Assessing Stable Isotope Data from Archaeological White-Tailed

ASSESSING STABLE ISOTOPE DATA FROM ARCHAEOLOGICAL WHITE-TAILED

DEER REMAINS AS A PALAEOENVIRONMENTAL PROXY AT THE SITE OF LA

JOYANCA, NORTHWESTERN PETEN, YUCATAN PENINSULA

MARIA JOSE RIVERA ARAYA

(Under the Direction of Suzanne Pilaar Birch)

ABSTRACT

Carbon and oxygen isotopes from herbivore teeth have previously been used as paleoenvironmental proxies in temperate zones. However, their application in tropical zones remains uncertain. In this study, sequential sub-samples from white-tailed deer (Odocoileus virginianus) teeth (second and third molars) from the Maya archaeological site of La Joyanca, located in northwestern Peten, Guatemala, show that enamel carbonate δ18O corresponds broadly to the observed precipitation δ18O over a the 10-month period of tooth formation, capturing the rainfall seasonality. The analyses also detect significant diachronic differences in the δ18O values between the periods 1100-1000 and 1000-900 BP at La Joyanca. The δ13C values were indicative of a C3-plant based diet. Isotopic analyses were also carried out on modern white-tailed deer from Georgia, in the southeastern United States, in order to validate the potential of this species to track changes in seasonal rainfall.

INDEX WORDS: Tooth enamel, Odocoileus virginianus, Bioapatite δ18O, δ13C, Seasonality,

Tropical paleoclimate ASSESSING STABLE ISOTOPE DATA FROM ARCHAEOLOGICAL WHITE-TAILED

DEER REMAINS AS A PALAEOENVIRONMENTAL PROXY AT THE SITE OF LA

JOYANCA, NORTHWESTERN PETEN, YUCATAN PENINSULA

MARIA JOSE RIVERA ARAYA

B.S., University of Costa Rica, Costa Rica, 2014

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment

of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

Maria Jose Rivera Araya

DEER REMAINS AS A PALAEOENVIRONMENTAL PROXY AT THE SITE OF LA

JOYANCA, NORTHWESTERN PETEN, YUCATAN PENINSULA

MARIA JOSE RIVERA ARAYA

Major Professor: Suzanne E. Pilaar Birch

Committee: Elizabeth J. Reitz David F. Porinchu

Electronic Version Approved:

Suzanne Barbour Dean of the Graduate School The University of Georgia May 2017 DEDICATION

This work is dedicated to my family and friends who have supported me throughout this process.

iv ACKNOWLEDGEMENTS

I would like to thank a number of individuals who have played a part in the completion of this thesis. First, I would like to give my greatest thanks to my advisor, Dr. Suzanne Pilaar Birch.

I also owe great thanks to my committee members, Dr. David Porinchu, and Dr. Elizabeth Reitz, whose help assisted me throughout the research. This thesis would not have been completed without any of you.

Many thanks also go to Dr. Kitty Emery for help in obtaining the archaeological samples that were analyzed in this project. I also sincerely appreciate the help of Dr. David Osborn from the Deer Laboratory at the University of Georgia, who kindly provided me with the modern deer samples. Finally, I would like to thank the staff of the Center for Applied Isotope Studies (CAIS) at UGA for their technical support while running the stable isotope analysis.

I would really like to thank the Fulbright Foreign Student Program for giving me the opportunity to study my master program at UGA. The Delta Kappa Gamma International Society for Key Women Educators, the Center for Archaeological Sciences, and University of Costa

Rica provided me with the funding for the laboratory analyses required for my thesis. Many thanks to these organizations.

v TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... v

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

1 INTRODUCTION, RESEARCH OBJECTIVES AND STUDY AREA ...... 1

1.2 Study areas ...... 5

2 LITERATURE REVIEW ...... 10

2.1 Paleoenvironmental reconstructions ...... 10

2.2 Stable isotope analysis of tooth enamel ...... 18

2.3 Stable isotope analysis of bone collagen ...... 24

2.4 White-tailed deer ecology ...... 26

3 METHODOLOGY ...... 30

3.1 Standards ...... 30

3.2 Materials ...... 31

3.3 Laboratory analyses...... 34

3.4 Data analysis ...... 35

4 RESULTS ...... 41

4.1 Standards ...... 41

4.2 Archaeological deer...... 42

vi 4.3 Modern deer ...... 48

5 DISCUSSION ...... 62

5.1 Methodology and precision ...... 62

5.2 Archaeological deer...... 64

5.3 Modern deer ...... 73

6 CONCLUSIONS...... 82

REFERENCES ...... 85

APPENDIX ...... 105

vii LIST OF TABLES

Table 1. Evidence of dry periods in the Maya lowlands during the Terminal Classic ...... 13

