References ...... 108

CHAPTER V COPROPHILOUS FUNGAL SPORE ACCUMULATION AT WHITE POND, SOUTH CAROLINA ...... 118

V.1 Background ...... 118 V.2 Palynological Methods ...... 121 V.3 Results ...... 124 V.4 Analysis and Discussion ...... 126 V.5 Conclusions and Future Research ...... 131 V.6 References ...... 132

CHAPTER VI CONCLUSIONS...... 136

APPENDIX PHOTOS OF COMMON PALYNOMORPHS IN THIS DISSERTATION RESEARCH ...... 140

LIST OF FIGURES

Page

Figure 1. Bryant and Holloway (2009) charcoal removal method (left) compared with the method detailed in this paper (right). We used a Branson© S450 sonifying disruptor horn in sample receptacle and plastic screw top base with a 10- micron mesh bottom and adjacent sample cup. Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis...... 26

Figure 2. Charcoal in a pollen sample before (top) and after (bottom) sonication at output setting 2 for three minutes. Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis...... 27

Figure 3. Branson S450 sonifying disruptor horn potential amplitude by output settings. Reprinted with permission from Perrotti et al. (2018), Palynology. Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis...... 32

Figure 4. Degradation classification system used in this study. Modified from Campbell and Campbell (1994). Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis...... 35

Figure 5. Class 2 and 3 pollen degradation of most fragile taxa after 3 minutes of sonication at increasing output settings, plotted against primary axis. 50 grains from each taxon were counted in this experiment. Black dashed line represents <10 micron debris removed from archaeological sediment sample after 3 minutes of sonication at increasing output settings, plotted on secondary axis. Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis...... 36

Figure 7. Class 2 and 3 pollen degradation of most fragile taxa after increased sonication duration at output setting 2, plotted against primary axis. 50 grains from each taxon were counted in this experiment. Black dashed line is <10 micron debris removed from archaeological sediment sample after sonication output setting 2 for increasing duration, plotted on secondary axis. Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis...... 38

Figure 8. Photos of each degradation class for the six most fragile pollen taxa presented in this study. Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis...... 41

Figure 9. Generalized lifecycle of Sporormiella...... 50

Figure 10. Sporormiella counts from the Page-Ladson site demonstrate how different ways of quantifying the fungal spores can change their representation...... 61

Figure 11. Map with Florida pollen studies discussed in the text. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd...... 79

Figure 12. Composite of cores 4A (left) and 3A (right) with uncalibrated AMS dates. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page- Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd...... 81

Figure 13. Spores of Sporormiella spp. from the Page-Ladson site. Note the variability in slit curvature, an important character in identifying Sporormiella. Scale bars are 10 microns. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd...... 83

Figure 14. Bayesian age model for Core 4A. Reprinted from with permission from Halligan et al. (2016). Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd...... 90 xii

Figure 15. Sporormiella concentration, accumulation and percentage of total pollen assemblage from Cores 4A and 3A, alongside pollen concentration and relative abundance of arboreal and herbaceous pollen types, with zones determined through visual inspection. Some of the areas of the core containing sediments aged ~15,000-13,000 cal BP with low Sporormiella accumulation rates coincide with relatively low pollen concentration values. Pollen concentration is also moderate to high during periods of Sporormiella absence (~12,700-10,750 cal BP and after 10,200 cal BP), suggesting the lack of Sporormiella during these periods is not a consequence of poor preservation. Measures of spore influx and concentration are vulnerable to uneven pollen preservation within a sedimentary record, because the conditions that degrade pollen may also degrade spores. As shown, the percentage diagram is not susceptible to this effect. However, there are still some areas of the Zone 1 that exhibit low Sporormiella abundance. It is possible that these represent times with relatively low megaherbivore abundance near the site. The consistent decline and disappearance of Sporormiella by the end of this zone (12,700 cal BP) likely signifies the disappearance of Pleistocene megaherbivores from the site. “Arboreal” includes pollen from trees and from taxa associated with mesic environments (such as Vitis). “Aboreal” also excludes Pinus pollen, which was probably transported to the site rather than locally present. This indicates a relative abundance of mesic forest vegetation until ~12,500 cal BP. After 12,500 cal BP, the vegetation at the site reflects a warmer, drier period resulting fewer trees and more open space. After 10,500 cal BP, it appears that conditions became even drier and warmer based on the dramatic increase in herbaceous pollen at the expense of arboreal types. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page- Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd...... 92

Figure 16. Selected important arboreal pollen types from Core 4A by modeled calibrated dates BP, with zones determined through visual inspection. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page- Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd...... 95

Figure 17. Selected important herbaceous taxa pollen from Core 4A, with zones determined through visual inspection. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd...... 96

Figure 18. White Pond, South Carolina...... 121

Figure 19. Bayesian age-depth model for White Pond, South Carolina (Bronk Ramsey, 2006) ...... 123

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Figure 20. Spore concentrations plotted by modelled year BP. Brown graphs indicate strongly coprophilous taxa. Green indicates fungi that do not strongly prefer dung as substrate. Zones are represented by black lines. Note scale change on the two “total spore” graphs...... 125

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LIST OF TABLES

...... Page

Table 1. Morphological characteristics of pollen types in this study...... 29

Table 2. Preferred substrate of commonly used dung fungal taxa...... 49

Table 3. AMS Dates from Cores 3A and 4A. Dates were calibrated using Calib 7.1...... 80

Table 4. Sporormiella zones from Page-Ladson site cores 3A and 4A, as discussed in text (Halligan et al. 2016)...... 89

Table 5. Dung fungi spore types in this study...... 119

Table 6. Generalized patterns from Watts (1980)...... 127

Table 7. Generalized dung fungal spore patterns from Core WP-2016-3...... 128

CHAPTER I

INTRODUCTION AND LITERATURE REVIEW

I.1 Overview

Despite decades of debate, the causes and effects of the extinction of over 35 large mammal species in Late Pleistocene North America remain ambiguous. This quandary exists primarily because environmental context of extinctions including climate and vegetation change is uncertain, especially in the southeastern United States. Potential causes include rapid overkill by humans, climate change, an extraterrestrial impact, hyperdisease, or some combination of these factors. Potential effects of megafaunal extinction include a proliferation of woody vegetation and increased fire frequency and intensity. However, research has yet to properly investigate such relationships in many parts of the Southeast at this time primarily due to a lack of high-resolution paleoenvironmental studies with reliable chronological control.

The objectives of this dissertation are threefold. First, I seek to pursue the best method for incorporating coprophilous fungal spores into more traditional palynological research. These spores are from fungi that strongly prefer dung as a substrate. They have been used over the past three decades with increasing frequency as a proxy for large herbivore abundance. However, many uncertainties exist surrounding the methods and applications for this data. Consequently, Chapter II of this dissertation is dedicated to better understanding the formation of the dung fungal record, and outlines applications and considerations for the use of this method. Second, Chapter III of this dissertation proposes a novel method for the extraction of fossil palynomorphs from sediments with high levels

of tiny organic debris. This became necessary because sediments from the two study sites in this dissertation contain high levels of organic debris that obscured pollen grains. Third, this dissertation research seeks to better understand ancient plant and herbivore communities in the southeastern United States, using case studies in northwestern Florida

(Chapter IV) and central South Carolina (Chapter B). To achieve this, I tested the central hypothesis that although vegetation was changing at the end of the Pleistocene due to warming climate with increased precipitation, the disappearance of large mammals intensified vegetation changes and increased the cover of woody vegetation in the

Southeast. I tested my central hypotheses by completing the following specific studies.

First, in Chapter IV I investigated the timing of herbivore extinction and terminal

Pleistocene vegetation change in northwestern Florida using dung fungal and pollen evidence from Page-Ladson. Second, in Chapter V, I analyzed dung fungal spores from a sediment core from White Pond, South Carolina to learn about the timing and process of terminal Pleistocene megafaunal extinction. These two studies are novel because they represent some of the best temporally-constrained terminal Pleistocene sedimentary records recovered from the Southeast.

I.2 Background

I.2.1 Theoretical Background of Extinctions

Disagreement over the causes of extinction concerns the relative impacts of human hunting (Martin, 1984; 2005; Alroy, 2001; Surovell and Waguespack, 2009; Abramson, et al. 2015; Araujo, et al. 2015; Frank et al., 2015; Surovell et al., 2016; Saltrè et al., 2016), climate change (Guthrie, 1984), disease (MacPhee and Marx, 1997) and a potential extraterrestrial impact (Firestone, 2007) on Pleistocene megafauna. Anthropologists are

particularly interested in this topic, because it has implications for understanding the sweeping effects that humans have on ecosystems. Anthropologists who argue that humans are responsible for the disappearance of megafauna cite examples of widespread, rapid extinctions on islands such as Madagascar and Australia directly after human colonization

(Burney, 2003; Burney and Flannery, 2005). Additionally, proponents of the overkill hypothesis point out that Late Pleistocene-aged Clovis technology appears to be specialized for hunting large game, and many archaeological sites contain Clovis tools in direct association with disarticulated and sometimes clearly butchered megafauna

(Surovell and Waguespack, 2009). Still others assert that the extinction of Pleistocene megafauna occurred so quickly, it was effectively geologically simultaneous (Faith and

Surovell, 2009; Surovell, 2016), implying a new external pressure such as exposure to human hunting.

Those who argue that climate was the driving cause do not readily accept that extinctions occurred so rapidly (Grayson, 2001; 2007; Grayson and Meltzer, 2002; 2003).

They emphasize climate change at the end of the Pleistocene and the effects that those changes would have had on animal communities by relegating them to small refugia of glacial vegetation. Conditions were variable across North America but were generally warming after the Last Glacial Maximum (26,000-20,000 cal BP). After the Bølling-

Allerød warm period, a reversal to cool, dry, glacial conditions (the Younger Dryas from

~12,900-11,700 cal BP) coincided with many of the last known occurrences of extinct fauna. Although paleoclimatic records for northwestern Florida are poor, records from south-central Florida document that the Younger Dryas brought an increase in precipitation along with the cooler temperatures (Grimm et al., 2006), as opposed to dry conditions

faced by the rest of North America. Recent research suggests that although climate change may have intensified the effects of human predation on megafauna, human hunting was the driving factor of extinction (Haynes, 2013; Araujo et al., 2015).

It is usually the causes of megafauna extinction that remain at the center of most archaeological research on the topic. However, recent discussions of the effects of megafauna extinction center around topics such as the impact of megaherbivore extinction on woody vegetation (Gill, 2014; Bakker et al., 2015), or effects on the global methane cycle (Smith et al., 2015). Large herbivores typically inhibit woody vegetation growth, so their disappearance may have caused an overgrowth of woody vegetation, increasing fuel for fires (Bakker et al., 2015). Particular species, such as gourds (Cucurbita sp.), may also have evolved after the extinction of megafauna, further changing plant communities

(Kistlera et al., 2015). In addition, Smith et al. (2015) suggest that the extinction of large herds of herbivores and the subsequent cessation of a great amount of dung production and deposition would have significantly reduced the release of methane, a heat-trapping greenhouse gas, into the atmosphere. This could have potentially contributed to the cooling climate of the Younger Dryas.

I.2.2 Pollen Reconstructions

This dissertation research uses pollen as proxy evidence for ancient vegetation assemblages. Pollen extracted from sediment samples can be used to reconstruct past vegetation because every plant species produces a unique pollen grain, and these pollen grains can be preserved for thousands of years. However, it is important to consider the relative pollen production of different plants. For example, Pinus (pine) and Quercus (oak) produce a large quantity of pollen grains that are often carried in the wind and deposited

far from their source. Therefore, they can be overrepresented in the pollen record. In contrast, certain plants such as Tilia (basswood) produce very few pollen grains, and the presence of that pollen indicates that it was a significant part of the ancient plant assemblage. Thus, it is important to understand the geography of the area and the ecological relationships between different plants to determine if sediment-sample pollen signatures truly reflect plant communities. After investigating these factors, if there is still doubt, additional analyses can be employed such as analyzing macrofossils, stomata, or phytoliths, which can strengthen pollen interpretations.

I.2.3 Coprophilous fungi in paleoecology

As previously mentioned, the timing of regional decline and disappearance of some species remains ambiguous. The faunal record is generally sparse, and entire regions in

North America harbor such poor preservational environments that there is virtually no record of extinct megfauna (Grayson, 2007). Even with each new discovery of additional megafaunal sites, it is unlikely that we will find or recognize the last existing member of any extinct species.

Therefore, the development of a proxy for large herbivore abundance was welcomed. Owen Davis (1987) first suggested using the fossil spores of the coprophilous fungus (used interchangeable with “dung fungus”), Sporormiella, as a proxy for

Pleistocene herbivore abundance. Davis (1977, 1987) recognized the potential of coprophilous fungi in paleoecology when he found Sporormiella spores in direct association with fossilized dung from Bechan Cave, Utah.

Some researchers have even suggested that declines in dung fungi can indicate human colonization, citing evidence that extinction events worldwide have followed

human settlement into a new area (Burney, 2003; Burney and Flannery, 2005; Gill, 2014).

However, Fiedel (2016) refutes that due to problems in both cultural chronology and inconsistencies between the dung fungi and faunal records. For example, declines in

Sporormiella observed in sediments younger than ~14,800 cal BP from Ohio and northern

Indiana (Gill et al., 2009; 2012) and New York (Robinson et al., 2005; Robinson and

Burney, 2008) were interpreted as demonstrating a sharp decline in megafaunal abundance, followed by extinction or near extinction within 1000 years. However, Fiedel (2016) points out that a number of megafaunal remains were recovered from the Northeastern United

States dating between 10,000 and 12,000 rcybp (~12,000-14,000 cal BP, suggesting the use of Sporormiella as a proxy for sharp decline in megafaunal abundance in this case is problematic.

Despite disparities such as this, in the nearly three decades that Sporormiella and other dung fungi have been used as proxy evidence for megafauna extinctions (Burney and

Flannery, 2005; Davis, 1987; Davis and Shafer, 2006; Gill et al., 2009), methodologies have become increasingly reliable, and dung fungi have been demonstrated to reflect relative large herbivore abundance and activity in many modern settings (Baker, 2016; Gill et al., 2013; Parker and Williams, 2012; Raper and Bush. 2009).

I.3 Research Objectives

The objective of this dissertation research is to understand the interactions between climate, vegetation and megafaunal extinction to the changing Late Quaternary plant communities in northwestern Florida. To further evaluate megafaunal extinction and environmental change in the southeastern United States, specific aims of this dissertation are to: (1) investigate the timing of herbivore extinction in northwestern Florida and

central South Carolina; (2) reconstruct the past vegetation assemblage by completing a pollen analysis on the same sediment core used for spore data from Page-Ladson, Florida.

To answer these questions, I tested the following hypotheses:

H1: Large herbivores disappeared from the Aucilla River region of Northern Florida by

about 12,600 cal BP.

H2: Climate became warmer and drier after the Pleistocene, leading to Aucilla River plant

communities shifting from a mesic hardwood forest toward the end of the Pleistocene,

to a xeric, disturbed oak woodland or savannah in the Holocene (after 12,000 cal BP).

H3: Megaherbivores played an important role in ecosystem regulation, so plant

communities will change after their disappearance.

H4: A similar pattern of megafaunal disappearance and vegetation change occurred at

White Pond, South Carolina.

In lieu of a traditional dissertation, I chose to prepare three papers for publication, with the addition of a research report, which will be used in a manuscript for publication after some additional research. The central questions driving this dissertation research surround the relationships between climate, plants, megaherbivores, and humans in the southeastern United States. However, this research used two different cutting-edge methods, which required further scientific exploration regarding the reliability and applications of each method. Therefore, Chapters II and III are dedicated to the further assessment of each of these methods.

Chapter II consists of a paper in press in the journal Palynology (Perrotti et al., in press). This paper, co-authored by Taylor Siskind, Mary Katherine Bryant, and Vaughn

Bryant, presents a novel method for the removal of tiny, unwanted organic debris in pollen

samples that can obscure pollen grains. These tiny debris are often similar size, physical consistency, and specific gravity as pollen, meaning they are not easily removed using traditional palynological techniques such as chemical maceration and mechanical separation. This method, sonication-assisted sieving, was demonstrated to be a useful method for debris removal, which I then used in my subsequent studies.

Chapter III is a paper in review for publication in the journal Vegetation History and Archaeobotany (Perrotti and Van Asperen, in review). This paper, co-authored by

Eline Van Asperen, reviews the current understanding of the use of dung fungal spores as a proxy for large herbivore abundance. This paper should prove particularly useful to those interested in the topic because it reviews the modern mycological literature and presents many environmental factors that may influence the formation of the dung fungal record.

The methods described in Chapters II and III enabled the exploration of the central research questions driving this dissertation research, detailed in chapters IV and V. First, chapter 4 presents the results of a pollen and dung fungal spore analysis at the archaeological site Page-Ladson, Florida (Perrotti, 2017). This is an original research paper published in the journal Quaternary International. This paper demonstrates that the

Pleistocene-Holocene transition brought about many drastic environmental changes in northwestern Florida, including fluctuating herbivore populations and transformation in the composition of vegetation communities.

Chapter V reports the results of a more comprehensive dung fungal analysis from

White Pond, South Carolina. This report will be a part of a larger publication, exploring the archaeology of the site. In addition, this site, which was originally the subject of a

pollen analysis by Watts in 1980, will be revisited for a more thorough, well-constrained palynological study.

Chapter VI concludes this dissertation by reiterating the conclusions drawn in each chapter. In addition, it demonstrates how this research produces a more comprehensive understanding of megafaunal extinctions and how methods from microscopic palynomorphs address the research objectives of this dissertation.

I.4 Study Site Descriptions

I.4.1 Page-Ladson, Florida (Chapter IV)

Page-Ladson, a submerged Paleoindian site in a sinkhole in the Aucilla River, northwestern Florida. Generally, submerged sites yield excellent preservation of plant materials because cycles of wetting and drying can destroy palynomorphs very quickly

(Campbell and Campbell, 1994). Thus, pollen and fungal spores within the Aucilla River are generally well preserved, as they have remained wet for almost the entire time since deposition. In addition, evidence of early occupation of the site (Halligan et al., 2016) reinforces the relativity of paleoecological information to archaeology, as we can be sure that the environmental reconstruction will provide information about what environmental conditions the first colonizers of the area actually faced.

Page-Ladson has long been the subject of archaeological research. James Dunbar and S. David Webb formed the Aucilla River Prehistory Project in the late 1990s to explore archaeological and paleontological sites within the Aucilla River system. These excavations revealed evidence of early human occupation at the Page-Ladson site. Early cultural material discovered during initial excavations at Page-Ladson include a mastodon tusk with cut marks and eleven lithic artifacts that have been confidently dated to older

than 13,950 cal BP (Dunbar, 2002; Dunbar, 2006; Webb, 2006). Additionally, thick mats of Mastodon digesta alongside Mastodon skeletal remains confirmed megafaunal activity at the site (Webb, 2006). The region is now under investigation by Michael Waters of the

Center for the Study of First Americans (CSFA) at Texas A&M University (TAMU) and

Jessi Halligan of Florida State University. These recent excavations recovered six more lithic artifacts from early contexts, including a biface, securely dated to ~14,550 cal BP

(Halligan et al., 2016).

Fluvial systems are not typically selected for palynological analyses; however, the sediments in many of the sinkholes of the Aucilla River are unique and reflect more of a ponding environment with episodes of colluvial sedimentation. Additionally, the superb preservation of plant material has allowed for the development of tight chronological control and thorough geoarchaeological investigations (Halligan, 2012). A sediment core was collected in 2015 using a modified Livingstone coring device, and is curated at the

LacCore facility at The University of Minnesota. This core demonstrates stratigraphic continuity, preservation of organic materials, and represents the sedimentary units from the

Pleistocene-Holocene transition, based on previous geoarchaeological investigations

(Halligan, 2012).

Few Late Pleistocene pollen records exist from the southeastern United States, particularly in Florida, because most lakes in the region became desiccated at some point in the Holocene. The pollen records that do exist lack the precision and resolution that the sinkhole deposits in the Aucilla River provides. The sedimentary record at Page-Ladson has a deep, continuous record with solid chronological control due to the collection of over

100 radiocarbon dates. The Aucilla River sediment sequences provide an unprecedented

opportunity to establish a high resolution, chronologically precise paleoenvironmental record.

I.4.2 Florida Environmental Chronology

The present day southeastern United States is primarily characterized by deciduous hardwood forests. Northwestern Florida is home to a warm mixed forest biome, interspersed with inland freshwater swamps and coastal salt-water swamps (Williams et al., 2004). However, climatic conditions were quite different in the Late Pleistocene. In general, the vegetation of Late Glacial Florida reflected a relatively dry, cool climate. For example, pollen and macrofossils from the first Page-Ladson pollen study record a dry forest prairie or savanna prior to 14,000 BP (Hansen, 2006). At this site, the pollen assemblage in deposits dated to 14,800-14,000 reflects a dominance of chenopods, grass, and drier arboreal taxa such as oak. A pine forest surrounded Camel Pond, sixty miles northwest of Page-Ladson, until 14,000 BP (Watts et al., 1992). Lake Sheelar, 130 miles southeast of Page-Ladson records pine and herb abundance, suggesting a cool climate until

14,600 BP, prior to the expansion of a mesic forest (Watts and Stuiver, 1980). In south- central Florida, both Lake Tulane and Lake Annie pollen sequences reflect a dry climate until at least 13,000 B.P. (Watts and Hansen, 1988; 1994). At Lake Annie, dry conditions are represented by a dominance of Ceratiola (rosemary) and Polygonella (jointweed) until

13,000 BP (Watts, 1975). Lake Tulane, however, may have received more precipitation than Lake Annie. From 13,700-10,900 BP, an open forest dominated by oak, hickory, juniper, red cedar, elm and ragweed surrounded Lake Tulane (Watts and Hansen, 1988).

The terminal Pleistocene in Florida brought about a general shift to more forested conditions across the majority of the state. By 12,500 BP at Page-Ladson, mesic forest and

floodplain trees such as maple, basswood and cypress increased. In addition, ragweed declined and disappeared from the pollen record by this time (Hansen, 2006). Camel pond saw an influx of spruce, which is representative of cool, moist conditions between 14,000-

12,600 BP (Watts et al., 1992). In addition, grass and ragweed decreased at this time, further indicating an increase in forest density. To the south, the Lake Sheelar pollen assemblage also reflects this shift, as a mesic forest emerged from 14,600-11,200 BP

(Watts and Stuiver, 1980). Lake Annie also saw evidence of increasing precipitation and had increased levels of oak after 13,000 BP. Watts (1975) suggests that the dominance of oak and ragweed at Lake Annie after 13,000 BP represents an upland oak and prairie scrubland, a vegetation community with no modern analog.

Mesic forests declined throughout Florida after the end of the Pleistocene, but the timing may be variable across the region. By 10,500 BP at Page-Ladson, mesic and floodplain trees were widely replaced by chenopods and Asteraceae, reflective of a warmer, drier, more open environment (Hansen, 2006). Spruce disappeared at Camel Pond prior to 12,600 BP, alongside expanding oak, beech and ironwood, suggesting warmer temperatures. Conditions warmed even more causing a hiatus in deposition at this site between 10,500-7,800 BP (Watts et al., 1992). Pollen records to the south reflect a similar pattern. Lake Sheelar pollen reflects an influx of grass, chenopods and ragweed at the expense of mesic forest taxa from 11,200-9,500 BP (Watts and Stuiver, 1980). Little Salt

Spring, also in peninsular Florida, shows evidence of dry conditions prior to 9,000 BP

(Brown and Cohen, 1985).

I.4.3 White Pond (Chapter V)

White Pond is a natural lake about 500 meters in diameter, located 40 kilometers northeast of Columbia, South Carolina. This lake is at the intersection of the Coastal Plain and the Piedmont. The current vegetation consists almost entirely of a commerically operated pine plantation, so the natural modern vegetation is largely unknown. This site has been subject to a pollen study in 1980 by Watts, but updated research is needed. Watts’

(1980) study divided the vegetation history at White Pond into three zones. First, from

19,100 BP-12,810 BP, the vegetation primarily consisted of pine, spruce, and various herbs, reflecting a cool, moist climate. After 12,810 BP, the vegetation shifted to reflect a warming climate. From 12,810 BP-9,500 BP, the vegetation was characterized by a decline of pine and a total disappearance of Picea. At this time, oak and other hardwoods encroached, forming a mesic hardwood forest. After 9,550 BP, vegetation reflected a drier climate. At this time, pine dominated the pollen assemblage alongside evidence of some patches of open prairie in the forest.

White Pond is an ideal location to extend understanding of the interactions of climate, plants and animals beyond northwestern Florida. Not only does this site demonstrate potential for the recovery of palynomorphs (Watts, 1980), but it also can provide a more regional look at the patterns of megafaunal extinction. Alongside the work at Page-Ladson, this research can help to inform the current relationships between warming climate, plants, animals, and humans.

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CHAPTER II

EFFICACY OF SONICATION-ASSISTED SIEVING ON QUATERNARY POLLEN

SAMPLES*

II.1 Introduction

Pollen studies of archaeological sites are recognized for their value in providing information about both the paleoenvironment and human activities. The nature of these archaeological pollen samples often requires the implementation of additional laboratory dispensation techniques to isolate pollen from unwanted organic debris. For example, many archaeologists are concerned with pollen in features such as hearths or floors, both of which can contain large amounts of charcoal. In these samples, carbon, in the form of charcoal flecks is inert and does not react with acids typically used to remove debris during pollen processing. These charcoal pieces can be abundant, and are often the same size as pollen grains. The overabundance of charcoal flecks in a sample can obscure pollen grains, making analysis difficult.

Using heavy density separation techniques can be effective in removing charcoal

(Bryant and Holloway, 2009), but is time consuming and requires the use of costly chemicals. Bryant and Holloway (2009) suggested using a sonifier to remove unwanted charcoal from archaeological pollen samples after attempting to sieve charcoal-rich archaeological samples through a 10-micron screen. After experimentation using mesh

* Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis. 23

screens with openings of five to ten microns in diameter, Bryant and Holloway (2009) found that the screen openings were too small, and the sample needed to be frequently agitated in order to facilitate its passing through the screen. They used a sonicating disruptor horn against the rim of a metal 850 mesh sieve (10 microns) to agitate the sample

(i.e. Figure 1). The sonicating disruptor horn emits ultrasonic vibrations to cause liquid sample agitation.

Sonication is commonly used in fossil pollen extraction (Caratini. 1980; Batten and

Morrison, 1983; Vaissiere, 1991; Daphne, 1992; Kannely, 2005; Laurence et al., 2011;

Bryant et al., 2012). Typically, pollen samples are placed in sonicating baths to mechanically separate pollen from surrounding organic and lithic material. The utility of ultrasonic probes to assist in geologic sample sieving was realized as early as the 1980’s

(Caratini, 1980; Batten and Morrison, 1983).

Sonication is also used for the extraction of other plant microfossils, and has been demonstrated to cause little degradation on both phytoliths (Lombardo et al., 2016) and starch grains (Cuthrell and Murch, 2016). Although researchers acknowledge that sonication may degrade fresh pollen (Jones, 2014), little is known about the precise effects of sonication on pollen degradation. In addition, most papers that suggest best sonicating practices including intensity and duration to avoid degradation in pollen samples are in reference to a sonic bath, not the use of a sonic horn (i.e. Laurence et al., 2011). Thus, it is necessary to empirically test the effects of sonication on pollen degradation in general and to also specifically measure pollen degradation using a sonicating disruptor horn. The horn distributes ultrasonic vibrations through the end of an attachment directly into the sample, rather than through a liquid bath in which the sample is submerged and sonicated. This

technique is preferable over a sonication bath because it can not only mechanically separate pollen from surrounding organic and lithic material, but can then help the small, unwanted material pass through a screen.

This paper presents a similar method, using a plastic screw top base with a 10- micron Nitex© mesh with the adjoining bottle as a receptacle for the pollen sample (Figure

1). Some pollen types are smaller than 10-microns, so it is important to consider using mesh with smaller openings. This is dependent on the research goals and origin of the samples. The removed debris can also be periodically examined to ensure small pollen types are not being lost. Qualitative observations suggest that the sample flows through the mesh with more ease when it is in Ethyl Alcohol (ETOH). The current study was done using a 20 kHz Branson© S450 analog sonicating disruptor horn and has been successful in ridding pollen samples of tiny debris (Figure 2).

Figure 1. Bryant and Holloway (2009) charcoal removal method (left) compared with the method detailed in this paper (right). We used a Branson© S450 sonifying disruptor horn in sample receptacle and plastic screw top base with a 10-micron mesh bottom and adjacent sample cup. Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis.

II.2 Methods

II.2.1 Processing

Twelve fresh pollen types were chosen based on their unique range of morphological characteristics demonstrated (Table 1). One-tenth of a gram of each taxon was combined and homogenized by manual stirring. One-tenth of a gram of that total assemblage was then placed in 15mL centrifuge tubes with 10 mL of glacial acetic acid.

Acetolysis was performed on each sample using a 9:1 acetic anhydride: sulfuric acid solution. Following acetolysis, all samples were washed in water several times. Sample 1 was then stained and curated in glycerin without sonication, as experimental control. The remaining samples were rinsed in ETOH and placed one at a time into a cup with a 10- micron Nitex mesh bottom. Each sample was sonicated using a 20 kHz Branson© S450

Sonicator with a disruptor horn.

Taxa Common Apertures Size Other Name Morphologica l Characteristic s Carya conformis Bitternut Triporate Diameter ~ 50 Psilate- hickory 휇m Scabrate Chenopodium Southern Periporate Diameter ~24 botrys magnolia 휇m Fraxinus Blue ash Tricolpate Diameter Scabrate quadrangulata ~22휇m Juniperus Pfitzer juniper Inaperturate Diameter Psilate pfitzeriana ~26휇m Magnolia Southern Monoculpate Length grandiflora magnolia ~100휇m; width and height ~60휇m Morus spp. Mulberry Diporate Diameter Psilate ~16휇m Pinus cembroides Mexican Inaperturate Body width Scabrate, pinyon ~40휇m, length Bisaccate, ~50휇m, Reticulate overall ~69휇m Bladders Poa pratensis Kentucky Monoporate Diamter Psilate, bluegrass ~22휇m annulated pore Populus Black Inaperturate Diameter Scabrate, thin trichocarpa cottonwood ~31휇m exine Prunus malus Tricolpate Width ~20휇m, Striate length ~27휇m Quercus geminata Sand live oak Tricolporate Diameter Verrucate ~21휇m Salix nigra Black willow Tricolporate Polar axis Reticulate ~21휇m, Equatorial diameter ~15휇m

Table 1. Morphological characteristics of pollen types in this study. Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis.

Three experiments were completed to demonstrate pollen degradation and the effectiveness of sonication-assisted sieving. In the first experiment, pollen samples were sonicated at increasing output settings for three minutes. This duration is based on a qualitative assessment suggesting that it was enough time to allow most of the <10-micron debris to pass through the screen. Output settings on the 20 kHz Branson© s450 sonifier control amplitude, therefore, increasing output settings increases intensity of the ultrasonic vibrations. Output settings tested were 1, 2, 3, 4, 5, and 6. The first 50 pollen grains of each taxon encountered during microscopic analysis from each taxon were counted. To test the effectiveness of this method, an archaeological sample from Arizona containing high amounts of charcoal was selected. Five grams of this unprocessed sediment was sonicated for three minutes at increasing output settings (1, 2, 3, 4, 5, and 6.) Both the >10 micron and <10 micron fractions were oven dried at 150 degrees Fahrenheit, then weighed.

After Experiment 1, it was still unclear if it was the ultrasonic vibrations or physical collisions within the sample that were more likely damaging pollen grains. To address this question, conditions of Experiment 1 were duplicated in Experiment 2 with the addition of sterile sand to the sample. The pollen count was increased to 150 grains from each taxon to increase sample size and reliability.

Experiment 3 tested the effect of sonication for different durations on pollen degradation. We selected output setting 2 because the previous experiments demonstrated that occurrences of pollen grain damage were relatively infrequent at this setting. The samples were sonicated for 1, 1.5, 2, 2.5, 3, 3.5 and 4 minutes. During microscopic analysis, the first 50 pollen grains encountered from each taxon were examined and recorded. To test the effectiveness sonication at output setting 3 for increasing durations,

five grams of the archaeological sample from Arizona containing high amounts of charcoal was sonicated at output setting 3 for 1, 1.5, 2, 2.5, 3. 3.5 and 4 minutes. Both the >10 micron and <10 micron fractions were oven dried at 150 degrees Fahrenheit, then weighed.

II.2.2 Analysis

II.2.2.1 Quantification of sonication intensity. Intensity of sonication was quantified by output settings, because each output setting can produce a range of amplitude

(or intensity) it can deliver based on a number of factors including the volume and viscosity of the sample. The output control settings regulate the amplitude of the ½” disrupter horn attached to the Branson© 450 Analog Sonifier, which can produce up to 400 watts. For each experiment, samples were processed on a constant duty cycle at output settings 1-6. Each output setting can produce a maximum percentage of watts, however, that does not necessarily mean that maximum percentage is always used. For example, output setting 3 allows 30% of 400 watts to be used. For a specific read on the wattage used, the dial on the meter is read on the analog sonifier. If on output setting 3, the specific wattage will be marked on the dial at 30% or less. This specific wattage can be easily read on an aqueous solution with a stable volume. In this paper, the tested method has a fluctuating volume; ETOH filtered into a waste beaker below the mesh and ETOH was periodically added to prevent aerosoling when the volume was too low, as aerosoling could cause additional pollen degradation. This fluctuating volume did not allow for one specific meter read per output setting. Provided is a figure from the Branson© instruction manual

(Figure 3), which gives the range of watts that could be used in each output setting.

The amplitude is also likely to vary between different brands of sonifiers, but generally will increase with higher output settings. There is a direct relationship between amplitude and intensity of sonication. Higher amplitude will result in more intense sonication, but the viscosity of the solution being sonicated must also be considered. More viscous solutions (i.e. honey and water) will require higher intensity sonication to produce

the same effects on the sample than pollen suspended in a less viscous solution such as

ETOH. In general, debris removal is more effective in a less viscous solution.

II.2.2.2 Quantifying pollen degradation. After processing, the samples were scanned at

400X magnification and the condition of the first 50-150 grains of each taxon were noted and categorized by the following degradation classification system after Campbell and

Campbell (1994) (Figure 4).

In presenting the results of the experiments measuring pollen degradation, the six most fragile pollen types are grouped together, due to the variability in degradation between the various taxa. The following taxa appeared to be the most fragile and effected by the sonication: Fraxinus quadrangulata, Magnolia grandiflora, Populus tricoparpa, Pinus cembroides, Prunus malus and Salix nigra. Chenopodium botrys, Juniperus phitzeriana,

Morus spp., Poa pratensis and Quercus geminate were more resilient to the effects of sonication. Results are also discussed in terms of classes of degradation. The pollen grains are clustered into groups based on identifiability. Classes 0 and 1 are grouped together because they are fairly easily to identify. Most of the time a small tear in a pollen grain does not inhibit an accurate identification, though there are instances where fossil pollen cannot be identified easily after one tear that does not exceed half the length of the grain.