Table 2. Summary of studies reporting stable isotope values of white-tailed deer bone apatite .. 16

Table 3. Studies reporting stable isotope values of white-tailed deer bone collagen (VPDB) ..... 16

Table 4. Contextual information for archaeological teeth selected for isotopic analysis ...... 32

Table 5. Contextual information for modern, wild teeth selected for isotopic analysis ...... 33

Table 6. Contextual information for modern, non-wild teeth selected for isotopic analysis ...... 33

Table 7. Minimum, maximum, range, and mean values of δ18O and δ13C from white-tailed deer teeth for 1100-1000 BP and 1000-900 BP...... 44

Table 8. Minimum, maximum, range, and mean values of δ18O and δ13C from wild white-tailed deer teeth...... 51

Table 9. Minimum, maximum, range, and mean values of δ18O and δ13C from non-wild white- tailed deer teeth...... 55

Table 10. Minimum, maximum, range, and mean values of δ18O and δ13C from archaeological and modern white-tailed deer teeth...... 58

Table 11. Carbon and nitrogen stable isotope collagen values, total nitrogen and carbon and carbon/nitrogen ratio of the deer mandibles analyzed ...... 59

13 15 Table 12. Mean values of δ C and δ N from modern white-tailed deer teeth ...... 59

Table A- 1. Details of archaeological deer teeth samples 105

Table A- 2. Details of individual tooth sub-sample isotopic measurements for archaeological deer ...... 106

Table A- 3. Details of individual tooth sub-sample isotopic measurements for modern, wild deer ...... 108

Table A- 4. Details of individual tooth sub-sample isotopic measurements for modern, non-wild deer ...... 110 viii LIST OF FIGURES

Figure 1. The subregions of the Maya territory in Central America...... 3

Figure 2. Map of Yucatan Peninsula. Location of La Joyanca and other sites mentioned in the text...... 7

Figure 3. Map of the Maya lowlands showing the locations of sites discussed in the text...... 11

Figure 4. Plot of standard values...... 42

Figure 5. Variation in modelled monthly precipitation δ18O in La Joyanca ...... 45

Figure 6. A. Intra-tooth oxygen and carbon isotope ratios of the second and third molars from three white-tailed deer for the period 1100-1000 BP...... 46

Figure 7. Intra-tooth oxygen and carbon isotope ratios of the second and third molars from three white-tailed deer for the period 1000-900 BP...... 47

Figure 8. Boxplot of medians and distribution of δ18O and δ13C values in white-tailed deer through time ...... 49

Figure 9. Intra-tooth oxygen and carbon isotope ratios of the second and third molars from modern wild, white-tailed deer...... 52

Figure 10. Intra-tooth oxygen and carbon isotope ratios of the second and third molars from modern wild, white-tailed deer (Individual 7)...... 53

Figure 11. Variation in modelled monthly precipitation δ18O in Georgia ...... 53

Figure 12. Intra-tooth oxygen and carbon isotope ratios of the second and third molars from modern, non-wild white-tailed deer ...... 56

Figure 13. Boxplot of medians and distribution of δ18O and δ13C values in non-wild and wild white- tailed deer ...... 57

Figure 14. Stable carbon (δ13C) and nitrogen (δ15N) ratios of Georgia deer jaw collagen...... 60

13 13 Figure 15. Stable carbon isotope data from apatite (δ apt) and collagen (δ Ccol) from Georgia deer mandibles ...... 60

ix Figure 16. δ13C against δ18O in the archaeological deer teeth dataset ...... 71

18 Figure 18. Range of calculated drinking water δ OSMOW values based on intra-tooth M2 and M3 enamel carbonate measurements for the population of wild and non-wild deer teeth whose isotope sequences cover both maxima and minima rainfall ...... 77

18 Figure 19. Range of calculated drinking water δ OSMOW values based on intra-tooth M2 and M3 enamel carbonate measurements for each of the deer teeth whose isotope sequences cover both maxima and minima rainfall ...... 78