Classes 2 and 3 have been grouped together because those classes represent damage to the pollen grains that is severe enough to potentially impede identification. Class 4 only applies to Pinus cembroides pollen, because it is the only grain that can be easily separated into “thirds”, as it has two bladders and a body. Though pine bladders and bodies can typically be identified to the genus level even when separated from other parts of the grain,

Class 4 Pinus degradation creates a quantification problem, because two bladders might

represent only one pollen grain, or two. Class 4 is considered serious degradation and should be avoided.

II.3.2 Experiment 2: Pollen degradation after sonication at different output settings with sand

No noticeable increase in pollen degradation resulted from sonication with sand

(Figure 6). Thus, it is likely that ultrasonic vibrations are causing the most damage to pollen grains. In addition, it does not appear that increasing the sample size considerably changed the outcome of the experiment.

II.3.3 Experiment 3: Pollen degradation after sonication at various durations

Increasing duration of sonication resulted in noticeably more damage among the very fragile pollen types such as Magnolia and Populus (Figure 7). However, most taxa did not show substantial damage with increasing duration, suggesting increasing the time of sonication at low output settings may be a viable option over raising output.

In the archaeological sample, more than twice the amount of tiny debris was removed after sonication for 4 minutes than one minute (Figure 7). However, output setting 2 still did remove as much tiny debris as the higher output settings, as demonstrated in Experiment 1.

Figure 6. Class 2 and 3 pollen degradation of most fragile taxa after 3 minutes of sonication at increasing output settings with the addition of sterile sand. 150 grains from each taxon were counted. Reprinted with permission from “Efficacy of sonication-assisted sieving on Quarternary pollen samples”, by Angelina G. Perrotti, Taylor Siskind, Mary Katherine Bryant, and Vaughn M. Bryant, 2018. Palynology. Copyright 2018 by Taylor and Francis.

Archaeologists are often most interested in the pollen from burned contexts within archaeological sites. Research questions involve topics such as what type of wood was used for fires, plants and animals cooked in fires, and variable use of different spaces within dwellings. To answer these questions, archaeologists collect the contents of pits, fire hearths and floor surfaces. These burned locales often contain extensive deposits of charcoal. Palynologists experience difficulty when examining these samples because reducing charcoal and ash in pollen samples is necessary to concentrate and examine the pollen. As previously discussed, charcoal removal is often not easy because it is inert and is not easily degraded with acids that are commonly used in pollen processing. Further complicating the removal of charcoal is the fact that it often has the same specific gravity of pollen, making heavy density separation inadequate method for maximum charcoal removal from some samples.

Sonication-assisted screening of samples through small mesh with openings of <10 microns can be more useful to remove small charcoal pieces that tend to float along with pollen in specific gravity solutions. Although pollen degradation can occur, particularly at the highest output settings, using lower output settings (and therefore amplitude) for increasing time can be a viable solution. More than twice the amount of <10 micron charcoal debris can be removed from a sample after using the sonication probe for four minutes rather than one minute (Figure 7). If pollen preservation is deemed to be very good in a sample, higher amplitude can be used to increase agitation, more debris can be removed (Figure 5). Different sonifiers will vary in the relationship between output settings and sonication intensity so replications of this method should take the relationship

of output setting and amplitude on each device into account.

II.5 Conclusions

The results of this study suggest that sonication is effective at removing small debris from archaeological pollen samples, which can help enable palynologists examine samples with large amounts of tiny debris that might otherwise obscure pollen grains.

Sonication at higher intensity is more effective at ridding samples of small organic debris.

This study does suggest that sonication at the highest output settings can result in the degradation of fragile pollen types (Figure 8). Therefore, the best practice is to use the lowest output setting possible that still effectively facilitates the sample’s debris passing through the screen.

In conclusion, sonication-assisted sieving is a relatively cost-effective and efficient way to remove tiny debris from a pollen sample. However, because each sample has a unique assemblage of pollen grains and exhibits varying levels of degradation, palynologists who want to use this technique should briefly experiment with their own samples to find the lowest effective output setting and duration possible. Future work investigating the use and importance of sonication within palynological methods might include experiments on the efficacy and effects of a sonication bath, different models of sonicating devices, and the effects of sonication on different plant microfossils.

II.6 References

Batten, D.J., Morrison, L., 1983. Methods of palynological preparation for

palaeoenvironmental, source potential and organic maturation

studies. Palynology-Micropalaeontology: laboratories, equipment and methods

(Costa, LI; editor). Bulletin of the Norwegian Petroleoum Directorate 2, 35-53.

Bryant, V.M., Holloway, R.G., 2009. Reducing charcoal abundance in archaeological

pollen samples. Palynology 33 (2), 63-72.

Bryant V.M., Kampbell S.M., Hall J.L., 2012. Tobacco pollen: archaeological and

forensic applications. Palynology 36 (2), 208-223.

Campbell I.D., Campbell, C., 1994. Pollen preservation: Experimental wet‐dry cycles in

saline and desalinated sediments. Palynology 18 (1), 5-10.

Caratini C., 1980. Ultrasonic sieving to improve palynological processing of sediments:

a new device. International Commission for Palynology Newsletter 3 (1), 4.

Cuthrell R.Q., Murch L.V., 2016. Archaeological laboratory extraction procedures and

starch degradation: Effects of sonication, deflocculation, and hydrochloric acid

on starch granule morphology. Journal of Archaeological Science: Reports 9,

695-704.

Dafne A., 1992. Pollination ecology. Oxford University Press, Oxford.

Holloway R.G., 1989. Experimental mechanical pollen degradation and its application

to Quaternary age deposits. Texas Journal of Science 41, 131-145.

Jones G.D., 2014. Pollen analyses for pollination research, acetolysis. Journal of

Pollination Ecology 13, 203-217.

Kannely A., 2005 Preparation and quantification of entomophilous pollen using

sonication and an area-counting technique. Madroño 52 (4), 67-269.

Laurence A.R., Thoms A.V., Bryant V.M., McDonough C., 2011. Airborne starch

granules as a potential contamination source at archaeological sites. Journal of

Ethnobiology 31 (2), 213-232.

Lebreton V., Messager E., Marquer L., Renault-Miskovsky J., 2010. A neotaphonomic

experiment in pollen oxidation and its implications for

archaeopalynology. Review of Palaeobotany and Palynology 162 (1), 29-38.

Lombardo U., Ruiz-Pérez J., Madella M., 2016. Sonication improves the efficiency,

efficacy and safety of phytolith extraction. Review of Palaeobotany and

Palynology 235, 1-5.

Vaissiere B.E., 1991. Honey bees, Apis mellifera L. (Hymenoptera: Apidae), as

pollinators of upland cotton, Gossypium hirsutum L. (Malvaceae), for hybrid

seed production. Ph.D. thesis. Texas A. and M. University. College Station

CHAPTER III

DUNG FUNGI AS A PROXY FOR LARGER HEBIVORE ABUNDANCE:

CONSIDERATIONS AND LIMITATIONS FOR ARCHAEOLOGICAL APPLICATION

III.1 Introduction

The use of Sporormiella spores from sedimentary sequences as a proxy for large herbivore abundance has garnered pronounced attention and scrutiny over the past three decades. Since it was first proposed as a proxy for Pleistocene megaherbivore abundance in the 1980s (Davis, 1987), increasing research has been devoted to developing sampling, recovery and quantification techniques, as well as understanding the applications and limitations of this method. In response to the rapid rate at which new information is being discovered on this topic, this paper presents a brief review of the archaeological applications so far, and outlines opportunities and limitations of using Sporormiella as a proxy for megaherbivore abundance.

Sporormiella is one of a number of genera of coprophilous fungi, also known as dung fungi. These are fungi that show a strong preference for (mainly herbivore) dung as their primary substrate. Sporormiella is one of many organisms that participate in the breakdown of herbivore dung after it is evacuated. This fungus belongs to a group of most commonly used fungal indicators of herbivore abundance, Ascomycota, which reproduce on the dung two to four weeks after it is deposited (e.g. Harper and Webster, 1964). There are some indications that the spores produced by Sporormiella and similar genera need to pass through the digestive tract of an herbivore in order to activate germination, but evidence for this is limited (Janczewski, 1871; Massee and Salmon, 1902; Krug et al.,

2004). Regardless of these uncertainties, the abundance of Sporormiella spores in sedimentary sequences has been demonstrated repeatedly to reflect herbivore abundance in both modern (e.g. Baker et al., 2016; Gill et al., 2013; Parker and Williams, 2011; Raczka et al., 2016; Wood and Wilmhurst et al. 2013) and ancient settings (e.g. Burney et al.,

2003; Davis, 1987; Davis and Shafer, 2006; Doyen and Etienne, 2017; Gill et al., 2009).

III.2 Applications for Archaeologists

Dung fungal records are important datasets for archaeologists because of the frequency of interactions between ancient people and large herbivores. First, Sporormiella continues to be used as a proxy to detect declines in late Pleistocene megaherbivore communities (e.g. Davis, 1987; Gill et al., 2009, 2014; Graham et al., 2016; Johnson et al.,

2015; Perrotti, 2017). The North American extinction of over 30 species of large mammals at the end of Pleistocene is of particular interest to archaeologists because it roughly coincides with the earliest human colonization of the continent. The cause of these extinctions remains the subject of intense debate. Disagreement concerns the relative impacts of human hunting (Abramson et al., 2015; Alroy, 2001; Araujo et al., 2015; Frank et al., 2015; Martin, 1984; Surovell et al., 2016; Surovell and Waguespack, 2009), climate change (Guthrie, 1984; Grayson and Meltzer, 2004), disease (MacPhee, 1997) and a potential extraterrestrial impact (Firestone, 2007) on Pleistocene megafauna.

Because faunal remains are sparse and can be difficult to date, it is hard to reliably establish extinction dates. The use of the Sporormiella proxy allows researchers to fill in geographic gaps where there may be no dateable fauna. Since Sporormiella spores occur in the same deposits as pollen and plant macrofossils, patterns in their abundance can be linked directly with trends and changes in the vegetation record, as well as with absolute

dates obtained for these deposits. The fungus has been found in sedimentary records across

North America, and is aiding in the understanding of the timing and process of megafaunal extinction, and its effects on vegetation communities (Gill et al., 2009, 2014; Perrotti,

2017).

If humans are unequivocally tied to megaherbivore extinction, it is possible that the timing of local extinctions could be used as a signal of human colonization in different regions, particularly when complementary with archaeological evidence. Studies incorporating Sporormiella in western North America are rare, but declines in

Sporormiella coincide with generally accepted dates of human colonization in the northeastern United States (Davis and Shafer, 2006; Gill et al., 2009; 2014). However, the

Sporormiella record and archaeological evidence from Page-Ladson, Florida (Halligan et al., 2016; Perrotti, 2017) indicate that humans and megaherbivores coexisted in the region for ~2,000 years. Fiedel (2016) suggests that Sporormiella may not be a reliable indicator of megaherbivore extinction in eastern North America because current Sporormiella records (i.e. Gill et al., 2009, 2014) point to a decline in megaherbivores around 14,800

BP, while some mammoth remains in the region are dated to as late as ~12,000 BP.

Furthermore, it is unclear whether absence of spores is equally informative as spore presence (e.g. see below for factors other than herbivore abundance influencing fungal growth, and thus, potential spore presence; see also Raper and Bush, 2009; Jones et al.,

2017). However, researchers using Sporormiella as a proxy for large herbivore abundance acknowledge that declines in Sporormiella do not necessarily indicate a complete extinction of all megaherbivores; but rather, a functional decline in grazing pressure that represents shrinking herbivore populations relegated to patchy environments prior to

extinction. Nonetheless, because of the discrepancies between regions and the inconclusive evidence for a human driven extinction of North American megafauna, at present

Sporormiella or other coprophilous fungi cannot be used as a proxy for human activity and migration in this region (Fiedel, 2016).

Human colonization seems to coincide with a decline in coprophilous fungi in other parts of the globe, including Australia (Rule et al., 2012), New Zealand (Wood et al., 2011) and Madagascar (Burney et al., 2003). In some cases, the initial fungal spore decline is followed by an increase after the introduction of domesticated animals (Burney et al.,

2003; Davis and Shafer, 2006; Graham et al., 2016) or other non-native herbivores (Wood et al., 2011).

Second, many archaeologists use Sporormiella and other coprophilous fungi as markers of pastoral and other human land use activities across Europe, Africa and the

Middle East (Ghosh et al., 2017; Shumilovskikh et al., 2016; Szymanski et al., 2017).

Coprophilous ascomycetes other than Sporormiella have also been verified to reflect pastoral activities in mountainous, pasture-woodland landscapes (Cugny et al., 2010), in upland grasslands and bogs (Feeser and O’Connell, 2010) and in boreal forest (Kamerling et al., 2017). Evidence of direct domestication of herbivores in the pre-Columbian

Americas is rare. However, dung fungi could potentially be used to provide more information about communal hunting in the Great Basin region of North America, where pronghorn and mountain sheep were rounded up and potentially kept in stone and brush pens (Hockett, 2005; Hockett and Murphey, 2009).

III.3 Opportunities and Limitations

III.3.1 Dung Fungus Reproduction

Interpreting the abundance of dung fungal spores in a sedimentary record requires an understanding of the different factors influencing fungal reproduction. Sporormiella is strongly coprophilous and is observed almost entirely in association with herbivore dung.

Gelorini et al. (2012) emphasized that only genera that are obligate to herbivore dung such as Sporormiella and Podospora could serve as a reliable signal of herbivore presence.

However, the precise lifecycle and substrate preferences remain ambiguous for even the most commonly noted dung fungal spores. Coniochaeta and Apiosordaria are two taxa that are often taken to indicate herbivore presence, but are found to be poor indicators of herbivore abundance (Doyen and Etienne, 2017). Other commonly observed semi- coprophilous ascomycetes such as Cercophora, Coniochaeta, and Sordaria are also found on other organic substrates, such as plant debris, decaying wood, or soil, with some frequency (Table 2) (Bell, 1983; Doveri, 2007; Guarro et al., 2012; Hanlin, 1990; Kruys and Wedin, 2009). Newcombe et al. (2016) found evidence that Sordaria, Preussia, and even Sporormiella may be epiphytic and concluded that the presence of these spores is not undisputable evidence of herbivore abundance. Because herbivore dung consists largely of partly digested plant remains, the ability of some dung fungi to opportunistically grow on plants is not surprising. However, coprophilous fungi taxa Sporormiella and Podospora have a strong preference for dung as a substrate and therefore typically reflect the presence of large herbivores. Despite its strong preference for dung as a substrate, caution still must be applied when observing Sporormiella in sediment samples, as Sporormiella spores can be indistinguishable from the spores of Preussia (Barr, 2001; Cain, 1961; Kruys and

Wedin, 2009). Though these two fungi are closely related, only Sporormiella depends on dung as a growth substrate (von Arx and von de Vera, 1987).

TYPE HABITAT Ascodesmis Herbivore dung (Basumatary and McDonald, 2017) Cercophora Herbivore dung, decaying wood (Bell, 1983; Hanlin, 1990) Coniochaeta Herbivore dung, decaying wood (Hanlin, 1990) Delitschia Herbivore dung, decaying wood (Bell, 1983) Podospora Herbivore dung Sordaria Herbivore and omnivore dung, decaying wood (Bell, 1983; Mighall et al., 2006) Sporormiella Herbivore dung (Ahmed and Cain, 1972)

Table 2. Preferred substrate of commonly used dung fungal taxa.

Even if dung fungal spores may not necessarily have to pass through the gut of an herbivore to complete reproduction, many fungal spores are consumed and move through the digestive system. After consumption by an herbivore, dung fungal spores are expelled with the dung after which they germinate and propel spores away from the dung. Some of these spores are covered in a gelatinous sheath. The spores typically adhere to nearby vegetation, and are inadvertently consumed along with the vegetation. After passing through the digestive tract, the spores are expelled along with the dung to complete the lifecycle again (Figure 9).

Figure 9. Generalized lifecycle of Sporormiella.

Extensive research exists on the fungal community composition of different types of herbivore dung (e.g. Ebersohn and Eicker, 1992; Mungai et al., 2011, 2012; Nyberg and

Persson, 2002; Piontelli et al., 1981; Richardson, 2001; Wicklow et al., 1980). Often, a few species are abundant, alongside a large number of rare species (Ebersohn and Eicker, 1992;

Krug et al., 2004; Richardson, 2001; Nyberg and Persson, 2002). Most coprophilous fungi occur on a wide range of dung types (Angel and Wicklow, 1983; Richardson, 1972, 2001), but many genera show a preference for certain types of dung. While these genera also

occur on other dung types, they occur more often and more abundantly on their preferred dung type (Bell, 2005; Lundqvist, 1972; Richardson, 1972, 2001; Van Asperen, 2017).

Spores of coprophilous fungi are typically a very local indicator of herbivore dung

(Graf and Chmura, 2006) due to their short dispersal distances (Ingold, 1961; Ingold and

Hadland, 1959; Trail, 2007; Yafetto et al., 2008). They can become airborne, but are likely deposited within 100 meters of the dung source (Gill et al., 2013). However, it is possible to get a more regional assemblage of dung fungi if water is present because spores can enter a river, pond, or lake via slopewash. The spores tend to settle out fairly rapidly

(Raczka et al., 2016), so spore concentration declines toward the center of lakes and ponds

(Raper and Bush, 2009). This discrepancy could be addressed by analyzing multiple cores from various locations within the same site. Overall, spores within smaller bodies of water are more likely to reflect herbivore abundance (Johnson, 2015).

Dung fungi have species or genera-specific responses to microenvironmental factors that affect the success of reproduction (Dix and Webster, 1995). The species- specific responses to environmental changes could potentially encourage or limit growth of a particular fungal type, which leads to a fluctuation in spores that is not actually representative of megafaunal activity. In part, this issue can be combatted by completing a comprehensive palynological study, including multiple coprophilous fungi taxa.

Presence, abundance and succession of specific fungal genera and species on dung incubated in the laboratory is known to differ from that on dung in field conditions (Angel and Wicklow, 1983; Harper and Webster, 1964; Richardson, 2001). Laboratory conditions are highly artificial, with relatively constant temperatures and humidity. In contrast, under field conditions, temperatures are generally lower and display daily fluctuations, and

waterlogging and precipitation vary in frequency and intensity. Although the growth of dung fungi in the laboratory cannot be used as a direct analog to fungal growth in nature, research suggests that coprophilous ascomycetes are not as successful when temperature or relative humidity becomes too high or too low (Asina et al., 1977; Kuthubutheen and

Webster, 1986a). The effects of low relative humidity can be compounded by competition between ascomycetes (Kuthubutheen and Webster, 1986b). Soil hydrology is also likely to affect spore reproduction (Wood and Wilmhurst, 2013).

Although at varying levels of success, spores can often germinate and grow across a wide range of temperatures. For example, Asina et al. (1977) found that three Sporormiella species could germinate at temperatures within a range of 10-30°C., although the range of temperatures at which germination was maximal was smaller (a range of 5-15 degrees).

Dung fungal development is generally slower at lower temperatures, and although the abundant genera tend to be present across a range of temperatures, they produce fewer fruitbodies at lower temperatures (Krug et al., 2004; Wicklow and Moore, 1974). However, dung could provide warmer conditions than are prevalent in the surrounding environment as long as decomposing organisms are still capable of growth (Lundqvist, 1972; Webster,

1970).

Most dung fungi germinate, grow and produce fruitbodies more slowly when water availability is low, and fruit for a shorter period of time, although some species produce fruitbodies more quickly (Dickinson and Underhay, 1977; Harrower and Nagy, 1979;

Kuthubutheen and Webster, 1986a, 1986b). Dickinson and Underhay (1977) suggest that the rapid decline in water content common in the warm and/or dry season soon inhibits fungal growth, whereas in the cold and/or wet season, growth may be limited or slowed by high

water content. Kuthubutheen and Webster (1986b) found that Sporormiella was the most tolerant of low water availability of the genera they tested, which included Podospora. The interaction between the effects of temperature and moisture availability on dung fungal growth leads to higher dung fungal diversity during the wetter, cooler season in temperate latitudes than in the warmer, drier season (Krug et al., 2004; Wicklow, 1992; Richardson,

2001; Van Asperen, 2017). Although winter temperatures are lower, this temperature drop presents a stress factor which may have the effect of reducing the reproductive fitness of dominant species, thereby releasing less-specialized, more stress-tolerant species. In contrast, in summer the primary factor is the lower substrate humidity negatively affecting germination rate (Harrower and Nagy, 1979; Kuthubutheen and Webster, 1986a, 1986b).

Dung type and animal behavior may also affect the durability of the dung resource.

Some animals defecate in latrines to which they return regularly, leading to large accumulations of dung material. Larger dung pats are less susceptible to desiccation, while clusters of pellets create a wider range of microhabitats but are more prone to desiccation

(Beynon, 2012). Furthermore, dietary diversity and the quality of the vegetation consumed also leads to dung with different characteristics. For example, dung from cattle feeding on the new growth of grass in spring generally has a higher moisture content than later in the growth season.

Another factor that may reduce the number of fruitbodies produced and the duration of fruiting is competition between ascomycetes (Lussenhop & Wicklow, 1985;

Kuthubutheen and Webster, 1986b). Sporormiella species often appear relatively late in the incubation period (Angel and Wicklow, 1983), so perhaps they are more easily outcompeted by genera that appear earlier when environmental factors favor those genera. In addition to

direct effects of environmental factors, in temperate latitudes, the activity of other dung- inhabiting species, in particular dung beetles (both adults and larvae) and fly larvae, is much higher in wet and warm conditions than in dry or cool conditions (Davis 1994). Beetle activity greatly diminishes at temperatures below 10°C, as well as in dry, hot conditions or very wet spells, with the main period of activity ranging from May to July, with lower levels of activity extending from early March to late November (Bertone et al., 2005; Floate &

Gill, 1998; Kadiri et al., 2014; Wassmer, 2014).

Besides potential direct consumption of dung fungi, the grazing activity of these insects has several adverse effects on dung fungal growth: it reduces the amount of dung available for growth, it disrupts fungal hyphae, and it fragments the dung (Lussenhop et al.,

1980; Wicklow & Yocom, 1982). Fragmentation makes the dung more susceptible to moisture loss, and also removes the competitive advantage of fungal hyphal growth compared to bacterial growth (Lussenhop et al., 1980). Lussenhop et al. (1980) found that the presence of dung beetles reduced dung fungal hyphal density, especially at lower moisture content, but this did not lead to lower rates of fruiting and increased dung fungal diversity, possibly by dispersing the fungi more widely. The presence of fly larvae was observed to reduce the species diversity of dung fungi (Wicklow & Yocom, 1982). However, they note that Sporormiella abundance was not significantly affected, whereas there was a small negative effect on Podospora and Sordaria. In another study, species diversity was not affected, but there was a highly significant 68% reduction in spore production in the presence of fly larvae (Lussenhop & Wicklow, 1985).

III.3.3 Incorporation of fungal spores into sedimentary records

Local hydrology has the potential to produce fluctuations in a spore record that are not representative of herbivore abundance at the site. Wood and Wilmhurst (2012) demonstrated that spore fluctuations correlate with changes in local hydrology. However, these correlations were not consistent. Two bogs demonstrated an increase in Sporormiella when water levels were at their peak, while one bog exhibited an increased in Sporormiella while water levels were lower than usual. Because the apparent correlations are not consistent between the sites, it is possible that the changes in local hydrology could be affecting herbivore behavior. Animals could be preferentially utilizing the site based on water availability, or may be avoiding it when water levels are too high due to decreased ground stability.

Rainfall and the degree of storminess at a site can also affect a dung fungal record.

Spores can be flushed into waterways, but will not reproduce and deposit higher numbers of fungal spores into a record if the fecal material is washed away soon after it is deposited. If deposited in a very arid environment, herbivore feces may dry out more quickly, which will result in a lack of spore reproduction. Finally, high energy depositional environments could potentially transport spores further than lower energy systems. In these types of environments, the fungal spore record could be more regional than expected, given the local nature of spore deposition.

III.3.4 Laboratory Recovery

Dung fungal spores and other non-pollen palynomorphs are generally extracted from palaeoecological samples along with pollen, usually using the ‘standard’ pollen preparation method (Faegri and Iversen 1989; Moore et al. 1991). However, a range of alternative

preparation techniques are also available. Several studies have tested the effect of a number of chemicals and preparation techniques on the survival and preservation of fungal remains.

Clarke (1994) processed samples from a variety of substrates with three different techniques:

A. boiling in KOH (potassium hydroxide), HF (Hydroflouric Acid), acetolysis,

mounting using TBA (tertiary butyl alcohol) (similar to the ‘standard’ pollen

preparation method; Faegri and Iversen, 1989; Moore et al., 1991);

B. boiling in NH4OH (ammonium hydroxide), sieving, swirling, mounting using

TBA;

C. boiling in KOH, sieving, heavy liquid separation with ZnCl2 (zinc chloride),

mounting using TBA.

Her results indicated that small round to oval fungal spores behave in a similar way as pollen in terms of survival. Treatment A led to a loss of large, buoyant forms, whilst these were the only forms consistently present in samples treated with method B. Thick-walled forms were lost in treatment C. None of the treatments led to significant preservation issues.

In a study focusing specifically on dung fungal spores recovered from dung samples incubated in the lab, Van Asperen et al. (2016) tested five preparation methods:

A. boiling in NaOH (sodium hydroxide), sieving (125 and 6 µm mesh), treatment

with HCl (hydrochloric acid), acetolysis, mounting using TBA.

B1. boiling in NaOH, sieving, treatment with HCl, mounting using TBA.

B2. boiling in KOH, sieving, treatment with HCl, mounting using TBA.

C. boiling in KOH, sieving, density separation by swirling, treatment with HCl,

mounting using TBA.

D. sieving, mounting using TBA.

This allowed them to tease out the effects of the different chemicals used in standard pollen preparation procedures on dung fungal spore recovery and preservation. The use of corrosive chemicals, such as NaOH, KOH and acetolysis, led to a significant loss of hyaline spores (e.g. Cheilymenia, Coprotus, Iodophanus, Peziza and immature Cercophora spores), as well as spores that lose their epispores over time (e.g. Ascobolus and Saccobolus). Such spores are unlikely to be preserved in sediments, but since these spores dominate certain dung types (Lundqvist, 1972; Richardson, 1972), this can significantly bias spore counts.

Hyaline appendages were also lost (Sordaria and Cercophora/Podospora). Spores with thicker, pigmented spore walls (e.g. Sordaria, Sporormiella and Cercophora/Podospora, as well as basidiomycete spores) were more resistant to chemical degradation. Sordaria spores deteriorated and Sporormiella spores tended to swell when acetolysis was used.

All spores that were large enough to be retained in the mesh were recovered when samples were sieved but not submitted to any other treatment. This includes the vulnerable spores that were lost when chemicals were used. Spores small enough to pass through the mesh (e.g. small basidiomycete spores and single cells of Saccobolus) were lost, which is significant considering that Sporormiella spores often break up into their constituent cells

(Ahmed and Cain, 1972). The single cells of some Sporormiella species are so small that they would not be retained in a 10 or 6 μm mesh, leading to a potential loss of information.

Other alternative pollen preparation techniques are also available, although these have not been tested explicitly on dung fungal spores. Riding and Kyffin-Hughes (2004) used a treatment with sodium hexametaphosphate followed by density separation by means of swirling and centrifuging. With this simple method, they achieved equal or better

palynomorph recovery than with the standard preparation method for most lithologies, although they did not test their method on sediments high in organic material.

Given the clear adverse effect of some of the chemicals used in standard pollen preparation methods on dung fungal spore recovery and preservation, it is highly advisable to test and use alternative, non-chemical, techniques wherever possible (cf. Van Asperen et al., 2016).

III.3.5 Analysis and Quantification

Typically, dung fungal spores are counted alongside pollen. In some environments, however, it may be beneficial to count spores in relation to tracer spores. Etienne and

Jouffrey-Bapicot (2014) suggest counting 300-350 Lycopodium tracer spores to obtain an accurate evaluation of Sporormiella in a sample, but the number of tracer spores and sample sizes in this study were not reported. The amount of tracer spores counted should depend on the environment, the size of the sample, the concentration of pollen within a sample, and how many Lycopodium are added.

Examining pollen samples for dung fungal spores using different approaches can produce discrepancy in results between sites. In the southeastern United States, for example, two different methods for quantifying dung fungal spores have been used. To our knowledge, recent research at Cupola Pond, Missouri (Jones et al., 2017) and Page-

Ladson, Florida (Perrotti, 2017) are the only two published palynological studies to attempt to incorporate dung fungal spores within the analysis from that region. In contrast to Page-Ladson, no evidence of Sporormiella spores were found in pollen samples at

Cupola Pond, leading Jones et al. (2017) to conclude that herbivory was not a key factor in ecosystem regulation around the site.

This discrepancy between the observation of dung fungal spores at Cupola Pond and Page-Ladson is most likely a result of differences in noting and quantifying dung fungal spores. Perrotti (2017) analyzed dung fungal spores by counting them against a known number of tracer spores, rather than observing them during pollen counts. Most dung fungal spore studies have used the latter method (Davis, 1987; Davis and Shafer,

2006; Gill et al., 2009; Jones et al., 2017), but this method is vulnerable to fluctuations in pollen accumulation rates. In sediments with high pollen concentrations, fungal spores are less-well represented. It has been suggested that functional extinction of megaherbivores can be observed when fungal spores fall below 2% of the total pollen assemblage (TPA).

This was first suggested by Davis (1987), in his work in (mostly arid) western North

America. At Page-Ladson, Florida, Sporormiella spores never constitute more than 2% of the TPA (Perrotti, 2017) due to a high concentration of arboreal pollen at the site. If

Sporormiella was tallied as a percentage of that total pollen assemblage, dung fungal spores would have been far less represented than those from arid environments as in the desert west, where this method was first conceived. Pollen concentrations at Cupola Pond

(Jones et al., 2017) are similar to those at Page-Ladson, suggesting that a different method of searching for dung fungal spores may have resulted in the recovery of more spores.

Analyzing and quantifying dung fungal spores as %TPA can also produce fluctuations within a spore record that are not representative of megaherbivore abundance.

Parker and Williams (2012) found a negative relationship between mean annual precipitation and spore abundance in lake-center sediments that they attributed to a higher influx of arboreal pollen during wet years, which would drive down relative Sporormiella abundance. Wood and Wilmshurst (2013) confirm that Sporormiella as %TPA is subject to

fluctuations in pollen accumulation that may skew spore data, even when the spores are being consistently deposited.

Whenever possible, reporting dung fungal spore abundance using both accumulation or concentration and %TPA could be beneficial. A potential advantage of measuring spores as %TPA is that the same environmental conditions that degrade pollen may also degrade spores. Therefore, decreased pollen concentration values due to poor preservational conditions would also explain a decline in fungal spore abundance.

Although not an effective figure in all environments, 2% threshold for “background” spores has been consistently suggested to be below 2% of the total pollen assemblage in various modern environments (Baker et al., 2016; Gill et al., 2013; Raczka et al., 2016).

However, it is not likely that this threshold can truly be extrapolated between sites. Total pollen production varies greatly between different ecosystems and fungal spores and pollen have differing reproductive strategies, so future studies should avoid quantifying spores solely as %TPA. Calculating spore accumulation requires a well-dated core, but can be a useful illustration of how changes in sediment deposition at a site can affect the accumulation of fungal spores (Figure 10).

Figure 10. Sporormiella counts from the Page-Ladson site demonstrate how different ways of quantifying the fungal spores can change their representation.

III.3.6 Current Limitations

Typically, North American researchers focus on Sporormiella as an indicator of megaherbivore abundance without including other coprophilous fungi. Perhaps due to its distinct morphology or presence in many North American pollen samples, it has become the sole taxon reported in many studies (Gill et al., 2009, 2014; Halligan et al., 2016;

Perrotti, 2017). Though Sporormiella alone has been used as a proxy for herbivore

abundance, it is becoming more apparent that noting other coprophilous fungi can increase the robustness of interpretations regarding megaherbivore abundance.

For example, Johnson et al. (2015) found that the commonly encountered coprophilous and semi-coprophilous ascomycetes Cercophora, Coniochaeta, Podospora, and Sordaria contributed to a better overall understanding of dung fungi abundance. In addition, Van Asperen (2017) notes that Sporormiella can be rare on the dung of some modern large herbivores whilst other coprophilous fungi are found in abundance.

Therefore, lack of Sporormiella in sediments does not always indicate that herbivores were not present at the site (e.g. Jones et al., 2017). Incorporating all dung fungi counts can better indicate herbivore presence and abundance (Baker et al., 2016; Johnson et al., 2015,

Van Asperen et al., 2016, 2017), but the dependence on dung of each taxon must be considered (Table 2).

In addition to a lack of herbivore activity, an absence of dung fungi at a site may be the result of a number of factors, such as a lake location distant from the shoreline (Raper and Bush, 2009) or distance from the nearest stream discharge (Etienne et al., 2013). As previously discussed, little is known about the environmental preferences of each dung fungus, and more research is needed on the topic. Additionally, little is known about how spores preserve. It is widely assumed that they are more durable than pollen because of their thick cell walls and ubiquity in pollen samples exhibiting poor pollen preservation.

Overall, effects of microenvironmental fluctuations on spore records may be minimized with the incorporation of multiple fungal taxa.

Because of the factors discussed above, it is possible that an absence of dung fungi from a palaeoecological record may not always indicate a decline in megaherbivores.

However, by understanding the past environment, particularly in regard to hydrologic factors, more weight can be placed on the interpretation of the abundance or lack of dung fungal spores. Ultimately, dung fungal spores alone likely cannot be used to infer an extinction event for several reasons. First, dung fungi inhabit many different types of herbivore dung, in addition to decaying plant material. It is possible that noting particular species of fungi that are associated with the dung from specific animals could contribute to the understanding of extinctions. However, species-level dung fungus identification relies on the fruiting body in addition to the spores. These are rarely persevered in fossil samples and would almost certainly be destroyed during laboratory procedures. Second, because dung fungal spores typically represent a very local proxy, it is unlikely to be possible to draw any patterns regarding the demise of wide-ranging megafauna from spore abundance alone. Third, the previously mentioned factors affecting spore reproduction, deposition and preservation, suggest that many different environmental factors could be responsible for a decrease in spore abundance. When using spores to infer extinction or regional disappearance of large herbivores, it is best practice to utilize this proxy alongside other lines of evidence, such as faunal remains or macrofossils from herbivore dung (i.e.

Halligan et al., 2016; Perrotti, 2017).