Figure A- 1. Photographs of the isotopically analyzed teeth: (a) JY7; (b) JY1 and JY2 112

x CHAPTER 1

INTRODUCTION, RESEARCH OBJECTIVES AND STUDY AREA

As the impacts of climate change increase, including growing landscape modifications and food insecurity in modern societies at the global and local scales, it is critical that we improve our understanding of the long-term reciprocal influences among humans, the climate and the environment, particularly in tropical zones, which may be among the most affected biomes in coming years (Hawkins et al., 2014; Huang et al., 2013). These types of studies can significantly contribute to the construction of future-looking models related to issues of resilience and vulnerability in modern human societies, expand the available local paleoenvironmental proxies in tropical areas and also better constrain climate simulations and projections in these zones (Iannone, 2014).

The paleoenvironmental studies conducted in the Yucatan Peninsula to date have targeted reconstructions at the regional level (Hodell et al., 2007; Medina-Elizalde et al., 2010; Wahl et al., 2014). These analyses lack a local, direct relation to cultural and human activities. The Maya area, and within it, the Yucatan Peninsula, is one of the world’s most crucial places to pursue long-term studies on human-environment relationships. There is evidence of at least 13,000 years of human occupation, including urban centers characterized by majestic architecture. The Maya also had deep knowledge of astronomy and mathematics, and were skilled artisans who carved stone, created polychrome pottery, and established complex trade systems (Douglas et al.,

2016).The southern Maya lowlands, in what is now northern Peten, Guatemala, are well known 1 as the area in which the Maya civilization developed (Figure 1). While a number of sociopolitical groups reached their apogee during the Classic Period (1350 – 1150 BP) (Aimers et al., 2011), the period from 1200 – 900 BP, known as the Terminal Classic, was characterized by political balkanization and increased power-sharing (Aimers et al., 2011; Demarest, 2004). This social reorganization, which took diverse forms across different sites in the Maya area, has been strongly correlated to climate change, specifically droughts (Aimers et al., 2011; Douglas et al.,

2015). Due to the local nature of many of these transformations and the diversity of sociopolitical trajectories in the region, site-scale resolution is crucial in comprehending this sociopolitical change and its connections to environmental conditions in the Mayan societies.

This thesis project aims to increase our understanding of local environmental and social processes during 1100 – 900 BP at the archaeological site of La Joyanca, northern Peten,

Yucatan Peninsula by analyzing carbon and oxygen isotopic signatures in white-tailed deer

(Odocoileus virginianus) teeth. This proxy has great potential to provide seasonal paleoenvironmental information on precipitation and vegetation at the local scale (Fricke et al.,

1996; Repussard et al., 2014). Studying local paleoenvironmental data during the Terminal

Classic from this site will provide more data on the correlation between climate events, land use and sociopolitical dynamics in this Maya society during 1100 - 900 BP. Studying La Joyanca provides the additional advantage of access to nearby paleoenvironmental data derived from multiple proxies for comparison of regional and local trends.

2 Figure 1. The subregions of the Maya territory in Central America. (Nic.:Nicaragua; C.R.: Costa Rica).

1.1 Research objectives

This thesis contains six chapters. The first includes an introduction of the topic, study area, and research objectives. The second provides a review of the relevant literature; the third explains the methodology that was followed; the fourth presents the results; the fifth discusses the results; and chapter 6 provides conclusions. The goals of this thesis research project are two- fold:

1A. Develop a record of seasonal changes in precipitation and vegetation at the local scale in La Joyanca, northwestern Peten, during the period 1100-900 BP.

The sequence of environmental and sociopolitical factors leading to the Maya social reorganization during 1100 – 900 BP remains poorly understood. This study will generate additional data to aid in the comprehension of the environmental history and the human-

3 environment relationships at La Joyanca. It will test whether the rapid reforestation recorded by

Carozza et al. (2007) beginning at 1190 BP at this site is also recorded in the stable isotope data retrieved from white-tailed deer from La Joyanca and if this is correlated to changes in precipitation amount. This will also allow for an assessment of diachronic change in the oxygen and carbon isotopic values between these time periods.

1B. Examine the potential of white-tailed deer teeth as a paleoenvironmental proxies at the local scale at La Joyanca, northwestern Peten, during the periods 1100 – 1000 BP and 1000-

900 BP.

Although the extrapolation of regional data to understand local processes in the Yucatan

Peninsula is inadequate, studies targeting the local scale are very limited in quantity. More research is needed to expand our knowledge of these paleoenvironmental processes. This objective will assess whether there is any significant difference between the mean, maximum and minimum isotopic values retrieved from both time periods. By comparing these data with existing proxies, it will test whether δ13C and δ18O values from white-tailed deer teeth are also viable proxies for La Joyanca.