III.4 Future Directions and Conclusions

The methods for analysis and research of analogs for the interpretation of dung fungal records are improving, but additional research is still needed. First, more modern experiments need to be conducted to understand the relationship between dung fungi abundance, herd size, and other geographical and environmental factors. Much research has been devoted to this topic recently and modern studies have drawn correlations

between cattle (Wood and Wilmhurst, 2012) and bison (Gill et al. 2013) herd size and

Sporormiella abundance. Baker et al.’s (2016) informative study conducted in The

Netherlands was an excellent demonstration of the correlation between spore accumulation, taphonomic processes and herd size, but more studies incorporating a wider variety of large herbivores would be valuable (Van Asperen et al., in prep.). Because spore reproduction and deposition will differ between environments, a wider array of modern environments should also be explored. If a clearer correlation between dung fungal spore abundance and megaherbivore diversity and abundance could be established, dung fungal data could be incorporated into dynamic vegetation models, strengthening our interpretation of the effects of grazing on vegetation systems.

Second, still more research is needed on laboratory recovery and identification of dung fungal spores. Certain laboratory recovery procedures can alter the size of dung fungi, further inhibiting species identification (van Asperen et al., 2016). Minimizing harsh chemical extraction procedures could be made possible by the implementation of techniques such as sonication-assisted sieving through <5 micron mesh (i.e. Perrotti et al.,

2018), or enzymatic removal of unwanted organic material (O’keefe, 2017). Regardless, more research is needed on the effects of standard palynological procedures on the recovery of dung fungal spores. By developing more standardized processing and extraction, comparisons between studies would be made easier.

Although additional research is still needed before researchers can fully rely on the application of dung fungi to questions raised in archaeology, we believe that this type of research has proven its potential as a valuable tool both for understanding past megaherbivore abundances. Palaeoecologists and archaeologists alike should recognize the

value of using dung fungi as a proxy, and consider the limitations and applications of dung fungi data to archaeology.

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megafaunal extinction events. Quaternary Science Reviews 77, 1–3.

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doi:10.1371/journal.pone.0003237

CHAPTER IV

POLLEN AND SPORORMIELLA EVIDENCE FOR TERMINAL PLEISTOCENE

VEGETATION CHANGE AND MEGAFAUNAL EXTINCTION AT PAGE-LADSON,

FLORIDA*

IV.1 Introduction

The Late Quaternary transition from glacial to interglacial climate resulted in drastic changes in plant and animal communities, including the disappearance of over 35 large mammal taxa in North America. The changes that occurred in the terminal

Pleistocene are of particular interest to archaeologists to contextualize cultural adaptations of the first Americans. The exact causes, effects, and the precise timing of Plesistocene megafaunal extinction remain unresolved. The Terminal Pleistocene megafaunal extinction in North America by 10,900 B.P. appears to have coincided with the sudden, global transition from Allerød warming to Younger Dryas cooling (Haynes, 2008). Russell, et al.

(2009) hypothesized that a ‘thermal enclave’ in the Southeast resulted in a unique mix of tropical and temperate vegetation, which may have enabled Pleistocene megafauna to persist beyond the Allerød-Younger Dryas transition (Webb et al., 2003; Webb, 2006). The present day southeastern United States (hereafter Southeast) is one region where dateable faunal remains are uncommon as a result of poor preservation throughout the region.

* Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd. 77

However, the most current evidence suggests that large herbivores were extinct in the region by the end of the Pleistocene (Halligan et al., 2016).

The lack of paleoenvironmental data has implications for understanding the environmental context of the initial peopling of the Americas because some of the earliest evidence for human occupation of North America has been discovered in the region (e.g.,

Halligan et al., 2016). Although the available pollen records can provide a foundational understanding of the paleoenvironment, most of them are poorly temporally constrained

(e.g. Watts and Hansen, 1994), and provide a rather coarse understanding of the changing vegetation during the late Pleistocene (Watts, 1969; Watts, 1975; Watts and Stuiver, 1980;

Watts et al., 1992; Watts and Hansen, 1988,1994). Existing environmental data in Florida

(Figure 11) must be extrapolated over long distances to provide context for early human adaptation because archaeological sites in Florida are often not good candidates for paleoenvironmental study.

Analyses of sediment samples from the Page-Ladson archaeological site resulted in the identification of both pollen and Sporormiella dung fungal spores. In 2014, two cores were extracted from the site and capture a nearly continuous terminal Pleistocene- early Holocene sedimentary record constrained by 41 radiocarbon assays (Table 3, Figure

12) (Halligan et al., 2016). Excellent organic preservation enables the investigation of environmental proxies, pollen and Sporormiella spores, in primary contextual association with radiocarbon-dated materials.

UCIA Co Eleva Stratum Mate ID 14C age ± Calibrated Age 17666 3A 7.53 6c wood Unidentified 8835 2 9885-9924 MS17666 # 3Are 7.74tion 6c woodrial Unidentified 8740BP 2 9657BP (1-9744 σ) 176669 3A 7.97 6b twig Unidentifiedhardwood 9590 20 10790-10887 176667 3A 8.17 6b twig Unidentifiedhardwood 9275 20 11174-11199 176668 3A 9.12 4c wood Unidentifiedhardwood 11240 25 13072-13119 176666 3A infill/dis root Unidentifiedhardwood root 3940 20 176663 3A infill/dis root Unidentifiedhardwood root 3945 15 149024 4A infill/disturbed woodseed Taxodiumwood distichum 8170 30 149035 4A infill/disturbed woodroot Quercuswood (Oak) root 8275 35 149031 4A infill/disturbed Wood Taxodium(Bald cypress) distichum seed 11705 35 149047 4A infill/disturbed woodbark Peridermwood (bark) 8200 30 149049 4A infill/disturbed Wood (BaldTaxodium cypress) distichum wood 8120 35 149010 4A 6.41 turbed6c twig Taxodium distichum 8000 30 8862-8919 149021 4A 6.58 turbed6c twig (BaldTaxodium cypress) distichum wood 8155 30 9027-9093 153349 4A 7.03 6c wood Quercus(Bald cypress) (Oak) 8830 20 9780-9853 149020 4A 7.32 6c bark Periderm(Bald cypress) (bark) 8910 30 1007-10063 143532 4A 7.51 6b bark Periderm (bark) 8955 35 10156-10202 149022 4A 7.68 6b wood Quercus (Oak) 9210 30 10287-10413 149023 4A 7.78 6a root Quercus (Oak) 9225 35 10370-10429 143523 4A 7.8 6a root Unidentified 9200 30 10279-10398 143534 4A 7.8 6a woodbark Periderm (bark) 9205 30 10285-10407 149029 4A 7.85 6a woodroot Unidentifiedhardwood 9245 30 10384-10498 149020 4A 8.09 5b wood Taxodium distichum 10000 30 11527-11603 149785 4A 8.11 5a wood Unidentifiedhardwood 10995 40 12768-12915 149026 4A 8.15 5a Stem- Monocot(Bald cypress) stem - 10590 35 12547-12634 149022 4A 8.37 4c wood Unidentified 11020 30 12811-12950 143537 4A 8.5 4c charrtwig Taxodiumcharred distichum 11060 35 12857-12995 149028 4A 8.51 4c wood Quercushardwood (Oak) 11120 45 12962-13071 ed 149034 4A 8.51 4c twig Taxodium(Bald cypress) distichum 11095 35 12919-13048 149039 4A 8.79 4c wood Morus rubra (Red 11875 30 13613-13679 149030 4A 8.86 4c woodseed Taxodium(Bald cypress) distichum 12230 45 14057-14185 149031 4A 9.09 4c wood Morusmulberry) rubra (Red 11810 35 13590-13654 149032 4A 9.24 4c twig Morus(Bald rubra cypress) (Red 11895 40 13703-13770 149033 4A 9.35 4b wood Quercusmulberry) (Oak) 12235 45 14061-14192 149034 4A 9.49 4b wood Quercusmulberry) (Oak) 12175 40 14003-14125 149035 4A 9.58 4a twig Quercus (Oak) 12385 40 14236-14571 143536 4A 9.95 3c wood Unidentified 12410 30 14312-14646 149048 4A 10.01 3c wood Diospyros virginiana 12465 40 14427-14812 143531 4A 10.08 3c twig Unidentifiablehardwood twig 12375 35 14215-14527 149042 4A 10.2 3c twig Morus(Common rubra (Red 12335 40 14149-14414 149042 4A 10.38 3a branc Taxodium distichum 13625 45 16308-16511 3 persimmon)mulberry) 0 Table4 3 . AMS Dates from Cores 3A andh 4A. Dates(Bald were cypress) calibrated using Calib0 7.1. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson,wood Florida ” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd.

Figure 12. Composite of cores 4A (left) and 3A (right) with uncalibrated AMS dates. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd.

Sporormiella is a genus of coprophilous fungus that has been established as a reliable proxy for large herbivore abundance (Figure 13) (e.g. Davis ,1987; Burney et al.,

2003; Burney and Flannery, 2005; Davis and Shafer, 2006; Gill et al., 2009; Baker et al.,

2013). The spores are obligate to herbivore dung and can be used as a measure of local herbivore populations (Raper and Bush, 2009; Gill et al., 2013; Baker et al., 2016). Dung fungal spores are discharged from the fruiting bodies within a few centimeters (e.g.

Yafetto, et al. 2008), and wind can carry the spores up to 100 meters (Gill et al., 2013). A low amount of spores (known as “threshold” levels, contributed by small mammals) are also deposited into sediments. Whether transported by wind or runoff, dung fungal spores typically remain local to the dung source (Baker, et al. 2016). Thus, this proxy can only reliably infer changes in the large herbivore community at the site and in the immediate vicinity.

It has even been suggested that declines in dung fungi can indicate human colonization, citing evidence that extinction events worldwide have followed human settlement into a new area (Burney et al., 2003; Burney and Flannery, 2005; Gill, 2014).

However, Fiedel (2016) refutes this idea due to problems in both cultural chronology and inconsistencies between the dung fungi and faunal records. Despite such disparities, methodologies have become increasingly reliable, and dung fungi have been demonstrated to reflect relative large herbivore abundance and activity in many modern settings (Raper and Bush, 2009; Parker and Williams 2012; Gill et al. 2013; Baker et al. 2016).

There are factors aside from herbivore abundance that can influence the

Sporormiella record, such as hydrologic activity. Fluctuating water levels and changes in relative humidity have the potential to skew a Sporormiella record (Raper and Bush, 2009;

Baker et al., 2013; Wood and Wilmhurst, 2013). Analyzing multiple cores from different locations within the same site, however, can help reduce these potentially misleading

signatures caused by increased spore deposition that occurs on the shoreline in comparison to the center of ponds and lakes (Johnson et al., 2015).

IV.2 Study Site

IV.2.1 Regional Settings

Most of the Southeast was characterized by boreal forests until about 15,000 cal

BP, after which warmer temperatures resulted in the emergence of mixed deciduous forests and open park and scrublands (Watts, 1980; Delcourt and Delcourt, 1988; Delcourt, 2002).

This period was followed by the Younger Dryas (YD), beginning around 12,900 cal BP, with cooler temperatures across most of North America. An increase in boreal vegetation at lower latitudes, corresponding to the YD, indicates regional cooling in the Southeast

(Watts, 1980). For example, Picea (Spruce) appeared to flourish as far south as northern

Florida at Camel Lake around 12,600 BP (Watts et al., 1992). Other evidence of relatively cool, wet conditions in the region comes from Page-Ladson and Sheelar Lake with the expansion of Pinus (pine) and mesic forest during the beginning of the YD (Watts and

Stuiver, 1980; Grimm et al., 2006; Hansen, 2006). Regional cooling indicated by records from north Florida is consistent with other southeastern records, such as Dust Cave Goshen

Springs, Alabama (Delcourt, 1980), where an expanse of boreal forest occurred at the onset of the YD.

South Florida was also likely cool, but wetter during the YD (Dunbar, 2006a).

Records from Lake Annie demonstrate an increase in precipitation after 13,000 BP with the expansion of oak scrub at the expense of more drought tolerant plants such as Ceratolia

(sand-hill rosemary) (Watts, 1975). Vegetation responses to the YD in peninsular Florida diverge from trends apparent elsewhere in North America. Both Little Salt and Warm

Mineral Springs reflect an earlier warming trend with the formation and preservation of peats during the middle and late YD (Cockrell and Murphy, 1978; Brown and Cohen,

1985)

IV.2.2 Page-Ladson site

The Page-Ladson site occurs in an inundated, mid-river sinkhole in the Aucilla

River, a karst-entrenched drainage system characterized by alternating reaches of subaerial and subsurface flow. A nearly continuous terminal Pleistocene-early Holocene sedimentary record accumulated with rising water levels in response to post-glacial sea-level rise.

Because these sinkholes are perennially wet, the organic materials within them are not subject to destructive cycles of wetting and drying (Campbell and Campbell, 1994).

Early excavations at the site revealed evidence of terminal Pleistocene human occupation (Dunbar et al., 1988). Early cultural materials discovered during initial excavations at Page-Ladson include a mastodon tusk with cut marks and flaked-stone artifacts older than 13,950 cal BP (Dunbar, 2002, 2006b; Webb, 2006). Thick mats of mastodon digesta alongside mastodon skeletal remains indicate frequent and extensive use of the sinkhole, probably as a water source (Webb, 2006). Recent excavations recovered an additional flaked-stone artifact from deposits dated to ~14,550 cal BP (Halligan et al.,

2016).

Previous paleoenvironmental analyses at Page-Ladson include a study of pollen and charcoal (Hansen, 2006). Abundances of Amaranthaceae (amaranth family) and prairie or dry forest taxa indicate a cool and dry climate between 14,800-14,000 cal BP. An increase in mesic forest taxa pollen such as Quercus (oak), Carya (hickory), Acer (maple),

Tilia (basswood), and Taxodium (bald cypress) alongside a decrease in herbacaeous pollen

in sediments younger than 12,500 cal BP suggests a shift to a wetter climate by the beginning of the YD. By about 10,500 cal BP, herbaceous pollen, charcoal and degraded pollen increased at the expense of mesic forest taxa, suggesting warmer and drier conditions at the site. Previous pollen studies at Page-Ladson provide a foundational understanding of local vegetation. However, additional research is needed with a better- constrained chronology. Because the previous study by Hansen (2006) was completed using samples from various excavation sidewalls, analyzing one continuous, well constrained, stratified record can provide better chronological control and sampling resolution than was previously possible.

One core (Core 4A) was subject to previous research, including an analysis of

Sporormiella abundance (Halligan et al., 2016). During analysis, the patterns that emerged allowed the core to be divided into four distinct zones based on Sporormiella accumulation patterns. Sporormiella Zone 1 spans 15,050-12,700 cal BP. A Sporormiella peak at

~13,000 cal BP may indicate the last abundance of proboscideans in the region, as spore accumulation gradually declines toward 12,700 cal BP, the end of Zone 1. Zone 2 begins after ~12,700 cal BP and is characterized by slow to nonexistent Sporormiella accumulation that likely signifies the decline and disappearance of megaherbivores at

Page-Ladson. The beginning of Zone 3 is characterized by an increase in Sporormiella after 10,775 cal BP, with a peak at ~10,400 cal BP. Because it is widely accepted that megafaunal extinctions in North America occurred prior to the beginning of the Holocene, this peak is anomalous. It is commonly accepted that the dung of smaller herbivores is less represented in the dung fungal spore record than larger herbivores (Davis, 1987; Davis and

Shafer, 2006; Gill et al., 2013; Baker et al., 2016); therefore, this may represent a time

when larger herbivores such as bison ranged south into Florida. Zone 4 begins at ~10,100 cal BP and is capped by a date of 8,700 cal BP at the top of the core. Sporormiella is completely absent from this zone. Palynomorph preservation in this zone of the core is good, and pollen concentration is high, ruling out the possibility that a lack of

Sporormiella stems from preservation issues.

IV.3 Methods

Cores 3A and 4A were collected off of a pontoon boat using a modified

Livingstone coring device, with the assistance of SCUBA divers. These cores were consistently refrigerated to inhibit microbial activity. Bulk sediment samples were extracted systematically at 10- and 20-cm intervals from Core 4A and 3A, respectively.

More intensive sampling occurred along intervals of drastic changes in Sporormiella concentration as determined by the results of the previous analysis. Pollen and

Sporormiella analyses were conducted on paired 1-cm3 subsamples. Different processes were used to extract pollen and Sporormiella from the subsamples, given the dissimilarity in size between pollen and the smaller Sporormiella subspores, which are typically about

10 microns (Ahmed and Cain, 1972).

IV.3.1 Sporormiella Processing and Analysis

After adding five tablets with ~18,584 Lycopodium spores as tracers, the

Sporormiella subsamples were processed using 5% KOH, 15% HCl, 48% HF, acetolysis solution and a series of screens with no smaller than 70-micron mesh (Faegri and Iversen,

1989). The samples were then curated in glycerine and mounted on slides. The slides were scanned at 400X magnification and Sporormiella were counted until 350 Lycopodium spores were identified following the methods of Etienne and Bapicot (2014).

IV.3.2 Pollen Processing and Analysis

Subsamples for pollen analysis were subject to the same processes used for

Sporormiella sample (Faegri and Iversen, 1989), with further measures such as sonication- assisted screening to remove unwanted organic debris smaller than 10 microns. Multiple rounds of heavy density separation using both 1.8 and 2.0 specific gravity zinc bromide solution were used to further rid the samples of organic debris. The samples were curated in glycerine and mounted on slides, which were scanned at 400X magnification until a total of 200 pollen grains were counted (Barkley, 1934; Bryant and Holloway, 1983).

IV.3.3 Analysis and Interpretations

Pollen and Sporormiella concentrations were determined by comparing the total fossil palynomorphs to the known number of Lycopodium in each sample (five tablets,

18,584 spores/tablet). Pollen data are quantified in terms of relative abundance and total pollen concentration, a measure that provides important information regarding increases in sediment deposition, preservation, and pollen rain. Sporormiella data are presented as concentration, accumulation rates and as a percentage of the total pollen assemblage. Other coprophilous fungi including Cercophora, Coniochaeta, Podospora and Sordaria were all noted but were not quantified in this study due to a lack of proper reference materials required to consistently and reliably identify each taxon. To calculate Sporormiella accumulation, Sporormiella concentrations were multiplied by a Bayesian deposition model (Figure 14), which estimates the rate of sediment deposition for the entire core

(Ramsey et al., 2006). These radiocarbon assays were acquired primarily from terrestrial plants. Carbonized remains were preferentially chosen. Age-depth relationships for core

3A are not modeled due to insufficient radiocarbon assays, but based on thicknesses of

correlated depositional units in 4A, the same sedimentation rate is assumed. Sporormiella data from both cores is presented as concentration as well as accumulation (Table 4).

Sporormiella was calculated as a percentage of the total pollen assemblage based on the amount of Lycopodium spores encountered from both pollen and Sporormiella samples.

Sporormiella was not included in calculations of relative abundance of different pollen types.

1 before Pleistocene Sporormiella reflects populations of Mastodon 12,600 etc. Spore accumulation is variable throughout the lowest part of core 4A, likely as a reflection of sandy nature of sediments. 2 12,599- A total absence of Sporormiella during this period represents 10,750 the disappearance of large herbivores. 3 10,749- A resurgence of Sporormiella in Holocene-aged sediments 10,200 demonstrates a reappearance of extant megaherbivores (perhaps Bison bison) in the area. 4 after 10,199 Absence of Sporormiella until the top of core reflects a second reduction or disappearance of megaherbivores in the region.

Table 4. Sporormiella zones from Page-Ladson site cores 3A and 4A, as discussed in text (Halligan et al. 2016). Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd.

Figure 14. Bayesian age model for Core 4A. Reprinted from with permission from “Pre- Clovis occupation 14,550 years ago at the Page-Ladson site, Florida, and the peopling of the Americas”, by Halligan, J.J., Waters, M.R., Perrotti, A.G., et al., 2016. Science Advances 2, e1600375–e1600375. doi:10.1126/sciadv.1600375, copyright by AAAS. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd.

IV.4 Results 90

IV.4.1 Sporormiella Results

IV.1.1 Zone 1 (Before 12,700 cal BP). The Sporormiella recovered from Core 3A generally confirms the patterns observed in Core 4A and allowed for the establishment of biogenic zones based on these patterns observed by visual inspection (Table 4, Figure 15).

This zone extends to the bottom of both cores, though the bottom of core 3A dates to approximately 14,100 cal BP, while the bottom of core 4A is older than 15,050 cal BP.

Some of the areas of this zone have low Sporormiella accumulation rates, which coincide with relatively low pollen concentration values. Pollen concentration is also moderate to high during periods of Sporormiella absence (~12,700-10,750 cal BP and after 10,200 cal

BP), suggesting the lack of Sporormiella during these periods is not a consequence of poor preservation. Both cores exhibit a final peak of Sporormiella around 13,000 cal BP, followed by a decline and disappearance by 12,700 cal BP.

Figure 15. Sporormiella concentration, accumulation and percentage of total pollen assemblage from Cores 4A and 3A, alongside pollen concentration and relative abundance of arboreal and herbaceous pollen types, with zones determined through visual inspection. Some of the areas of the core containing sediments aged ~15,000-13,000 cal BP with low Sporormiella accumulation rates coincide with relatively low pollen concentration values. Pollen concentration is also moderate to high during periods of Sporormiella absence (~12,700-10,750 cal BP and after 10,200 cal BP), suggesting the lack of Sporormiella during these periods is not a consequence of poor preservation. Measures of spore influx and concentration are vulnerable to uneven pollen preservation within a sedimentary record, because the conditions that degrade pollen may also degrade spores. As shown, the percentage diagram is not susceptible to this effect. However, there are still some areas of the Zone 1 that exhibit low Sporormiella abundance. It is possible that these represent times with relatively low megaherbivore abundance near the site. The consistent decline and disappearance of Sporormiella by the end of this zone (12,700 cal BP) likely signifies the disappearance of Pleistocene megaherbivores from the site. “Arboreal” includes pollen from trees and from taxa associated with mesic environments (such as Vitis). “Aboreal” also excludes Pinus pollen, which was probably transported to the site rather than locally present. This indicates a relative abundance of mesic forest vegetation until ~12,500 cal BP. After 12,500 cal BP, the vegetation at the site reflects a warmer, drier period resulting fewer trees and more open space. After 10,500 cal BP, it appears that conditions became even drier and warmer based on the dramatic increase in herbaceous pollen at the expense of arboreal types. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd.

IV.4.1.2 Zone 2 (~12,700-10,750 cal BP). This zone is characterized by the absence or near absence of Sporormiella in both cores and dates to about 12,700 cal BP until 10,750 cal BP.

IV.4.1.2 Zone 3 (10,749-10,200 cal BP). This zone consists of Sporormiella accumulation at levels comparable to or even higher than terminal Pleistocene levels.

Beginning after 10,750 cal BP, Sporormiella accumulation rises until it peaks between

10,675 and 10,400 cal BP. In core 3A, the peak of about 860 spores per centimeter per year at about 10,675 cal BP exceeds the spore accumulation rate demonstrated in the late

Pleistocene spore peak of 216 spores per centimeter per year around 13,000 cal BP.

Sporormiella accumulation in both cores rapidly declines until about 10,200 cal BP when it nearly disappears.

IV.4.1.4 Zone 4 (after 10,200 cal BP). Sporormiella is absent in both cores in sediments dated younger than 10,200 cal BP. Pollen preservation is good in this section of the core, and pollen concentration levels are high (Figure 15), ruling out the possibility that the lack of Sporormiella stems from taphonomic issues.

IV.4.2 Pollen Results

The pollen assemblage from core 4A demonstrates variability in the vegetation in the region throughout the Pleistocene-Holocene transition (PHT) (Figures 16, 17). Both plant and animal communities are likely responding to changes in climate during this time period. Visual inspection suggests a strong relationship between the pollen assemblage and the Sporormiella record also exists. Zones delineated by changes in pollen frequencies co- vary with changes in the concentration of Sporormiella (Figures 16, 17).

Figure 16. Selected important arboreal pollen types from Core 4A by modeled calibrated dates BP, with zones determined through visual inspection. Reprinted with permission from “Pollen and Sporormiella evidence for terminal Pleistocene vegetation change and megafaunal extinction at Page-Ladson, Florida” by Angelina G. Perrotti, 2017. Quaternary International, Copyright 2017 by Elsevier Ltd.

IV.4.2.1 Zone 1 (before 12,700 cal BP). This zone largely consists of pollen representing a mesic hardwood forest (a North American forest ecosystem containing elements of boreal and oak-hickory forests) or a mesic parkland with the exception of the lowest parts of the core. Sediments older than ~14,500 cal BP contain fewer hardwood deciduous forest pollen such as Betula (Birch), Fraxinus (Ash), Liquidambar (Pink Gum),

Ostrya/ Carpinus (Hornbeam), and Salix (Willow). The younger sediments in this zone exhibit an overall increase in mesic hardwood deciduous forest types, particularly between

~14,500 cal BP-13,500 cal BP (Figure 16).

IV.4.2.2. Zone 2 (12,700-10,750 cal BP). Warmer, drier conditions during this period are indicated by a decrease in hardwood forest taxa. Betula, Carya, Celtis

(hackberry), Fraxinus, Ostrya/Carpinus, Morella (wax myrtle) and Taxodium (bald cypress) pollen all decline at the end of this zone. This zone includes a dramatic increase in the amount of Poaceae pollen, which peaks at nearly 30% of the total pollen assemblage around 11,900 cal BP. Pinus pollen also peaks during this time.

IV.4.2.3 Zone 3 (10,750-10,200 cal BP). This zone includes the resurgence of both

Sporormiella and Ambrosia pollen into the record. It only represents about 500 years, and the vegetation remained somewhat unchanged throughout this time. The pollen assemblage is similar to the pollen from Zone 2 (see section IV.4.2.2).

IV.4.2.4 Zone 4 (after 10,200 cal BP). This zone represents an absence of large megaherbivores in the region after 10,200 cal BP. Around 10,000 cal BP, a drastic vegetation transition occurred. From 10,000 cal BP until at least 8,700 cal BP (at the top of the core), abundance of herbaceous and Poaceae pollen dominate the record at ~35-55% in comparison to arboreal types which represent ~27-42% of the pollen assembage (Figure

15). The only abundant arboreal taxon after 10,200 cal BP is drought-tolereant Quercus, which makes up between 22-45% of the total pollen assemblage. Amaranthaceae pollen is the most abundant type during this time.

IV.5 Discussion

This study is one of the first major dual investigation of PHT plant and animal communities in the Southeast using palynological proxy measures. In addition, the sedimentary record from Page-Ladson has tight chronological control, allowing finer resolution regarding both megaherbivore population and vegetation changes than any previous record in Florida for the PHT.

IV.5.1 Zone 1 (Before 12,700 cal BP)

In general, the pollen from Page-Ladson confirms the previous pollen investigation from this site (Hansen, 2006). Cooler, drier regional climate prior to 14,500 cal BP is indicated in the pollen evidence presented in this study (see section IV.4.2.1) by reduced deciduous hardwood taxa, and from a pine forest at Camel Pond (Figure 11), about 100 km northwest of Page-Ladson (Watts et al., 1992). Furthermore, a relatively cool and dry climate in peninsular Florida is also represented by an abundance of Pinus and herb pollen at Sheelar Lake (Watts et al., 1982), and high levels of Ceratolia (sand-hill rosemary) and

Polygonella (jointweed) at Lake Annie (Watts, 1975).

This pollen investigation demonstrates that Page-Ladson became slightly warmer after 14,500 cal BP, based on an increase in hardwood forest taxa. During this time, pollen from plants comprising a mesic forest such as Betula, Carya, Fraxinus, Morella,

Liquidambar and Ulmus (Elm) are abundant. In addition, other mesic and riparian plants such as Vitis (Grape), Salix and Cyperaceae (sedges) are present as well. The abundance of

arboreal pollen certainly suggests many hardwood deciduous trees were present near the site, but it is important to note that these could have been growing in a patchy environment surrounding the sinkholes in the Aucilla River. Poacaeae (grass) pollen reaches levels of

10% abundance, suggesting that some open patches existed in the region.

This terminal Pleistocene transition from a cool, dry climate to a warmer, more mesic climate is also demonstrated by previous investigations at Page-Ladson noting an increase in Quercus, Carya, Acer, Tilia and floodplain taxa such as Taxodium by 12,500 cal BP (Hansen, 2006). Although the abundance of the aforementioned taxa is variable in this current study, the general increase in arboreal pollen types (Figure 16) supports

Hansen’s (2006) interpretation of a wetter climate. These changes in the pollen assemblage coincide with a decrease in herbaceous pollen such as Ambrosia (Hansen, 2006). Camel

Pond pollen also reflects this pattern by a dominance of Quercus, Fagus (Beech), and

Ostrya, suggesting warming temperatures by 12,600 cal BP. Pollen studies from Sheelar

Lake (Watts and Stuiver, 1980) and Lake Tulane (Watts and Hansen, 1988, 1994) in peninsular Florida demonstrate similar patterns with an increase in mesic forest taxa such as Carya, Cupressaceae (cypress family, i.e. juniper and bald cypress) and Ulmus.

Sporormiella in this zone attests to consistent presence of megaherbivores in the vicinity of the Page-Ladson site. Sporormiella accumulation rates are variable throughout the bottom of both cores (4A in particular), but it is likely that reduction in the accumulation of spores is due to depositional factors. The samples with low levels of

Sporormiella are predominately sand and have low pollen concentration (Figure 15). In these situations, it is useful to look at Sporormiella as a percentage of the total pollen assemblage (Figure 15). Measures of spore influx and concentration are vulnerable to

uneven pollen preservation within a sedimentary record, because the conditions that degrade pollen may also degrade spores. Percentage diagrams are not subject to this effect.

However, there are still some areas of the Zone 1 that exhibit low Sporormiella abundance.

It is possible that these represent times with relatively low megaherbivore abundance near the site. The consistent decline and disappearance of Sporormiella by the end of this zone

(12,700 cal BP) likely signifies the disappearance of Pleistocene megaherbivores from the site.

Pollen from plants that thrive in dry climate or recently disturbed soil remains fairly low during this time period, with the exception of Ambrosia (ragweed). However,

Ambrosia has been cited as a potential grazing indicator because of its abundance alongside Sporormiella in modern studies of bison pastures (Gill et al., 2013). The relationship between Sporormiella and Ambrosia observed in this record is dramatic, supporting that the presence and abundance of this disturbance plant may an indicator of herbivore activity. Ambrosia pollen remains between 10 and 20% of the total pollen assemblage until about 12,700 cal BP. At this time, both Ambrosia and Sporormiella decline and disappear, indicating the decline of herbivores in this region at this time.

IV.5.2 Zone 2 (12,700-10,750 cal BP)

Evidence of drier conditions is demonstrated by the pollen assemblage in this zone

(section IV.4.2.2). At ~23%, Pinus pollen is abundant in this part of the core, but it is probable that this increase is a result of pollen overrepresentation, rather than real changes in the vegetation community. For example, Asteraceaes (including Ambrosia) face a marked decrease during this period. It is probable that, due to the slowing of colluvial deposition and the decrease in pollen production by arboreal plants, background levels of

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Pinus became overrepresented. Pinus pollen can travel over 100 kilometers (Mack and

Bryant, 1974). Most of the Pinus pollen recovered from this core was severely degraded, which suggests long distance transportation of non-local pollen grains (Erdtman, 1969, pp.486). In addition, Pinus macrobotanical remains have not been recovered from Page-

Ladson (Newsome, 2006). If Pinus is removed from the relative comparison of herbaceous and arboreal pollen abundance (Figure 15), herbaceous pollen is about equally represented during this period. A drastic increase in Amaranthaceae pollen to 30% abundance toward the end of this zone also suggests that conditions became warmer and drier during this time. Because of the absence of large herbivores, which are known ecosystem regulators, fires may have become more frequent (also indicated by an increase in charcoal observed by Hansen, 2006), further contributing to the rise in plants that colonize disturbed ground,

This zone coincides with the majority of the YD cool period and the absence of

Sporormiella from these sediments probably signifies the absence of megaherbivores in the region at this time. Sporormiella is robust with a thick cell wall, and is resistant to degradation in both soils and in chemical laboratory procedures (van Asperen et al., 2016).

Because pollen is also present and well preserved during this zone (Figure 15), it is not expected that the low abundance of Sporormiella in this zone is a result of a taphonomic bias.

IV.5.3 Zone 3 (10,750-10,200 cal BP)

Further warming and drying in the beginning of the Holocene is indicated by pollen that suggests a transition from forest or parkland to a more open environment, such as an oak savanna. Climatic conditions were warmer and drier than those indicated by

Pleistocene sediments based on the lack of mesic hardwood taxa. The end of this zone

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indicates conditions became even drier, warmer and more disturbed due to an increase in fire frequency around 10,200 cal BP, which is also represented by an increase in charcoal observed during previous paleoenvironmental analyses at Page-Ladson (Hansen, 2006).

This interpretation is also based on an increase in Astereaceae to over 20% and a decrease in arboreal taxa with the exception of Quercus, which is relatively drought tolerant in comparison to other trees. Salix and Ulmus pollen also increase toward the end of this zone, which may reflect increased riparian activity at the site during this time, rather than directly indicating an increase in precipitation.

The Sporormiella resurgence in this zone is anomalous because it occurs after the period of extinction suggested by faunal evidence. It is possible that this zone could reflect the incursion of large herbivores into the area during this time. Previous research implies that the dung of small herbivores is less represented in the dung fungal record in comparison to the dung of large herbivores (Davis, 1987; Davis and Shafer, 2006; Gill et al., 2013) and are unlikely to provide as significant a contribution to Sporormiella abundance; therefore, this may represent a time when bison herds ranged into Florida. Both archaeological and historic evidence support the plausibility that bison may have inhabited

Florida during the early Holocene. Despite a lack of dated bison remains throughout the

Holocene in Florida (Rucker, 1992), bison have been archaeologically and historically documented as much as ~450 km south of Page-Ladson, near present day Tampa Bay

(Rostlund, 1960).

IV.5.4 Zone 4 (after 10,200 cal BP)

Conditions continued to become more xeric after 10,200 cal BP as evidenced by the increase in pollen from more shade intolerant plants, suggesting an oak savanna-like

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environment. The abundance of herbaceous and Poaceae pollen become more abundant in the record in comparison to arboreal types (Figure 15). The decrease in arboreal taxa after

10,200 cal BP is particularly notable because trees often produce much more pollen that travels longer distances than herbaceous plants (Tauber, 1965)

The warming and drying at Page-Ladson around 10,200 cal BP is also established in previous studies at the site by decreased mesic taxa and an increase in charcoal, degraded pollen, Amaranthaceae and Asteraceae pollen (Hansen, 2006). The decline of mesic hardwood types also occurs at Lake Tulane (Watts and Hansen, 1988, 1994) and

Sheelar Lake (Watts and Stuiver, 1980) by about 10,000 cal BP. In addition, a hiatus in deposition caused by lower water levels at Camel Lake beginning around 10,500 cal BP provides further evidence of a drier climate during this time (Watts et al., 1982).

The absence of Sporormiella in sediments dated younger than 10,200 cal BP indicates another disappearance of large herbivores in the region. Pollen preservation is good in this section of the core, and pollen concentration levels are high (Figure 15), ruling out the possibility that the lack of Sporormiella stems from depositional or taphonomic issues. It is currently unclear what may have caused the secondary abandonment of the site by large herbivores after 10,200 cal BP. The changing vegetation at Page-Ladson in the early Holocene demonstrated by an increase in herbaceous taxa and a decrease in mesic forest pollen types, may have been the result of climate change or a changing herbivore population given the following reasons. First, it is feasible that warmer temperatures and more arid conditions led to the reduction of the mesic forest vegetation. After the Younger

Dryas, most of North America became warmer, and changing vegetation suggesting a warmer, more arid climate is also seen throughout Florida, as previously discussed.

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Second, it is possible that the disappearance of large browsers contributed to a shifting vegetation regime. Large herbivores are considered ecosystem regulators and alter fire regimes and successional plant communities. Bison can inhibit woody vegetation from growing through grazing, trampling and wallowing (Hester et al., 2006). However, elephants (and mastodons) can maintain forest ecosystems by creating gaps in vegetation and disbursing seeds in their dung, allowing for the new saplings to grow.

The effects that large herbivores have on the environment are not always straightforward and the classic understanding of large grazers’ effects on the environment is oversimplified. For example, Gill et al. (2009, 2012) argue that the late Pleistocene decline of megaherbivores in the Midwest and Northeastern United States resulted in woody vegetation overgrowth and increased fires. Although this may be the case, the interpreted vegetation response to megaherbivore noted here cannot necessarily be ubiquitously applied to different sites. Declining plant competition and grazing interplay occurs differently in diverse environments based on various plants’ light requirements and herbivore foraging preferences. Grazing typically increases vegetation diversity, unless the soil is very dry or very wet (Hester et al., 2006).

Large herbivores also influence tree recruitment, typically by increasing open space within woodlands. Bark stripping by elephants (and mastodons) can make trees more vulnerable to fire, which can also reduce canopy density, leading to more vegetation growth at ground level and ultimately, more tree growth. In this situation, an open forest setting with more fire-resistant trees might emerge. Trampling is another factor to consider, and has mixed effects on plant communities. Although trampling can inhibit sapling and small plant growth, it can also rework hardened sediment and soil, allowing seeds to

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successfully germinate, thereby increasing plant diversity. Nutrient cycles can also be severely altered by grazing. Herbivore urine and feces typically enhance the nutrient cycle by increasing nitrogen content.

IV.5.5 Anthropogenic Relationship

The Sporormiella evidence from Page-Ladson suggests that large herbivores were extinct in the Aucilla River region by around 12,700 BP, which coincides with broader assumptions about the timing of continent-wide megafauna extinction in North America.

Whether this extinction is driven by climate or human hunting is still largely debated (see

Haynes, 2009).

Evidence for an anthropogenic cause of megafaunal extinction includes multiple archaeological examples of humans hunting and butchering megafauna in North America

(e.g. Haury et al., 1953, 1959; Leonhardy, 1966; Hester, 1972; Frison and Todd, 1986;

Haynes and Huckell, 2007; Sanchez et al., 2014). At Page-Ladson, a mastodon tusk exhibits cut marks that appear to suggest human butchering (Webb, 2006; Halligan et al.,

2016). Human colonization resulting in extinction is a worldwide phenomenon and has been documented on islands such as New Zealand and Madagascar (Burney et al., 2003;

Burney and Flannery, 2005). A recent surge of model- and simulation-based evidence has also demonstrated the likelihood that human hunting led to extinctions. Statistical analysis of the radiocarbon record has demonstrated that end Pleistocene extinctions in North

America occurred so rapidly; they were geologically simultaneous (Faith and Surovell,

2009). Additional mathematical models involving parameters such as prey choice, patch choice and mammal population stability consistently demonstrate that even a small increase in predatory behavior (such as that by humans) could increase death rates enough

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to decimate a population of slow breeding mammals (Grund et al., 2012; Frank et al.,

2015). Another simulation by Araujo et al. (2015) acknowledges that climate change probably stressed large mammal communities, but that human arrival was a necessary factor to cause extinctions.

The discovery of a bifacially flaked stone tool in a stratum dating to 14,550 cal BP suggests that humans and megafauna coexisted at Page-Ladson for over 2,000 years

(Halligan et al., 2016). This long period of coexistence indicates that if humans did cause extinction from overhunting, it did not occur as rapidly as previously suggested (Martin and Klein, 1986). However, Sporormiella and pollen evidence at Page-Ladson does not suggest that megaherbivore disappearance from the site was immediately preceded by a drastic shift in vegetation. It is possible that a combination of anthropogenic and climatic influence resulted in megafaunal extinction at the site. General warming during the terminal Pleistocene would have stressed animals, and a return to dry, glacial-like conditions with the onset of the YD around 12,900 cal BP may have shocked these mammal populations and placed too much stress on breeding populations. Then, an intensification of Clovis hunting further decimated the megaherbivore communities

(Haynes, 2013).

Humans in the area would have fairly rapidly adapted to both the changes in potential game, but also in climate and vegetation. The PHT is characterized by an increase in diet breadth amongst humans in Florida and a shift from large lanceolate Paleoindian points to smaller, corner- and side-notched Archaic points. Despite this shift, larger side and corner notched points such as Bolen (coinciding with the early Holocene bison peak at

Page-Ladson) could have been used to hunt bison. The post-Pleistocene sediments at Page-

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Ladson show evidence of regular usage by Early Archaic populations in the form of hearths and discarded projectile points, adzes, and flake tools (Carter and Dunbar, 2006).

IV.6 Conclusion

The pollen and Sporormiella record at Page-Ladson provides compelling evidence for dramatic environmental changes in late Pleistocene-early Holocene Northwestern

Florida. Based on the disappearance of Sporormiella from two sediment cores at the site, it is likely that megaherbivores disappeared from the region by ~12,700 cal BP. However, the anomalous spore peak between ~10,750-10,200 cal BP may indicate an incursion of large herbivores such as bison into the region. The decline and disappearance of

Sporormiella from sediments dated younger than 10,200 cal BP suggest that this incursion was brief, and large herbivores were no longer in abundance after this time. Based on the existing archaeological and faunal evidence, as well as the evidence presented in this paper, it is currently unclear which large herbivores contributed to the Early Holocene

Sporormiella increase.

Pollen evidence from the site also reflects dramatic vegetation changes, which are likely a response to both changing climate and fluctuating herbivore populations. Prior to

14,500 cal BP, the pollen assemblage reflects a relatively cool and dry climate. Between ca. 14,500-12,600 cal BP, the sediments are characterized by an increase in hardwood forest and mesic plant taxa, indicating an increase in both temperature and precipitation.

After 12,600 cal BP, a decrease in arboreal pollen, with the exception of oak, alongside an increase in herbaceous pollen, indicates drier, warmer conditions. These results contextualize changes in human behavior at the onset of the Holocene.

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Overall, the submerged sites in the Aucilla River yield not only a rich cultural record, but also microscopic organic remains that can help reconstruct the environment that some of the earliest inhabitants of North America would have been tied to. Future studies in the Aucilla River region should seek to better understand this Early Holocene

Sporormiella peak, as well as associated plant and animal remains. Other submerged sites in the Southeast also have the potential to further strengthen interpretations of megafaunal extinction as well as vegetation changes in response to changing climatic conditions and herbivore influence.

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Webb, S.D., Dunbar, J.S., 2006. Carbon dates, in: First Floridians and Last Mastodons:

The Page-Ladson Site in the Aucilla River. Springer, New York, pp. 83–101.

Webb, S. D., Graham, R.W, Barnosky, A.D., Bell, C.J., R., Hadly, E.A., Lundelius, H.,

McDonald, G., Martin R.A., Semken, J., Steadman, D.W., 2003. Vertebrate

paleontology. In Gillespie, A.R. (Eds.), Developments in Quaternary Sciences: The

Quaternary Period in the United States, Volume 1. Elsevier.

Wood, J.R., Wilmshurst, J.M., 2012. Wetland soil moisture complicates the use of

Sporormiella to trace past herbivore populations. Journal of Quaternary Science 27,

254–259.

Yafetto, L., Carroll, L., Cui, Y., Davis, D.J., Fischer, M.W.F., Henterly, A.C., Kessler,

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N.P., 2008. The Fastest Flights in Nature: High-Speed Spore Discharge

Mechanisms among Fungi. PLoS ONE 3(9), e3237. doi:10.1371/journal.pone.0003237.

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CHAPTER V

COPROPHILOUS FUNGAL SPORE ACCUMULATION AT WHITE POND, SOUTH

CAROLINA

V.1 Background

Owen Davis (1987) first suggested using the fossil spores of coprophilous fungi

(also referred to as “dung fungi”), Sporormiella, as a proxy for herbivore abundance and disappearance at the end of the Pleistocene. Davis (1977, 1987) recognized the potential of coprophilous fungi in paleoecology when he found Sporormiella spores in direct association with fossilized dung from Bechan Cave, Utah, and also noted the frequency of

Sporormiella spores increased with evidence of overgrazing of domestic livestock in historic-age deposits at Wildcat Lake in Washington State.

Some researchers have even suggested that declines in dung fungi can indicate human colonization, citing evidence that extinction events worldwide have followed human settlement into a new area (Burney, 2003; Burney and Flannery, 2005; Gill, 2014).

However, Fiedel (2016) refutes that due to problems in both cultural chronology and inconsistencies between the dung fungi and faunal records. For example, declines in

Sporormiella observed in sediments younger than ~14,800 cal BP from Ohio and northern

Indiana (Gill et al., 2009, 2012) and New York (Robinson et al., 2005; Robinson and

States dating between 10,000 and 12,000 rcybp (~12,000-14,000 cal BP).

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Despite disparities such as this, in the nearly three decades that Sporormiella and other dung fungi have been used as proxy evidence for megafauna extinctions (Burney and

Although less commonly incorporated into paleoecological studies, other coprophilous fungi taxa are frequently observed alongside in Sporormiella, often in even higher concentrations. These other fungi types, including Cercophora, Conichaeta,

Podospora, and Sordaria (see Table 5) are not necessarily obligate to herbivore dung

(Bell, 1983), but have still been deemed as indicators of herbivore populations (Johnson,

2015). Counting the abundance of these fungi alongside Sporormiella may provide a more complete understanding of ancient herbivore abundance and disappearance, as different fungi may have species-specific reactions to microenvironmental fluctuations.

TYPE HABITAT Cercophora Herbivore dung, decaying wood (Bell 1983; Hanlin 1990) Coniochaeta Herbivore dung, decaying wood (Hanlin 1990)

Podospora Herbivore dung Sordaria Herbivore and omnivore dung, decaying wood (Bell 1983; Mighall et al. 2006) Sporormiella Herbivore dung (Ahmed and Cain 1972)

Table 5. Dung fungi spore types in this study.

This paper presents the results of a dung fungal spore analysis from White Pond,

South Carolina (Figure 18). White Pond is a natural lake about 500 meters in diameter,

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located 40 kilometers northeast of Columbia, South Carolina. This lake is at the intersection of the Coastal Plain and the Piedmont. The current vegetation exists almost entirely of a commerically operated pine plantation, so the natural modern vegetation is largely unknown. This site has been subject to a pollen study in 1980 by Watts, but updated research is needed. Watts’ (1980) study divided the vegetation history at White

Pond into theones. First, from 19,100 BP-12,810 BP, the vegetation primarily consisted of

Pinus, Picea, and various herbs, reflecting a cool, moist climate. After 12,810 BP, the vegetation shifted to reflect a warming climate. From 12,810 BP-9,500 BP, the vegetation was characterized by a decline of Pinus and a total disappearance of Picea. At this time,

Quercus and other hardwoods encroached, forming a mesic hardwood forest. After 9,550

BP, vegetation reflected a drier climate. At this time, Pinus dominated the pollen assemblage alongside evidence of some patches of open prairie in the forest.

This site is an ideal location to extend understanding of the interactions of climate, plants and animals beyond northwestern Florida. Not only does this site demonstrate potential for the recovery of palynomorphs (Watts, 1980), but it also can provide a more regional look at the patterns of megafaunal extinction.

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Figure 18. White Pond, South Carolina.

V.2 Palynological Methods

One cubic centimeter was taken from 35 pollen samples, originally collected by C.

Moore in 2016 from Core WP 2016-3. After the addition of 3 Lycopodium tablets containing 9,666 tracer spores each, the samples were heated for 10 minutes to 80 degrees

C in 5% KOH to remove humates. Screening through 250 micron and 150 micron screen followed. After decanting supernatant liquid, 48% HF was added for 24 hours, followed by an HCl wash. The samples were then subject to Acetolysis with a 9:1 Acetic Anhydride:

Sulfuric Acid solution for 10 minutes at 80 degrees C. Each sample was then screened through 70-micron mesh, stained and curated in glycerine.

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Samples were mounted on slides and scanned using light microscopy at 40x for 20 transects. Lycopodium, Cercophora, Coniochaeta, Podospora, Sordaria and Sporormiella were tallied. Concentration (spores/cm3) of each dung fungi taxa was then calculated using the following formula:

Spores x (9,666 x 3) Lycopodium counted

All dates are presented and discussed in modelled calendar years before present, determined through a Bayesian age-depth model based off of 20 AMS dates on twigs and small aquatic seeds and two bulk-sediment dates (Figure 19). The modelled sample ages were calculated by adding the length of each sample cube to the starting core depth. Each sample age was then averaged and plotted using the age of the middle of the sample.

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Figure 19. Bayesian age-depth model for White Pond, South Carolina (Bronk Ramsey, 2006)

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When analyzing the data, I grouped fungal spores based on how strongly each taxon’s abundance reflects megaherbivore presence. Podospora and Sporormiella are strongly coprophilous and are good indicators of large herbivore abundance. The total spore concentration of these two taxa is presented in Figure 20. Other types such as

Sordaria, Coniochaeta and Cercophora are not strong indicators of megaherbivore presence (i.e. Perrotti and Van Asperen, in prep).

V.3 Results

Based on visual comparison of images of Core WP 2016-3 to the 2015 core and sedimentary units, I have divided up WP 2016-3 into 4 zones. Samples 35-24 are in Zone I, which dates from ~15,300-13,400 cal BP. Sample 23 straddles the contact of Zone I and

Zone II, which includes samples 23-15. This zone dates from ~13,400-12,600 cal BP.

Samples 14-10 are in Zone III, which dates to ~12,600-10,750 cal BP. Zone IV includes samples 9-1 and extends to the top of the section, dated to ~9,900 cal BP.

V.3.1 Zone I (>13,400 cal BP)

All fungal spores remain profuse throughout this zone. Strongly coprophilous

Sporormiella and Podospora are both abundant, with a total concentration of up to 16,000 spores/cm3. Total spore concentration reaches 36,000 spores/cm3.

V.3.2 Zone II (13,400-12,600 cal BP)

Fungi concentrations remain moderate to high throughout this zone. Total spore concentration remains high primarily due to a peak in Coniochaeta. Sporormiella and

Podosopora abundance is more variable than in Zone II. Total spore concentration declines toward the end of this zone, primarily as a result of declining Coniochaeta and Podospora.

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V.3.3 Zone III (<12,690-10,750 cal BP)

With the beginning of this zone corresponding to the Younger Dryas, total spore concentration declines and remains low throughout Zone III. Total fungal spore concentration reaches a high of only ~10,000 spores/cm3. At the end of this zone, strongly coprophilous fungal spores become nearly absent.

V.3.4 Zone IV (<10,750 cal BP)

This zone marks a slight increase in total spore concentration, but is likely due to an increase in Coniochaeta. Although strongly coprophilous fungal spore abundance remains relatively low, a small peak near the top of the section in conjunction with increased Coniochaeta and Cercophora may indicate an increase in herbivore population.

V.4 Analysis and Discussion

Dung fungal spore data as a proxy for large herbivore abundance is best understood in context of the ancient plant community, which can be inferred through the pollen record.

The existing pollen record from White Pond was analyzed by Watts (1980) and is poorly temporally constrained (Table 6). Nonetheless, it provides valuable foundational information. I recalibrated the three dates in Watts’ (1980) pollen report using Calib 7.1.

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Pollen Zone General Taxa Characteristics 23,000-15,260 Pinus/Picea/Herb Pinus=up to 90% of upland pollen Quercus and Carya frequent toward end (estimate ~17200 cal BP) Ambrosia, Artemisia, Polygonella common 15,260-10,920 Fagus-Carya “Mesic” trees=up to 30% Absence of pinus and picea Stable water level=fine algal gyttja with silt 10,920- Pinus-Liquidambar Pinus-Liquidambar an earlier subzone, later present subzone dominated by Pinus

Table 6. Generalized patterns from Watts (1980).

In Zone I, total spore concentration is high, despite each taxon having seemingly independent fluctuations in concentration (Table 7, Figure 20). Coniochaeta and

Sporormiella are most abundant, followed by Podospora. Watts’ (1980) pollen analysis at the site suggests that a major vegetation change from a Pinus (pine) and Picea (spruce)- herb forest to a Fagus-Carya (beech-hickory) forest. This change indicates a more mesic environment that also includes abundances of Quercus (oak), Acer (maple) and Betulaceae

(birch family) after 15,260 cal BP. According to the existing pollen evidence, little change in this Fagus-Carya forest occurred between 15,260 cal BP and 10,920 cal BP. Thus, the declining total spore concentration after the end of sedimentary Zone I does not appear to be immediately preceded by any major vegetation change.

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Sedimentary General Spore Interpretation and Zone-Dates Characteristics Relation to pollen record III Concentration high Abundance of megaherbivores (MHs) >13,400 Little vegetation change immediately preceding OR following decline of MHs II Concentration Possible a decline of MHs, but not 13,400-12,600 moderate-high disappearance Decline not likely result of changing water levels Further decline coincides with onset of YD II & I Concentration low Probable disappearance of MHs <12,600 Noteable Sporormiella peak after ~8700 cal BP

Table 7. Generalized dung fungal spore patterns from Core WP-2016-3.

Zone II is characterized by moderate-high total spore concentrations. Because total spore concentration is moderate, and Coniochaeta (which is also associated with decaying wood, in addition to herbivore dung) (Bell, 1983) is the only fungi with consistently high concentration, this could represent a decline in herbivore population during this zone. The further decline of total spore concentration after the end of this zone, at 12,600 cal BP does not appear to be preceded by vegetation change based on current pollen evidence.

However, this likely decline and potential disappearance of megaherbivores in the region does coincide with the beginning of the Younger Dryas. This is consistent with the timing of megafaunal extinction elsewhere in North America. For example, sediments from Page-

Ladson, Florida demonstrated a drastic decrease in Sporormiella between 12,600-12,700 cal BP (Halligan et al., 2016; Perrotti, 2017).

Sedimentary Zone III consists of low total spore concentration. After 12,600 cal

BP, total spore concentration does not rise above 10,000 spores/cm3. This likely indicates an absence or near absence of large herbivores at White Pond.

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Zone IV marks a slight increase in total spore concentration, but is likely due to an increase in Coniochaeta. Although strongly coprophilous fungal spore abundance remains relatively low, a small peak near the top of the section in conjunction with increased

Coniochaeta and Cercophora may indicate an increase in herbivore population. Although additional research is needed, it is possible that this is analogous to the early Holocene

Sporormiella resurgence at Page-Ladson, which occurred around 10,500 cal BP. It’s possible that analyzing the sediments above the currently analyzed section could provide more information about the cause of this pattern.

Because interest in the paleoenvironment of the Southeast is increasing, we can begin to orient the data from White Pond within other paleoenvironmental research in the region. Though researchers are conducting increasing amounts of paleovegetation research in this area, few are incorporating dung fungal spores into their analyses. To my knowledge, recent research at Cupola Pond, Missouri (Jones et al., 2017) and Page-

Ladson, Florida (Perrotti, 2017) are the only two existing palynological studies to attempt to incorporate dung fungal spores within the analysis from the region. Suprisingly, Jones et al. (2017) found no evidence of Sporormiella spores in their pollen samples and concluded that herbivory was not a key factor in ecosystem regulation in the region. The discrepancy between the observation of dung fungal spores in Jones et al. (2017) study with my own work at Page-Ladson (Halligan et al., 2016; Perrotti et al., 2017) and White Pond raises interesting questions that deserve further exploration.

First, it is possible that the identification of fungal spores and other non-pollen palynomorphs (NPPs) is so rarely taught in traditional North American palynological training, that these NPPs are being overlooked. This issue could be compounded by the

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fact that Sporormiella, the only commonly utilized dung fungal spore in North American studies, can be rare in certain environments. It is not clear exactly which environmental factors control the reproduction and distribution of Sporormiella spores, and they can be scarce even in modern settings with a high volume of herbivore activity (Van Asperen,

2017). The occurrence and abundance of other coprophilous and semi-coprophilous spores could better reflect abundances of Pleistocene megaherbivores, but they have yet to be systematically recorded and published from any site in North America.

Second, this discrepancy could be a result of differences in noting and quantifying dung fungal spores. I analyzed dung fungal spores by counting them against a known number of tracer spores, rather than observing them during pollen counts. Most dung fungal spore studies have used the latter method (Davis, 1987; Davis and Shafer, 2006;

Gill et al., 2009; Jones et al., 2017), but this method is vulnerable to fluctuations in pollen accumulation rates. In sediments with high pollen concentrations, fungal spores are less- well represented. It has been suggested that functional extinction of megaherbivores can be observed when fungal spores fall below 2% of the total pollen assemblage. This was first suggested by Davis (1987), in his work in (mostly arid) western North America. At Page-

Ladson, Florida, Sporormiella spores never constitute more than 2% of the total pollen assemblage (Perrotti, 2017). However, pollen concentration at that site reflects a mesic hardwood forest during many times. If Sporormiella was simply tallied as a percentage of the total pollen assemblage, these fungal spores would have been far less represented than those from arid environments such as those in the desert west, where this method was first conceived of. Pollen concentrations at Cupola Pond (Jones et al., 2017) are similar to those at Page-Ladson, suggesting that a different method of searching for and quantifying dung

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fungal spores may have provided a better understanding of the dung fungal record at the site. It is currently unclear why analyzing Sporormiella as a percentage of the total pollen assemblage has been successful in the northeastern United States.

Third, it is possible that this discrepancy is representative of the actual occurrence of Sporormiella spores. This could be an artifact of environmental factors that affected the reproduction, deposition, or preservation at Cupola Pond. Conversely, it could represent actual variations in the use of each site by megaherbivores. It is still unclear how browsers contribute to the dung fungal record in comparison to grazers. Seasonal migration of herbivores to the sites could also affect the formation of the dung fungal record.

V.5 Conclusions and Future Research

In conclusion, this study from White Pond reflects the need for more research both regarding the methodology and interpretation of dung fungal spore analyses. First, it is important that we begin to establish a more standardized methodology for the quantification and interpretation of dung fungal spores. Second, the application of the data to understanding megaherbivore abundance could be improved by better understanding the relative contributions of browsers and grazers to a dung fungal record. Increased understanding of the environmental factors that affect spore reproduction, deposition, and preservation is also needed. Third, it is important to collect more data throughout the region to both increase spatial and temporal resolution of the data as well as better develop the application of this method.

More research at White Pond is also desirable. Because fluctuations of dung fungi concentrations can sometimes be attributed to depositional biases, such as changing water level, additional research is needed to increase confidence that the spore patterns seen in

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the White Pond record are not a reflection of changing water levels. Though Watts (1980) interpreted fine algal gyttja with silt in Pollen Zone 2 to reflect stable water levels, overall, his study is too poorly temporally constrained to draw any solid conclusions about water level. Additional pollen analyses are needed at White Pond, primarily to increase chronological control of our understanding of vegetation change. Overall, the paleoecological record at White Pond is so rich, so other future analyses could include phytoliths, diatoms and dinoflagellates.

V.6 References

Ahmed, S.I., Cain, R., 1972. Revision of the genera Sporormia and Sporormiella. Canadian

Journal of Botany 50, 419–477.

Asperen, E.N. van, 2017. Fungal diversity on dung of tropical animals in temperate

environments: Implications for reconstructing past megafaunal populations. Fungal

Ecology 28, 25–32. doi:10.1016/j.funeco.2016.12.006

Baker, A.G., Cornelissen, P., Bhagwat, S.A., Vera, F.W.M., Willis, K.J., 2016.

Quantification of population sizes of large herbivores and their long-term

functional role in ecosystems using dung fungal spores. Methods in Ecology and

Evolution 7, 1273–1281. doi:10.1111/2041-210X.12580

Bell, A., 1983. Dung fungi: an illustrated guide to coprophilous fungi in New Zealand.

Victoria University Press, Wellington, NZ.

Burney, D., Robinson, G.S., Burney, L.P., 2003. Sporormiella and the late holocene

extinctions in Madagascar. Proceedings of the National Academy of Sciences of

the United States of America 100, 10800–10805. doi:10.1073/pnas.1534700100

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Davis, O.K., 1987. Spores of the dung fungus Sporormiella: Increased abundance in

historic sediments and before Pleistocene megafaunal extinction. Quaternary

Research 28, 290–294. doi:10.1016/0033-5894(87)90067-6

Davis, O.K., Shafer, D.S., 2006. Sporormiella fungal spores, a palynological means of

detecting herbivore density. Palaeogeography, Palaeoclimatology, Palaeoecology

237, 40–50. doi:10.1016/j.palaeo.2005.11.028

Faegri, K., Iversen, J., 1989. Textbook of pollen analysis. Caldwell, N.J. : Blackburn Press,

c1989; 4th ed. / by Knut Faegri, Peter Emil Kaland, and Knut Krzywinski.

Fiedel, S.J., n.d. The spore conundrum: Does a dung fungus decline signal humans’ arrival

in the Eastern United States? Quaternary International.

doi:10.1016/j.quaint.2015.11.130

Gill, Jacqueline L., Williams, J.W., Jackson, S.T., Lininger, K.B., Robinson, G.S., 2009.

Pleistocene megafaunal collapse, novel plant communities, and enhanced fire

regimes in North America. Science (New York, N.Y.) 326, 1100–1103.

doi:10.1126/science.1179504

Gill, J.L., Williams, J.W., Jackson, S.T., Donnelly, J.P., Schellinger, G.C., 2012. Climatic

and megaherbivory controls on late-glacial vegetation dynamics: a new, high-

resolution, multi-proxy record from Silver Lake, Ohio. Quaternary Science

Reviews 34, 66–80.

Gill, J.L., McLauchlan, K.K., Skibbe, A.M., Goring, S., Zirbel, C.R., Williams, J.W.,

2013. Linking abundances of the dung fungus Sporormiella to the density of bison:

implications for assessing grazing by megaherbivores in palaeorecords. Journal of

Ecology 101, 1125–1136. doi:10.1111/1365-2745.12130

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Halligan, J.J., Waters, M.R., Perrotti, A., Owens, I.J., Feinberg, J.M., Bourne, M.D.,

Fenerty, B., Winsborough, B., Carlson, D., Fisher, D.C., Stafford, T.W., Dunbar,

J.S., 2016. Pre-Clovis occupation 14,550 years ago at the Page-Ladson site,

Florida, and the peopling of the Americas. Science Advances 2, e1600375–

e1600375. doi:10.1126/sciadv.1600375

Johnson, C.N., Rule, S., Haberle, S.G., Turney, C.S.M., Kershaw, A.P., Brook, B.W.,

2015. Using dung fungi to interpret decline and extinction of megaherbivores:

problems and solutions. Quaternary Science Reviews 110, 107–113.

doi:10.1016/j.quascirev.2014.12.011

Jones, R.A., Williams, J.W., Jackson, S.T., 2017. Vegetation history since the last glacial

maximum in the Ozark highlands (USA): A new record from Cupola Pond,

Missouri. Quaternary Science Reviews 170, 174–187.

doi:10.1016/j.quascirev.2017.06.024

Perrotti, A.G., 2017. Pollen and Sporormiella evidence for terminal Pleistocene vegetation

change and megafaunal extinction at Page-Ladson, Florida. Quaternary

International. doi:10.1016/j.quaint.2017.10.015

Raper, D., Bush, M., 2009. A test of Sporormiella representation as a predictor of

megaherbivore presence and abundance. Quaternary Research 71, 490–496.

doi:10.1016/j.yqres.2009.01.010

Watts, W.A., 1980. The late Quaternary vegetation history of the southeastern United

States. Annual Review of Ecology and Systematics 11, 387–409.

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Wood, J.R., Wilmshurst, J.M., 2013. Accumulation rates or percentages? How to quantify

Sporormiella and other coprophilous fungal spores to detect late Quaternary

megafaunal extinction events. Quaternary Science Reviews 77, 1–3.

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CHAPTER VI

CONCLUSIONS

This dissertation research was driven by the central objective of developing a better understanding the interactions between terminal Pleistocene climate, plants, animals, and humans in the southeastern United States. This objective was addressed by completing a palynological analysis, including dung fungal spores, of two sites in the Southeast. It quickly became clear that pursuit of my research questions would require the use of two cutting-edge methods. Before they could be properly utilized, each method required proper scrutiny. This led to the development of two papers and constitute Chapters II and III of this dissertation.

Chapter II detailed the development and testing of a novel technique to remove unwanted organic debris in pollen samples. These debris in archaeological pollen samples can obscure pollen grains during microscopic analysis. When attempting to remove debris smaller than 10 microns from a pollen sample, screen openings can be too small, requiring the sample to be agitated in order to facilitate its passing through the screen. In this chapter, I tested this recently developed method that uses a Branson© S450 sonicating disruptor horn to agitate the sample, keeping the screen free from debris. Using this method, I was successful in ridding the pollen sample of debris smaller than 10 microns.

This research paper also demonstrated that caution must be applied when utilizing this method, because it has the potential to damage pollen grains. This study demonstrated that when used for 3 min at low to medium output intensities, a sonicating disruptor horn can

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aid in the removal of unwanted debris without considerably damaging pollen grains. I then used this method in Chapters IV and V of this dissertation.

Chapter III outlines the opportunities and limitations of using dung fungal spores as a proxy for large herbivore abundance in archaeological settings. Dung fungal spore analysis was one of the central methods I used in pursuing the central research objectives for the dissertation. Due to its importance in this dissertation, this chapter presented a brief review of the archaeological applications so far, and outlined opportunities and limitations of using Sporormiella as a proxy for megaherbivore abundance. Specific archaeological uses of this proxy explored in this paper include understanding megaherbivore extinctions and human land use patterns such as pastoralism and agriculture. I explained how dung fungal records are formed and outlined factors affecting spore reproduction and preservation. These elements include how strongly each commonly used dung fungal taxon relies on dung as a substrate, environmental factors affecting reproduction, and influences on fungal spore deposition. This paper is particularly significant because it includes a review of the modern mycological literature, which is rarely accessed by s. To conclude this chapter, I discussed limitations of the dung fungal spore proxy and propose directions for future research.

Chapter IV detailed the results of a comprehensive palynological study from the archaeological site Page-Ladson, Florida. Here, I analyzed two sediment cores containing stratified deposits spanning the Pleistocene-Holocene transition, a period characterized by the widespread extinction of Pleistocene megafauna and abrupt changes in vegetation type and distribution. The disappearance of Sporormiella, a well-established proxy for large herbivore abundance, by ~12,700 cal BP is consistent with the timing of Terminal

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Pleistocene megafaunal extinction elsewhere in North America. However, a resurgence of

Sporormiella between ~10,750-10,200 cal BP suggests an Early Holocene incursion of extant megaherbivores such as bison. Pollen evidence from the site also reflects dramatic vegetation changes, which are likely a response to both changing climate and fluctuating herbivore populations. Prior to 14,500 cal BP, the pollen assemblage reflects a relatively cool and dry climate. Between ca. 14,500e12,600 cal BP, the sediments are characterized by an increase in hardwood forest and mesic plant taxa, indicating an increase in both temperature and precipitation. After 12,600 cal BP, a decrease in arboreal pollen, with the exception of oak, alongside an increase in herbaceous pollen, indicates drier, warmer conditions. Ultimately, this study is significant because it contextualizes changes in human behavior at the onset of the Holocene.

In Chapter V, I expanded the spatial resolution of the initial dung fungal research completed at Page-Ladson with a fungal spore analysis from Pleistocene-aged sediments at

White Pond, South Carolina. The data collected in this research suggested that megaherbivores declined from the site around the same time as Page-Ladson, around

12,600 cal BP. In the context of previous palynological data collected at the site, it does not appear that the decline of megaherbivores was preceded by drastic environmental change. The significance of this study is primarily its implications for understanding the applications of using dung fungal spores as proxy evidence for herbivore abundance.

Overall, this dissertation research has many implications for both paleoecology and archaeology. First, it demonstrates the potential for incorporating new methods into our understanding of ancient humans and their relationships with the environment. Second, it establishes the need for these methods to be empirically studied. Specifically, Chapters III

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and V present the need for the development and utilization of more standardized methods in dung fungal analysis. As discussed in Chapter V, most dung fungal spore studies have quantified fungal spores as a percentage of the total pollen assemblage, but this method is vulnerable to fluctuations in pollen accumulation rates. In sediments with high pollen concentrations, fungal spores are less-well represented. The first studies to utilize this method suggest that a decrease of dung fungal spores below 2% of the total pollen assemblage can represent a functional extinction. However, this conclusion was drawn primarily from in his work in (mostly arid) western North America. In Chapter IV,

Sporormiella spores at Page-Ladson, Florida never constitute more than 2% of the total pollen assemblage. However, pollen concentration at that site reflects a mesic hardwood forest during many times. If Sporormiella was simply tallied as a percentage of the total pollen assemblage, these fungal spores would have been nearly undetectable compared those from arid environments such as those in the desert west, where this method was first conceived of.

In addition, future research that expands the temporal and spatial scale of paleoenvironmental data in the southeast would be valuable. The southeastern United

States holds a rich cultural history, from some of the earliest inhabitants of this continent, to the advanced agricultural civilizations of the Mississippian period. Although promising locations for paleoenvironmental study are rare in the region, further exploration and testing will surely reveal places ideal for more analyses.

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APPENDIX

PHOTOS OF COMMON PALYNOMORPHS IN THIS DISSERTATION RESEARCH

Figure A-1. Pinus sp. pollen from Page-Ladson. Scale bar is 10 microns.

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Figure A-2. Carya sp. pollen from Page-Ladson.

Figure A-3. Tilia sp. pollen from Page-Ladson. Photo taken at 400x magnification.

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Figure A-4. Cupressaceae pollen from Page-Ladson.

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Figure A-5. Quercus sp. pollen from Page-Ladson.

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Figure A-6. Fraxinus sp. pollen from Page-Ladson.

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Figure A-7. Fraxinus sp. pollen from Page-Ladson.

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Figure A-8. Vitis sp. pollen from Page-Ladson.

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Figure A-9. Poaceae pollen from Page-Ladson. No scale bar pictured, photo was taken at 400x magnification.

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Figure A-10. Liquidambar sp. pollen from Page-Ladson.

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Figure A-11. Coniochaeta sp. spore from White Pond.

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Figure A-12. Cercophora sp. spore from White Pond.

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Figure A-13. Sordaria-type spore from White Pond.

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Figure A-14. Podospora-type spore from White Pond.

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Figure A-15. Sporormiella sp. spore from Page-Ladson.

Figure A-16. Sporormiella sp. spore from White Pond. 153