2. Evaluate the relationship of modern precipitation, temperature and dietary factors in the stable isotope values of bones and teeth of white-tailed deer in order to validate their use as a proxy for δ18O in precipitation.

By conducting this research on a population of modern deer, our results will provide further information on the natural range in oxygen and carbon isotope signatures for this species, during a specific period of time for which monthly average temperature, precipitation amount and δ18O in precipitation records exist. This information will assist in the interpretation of 4 oxygen and carbon isotope values of white-tailed deer teeth from La Joyanca and validate their use as a proxy for rainfall δ18O, described above in aim 1.

In summary, this research will contribute to the understanding of local paleoenvironmental dynamics at La Joyanca by providing new on-site data that can be compared to previous local and regional studies in the area.

1.2 Study areas

This study is conducted on white-tailed deer teeth samples from two locations. The archaeological samples come from the archaeological site of La Joyanca, northwestern Peten,

Guatemala (Figure 2) and the modern ones are from Georgia, United States.

1.2.1 Archaeological site-La Joyanca

La Joyanca is located in the upland region south of the San Pedro Martir River, northwestern Peten, Guatemala, at an altitude of ~ 100 m. The nearest weather station, which contains 24 years of data, is around 70 km from La Joyanca in Flores, Peten. The average annual precipitation is ~ 1840 mm; average maximum and minimum temperatures are 32.4 and 20.6 °C respectively (http://www.insivumeh.gob.gt). The regional climate is strongly seasonal, with

~75% of annual rainfall occurring during the wet season from May to October and the remainder during the dry season from November to April.

The annual climate cycle in La Joyanca today is characterized by minor fluctuations in surface temperature, with a well-defined wet/dry cycle mainly determined by the position and strength of the Bermuda-Azores cell of the Subtropical High (Folan et al., 1983) and by the annual migration of the Intertropical Convergence Zone (ITCZ) (Giannini et al., 2000). The major part of precipitation usually falls in summer (from June to October), when the ITCZ is at 5

18 its northernmost position. The highest values of δ ORain are seen during the winter (February), when the ITCZ moves southwards, away from the Yucatan peninsula, associated with reduced

18 amounts of rainfall and low vapor pressure (Haug et al., 2003). Conversely, lowest δ ORain values are seen during late summer (September), when the ITCZ lies precisely on the peninsula, providing heavy rainfall. The weighted mean annual average δ18O value is −4.0‰ for Veracruz.

The seasonal cycle in δ18O ranges from +0.3 to −5.3 ‰ with lowest values in the June to

November wet season when monthly rainfall totals commonly exceed 300 mm (Lachniet et al.,

2009). Temporally speaking, the dominant control of precipitation δ18O values is the amount

18 effect (Dansgaard, 1964) in which ORain decreases with increasing amount of rainfall. Spatially, only two variables, distance from the coast and mean catchment altitude, explain 84 % of the surface water δ18O variability. This climate pattern suggests there should be a record of seasonal changes in the sequential sampling of tooth enamel (Repussard, 2009).

La Joyanca is located relatively close (tens of kilometers) to many large prehistoric urban centers, including El Mirador, Nakbe, and Xulnal. It is also in an ideal position to compare to previous local environmental and climate analyses at the sites of Piedras Negras and Motul de

San José (Emery et al., 2000, 2008). This also facilitates the comparison to regional trends.

This site was a secondary political center with a single public plaza and one stele with an inscription and calendar date of the early Classic period (1465 BP). At the regional level, La

Joyanca was an important settlement; it once had an estimated population of 1500 inhabitants. It shows an unusual, high density of structures, with 637 in 1.6 km2 (Arnauld, 2004; Arnauld et al.,

2013). This site reached its apogee by the Late-Terminal Classic but its population gradually declined between 1100 and 900 BP (Arnauld et al., 2013). A study of the land use at this site concluded that after 1190 BP, the absence of detrital inputs and rapid reforestation constitute the 6

local evidence of the regional collapse, which is in accordance with the archaeological data

(Carozza et al., 2007). Studying local paleoenvironmental data during this period can determine whether there is a correlation between political, social, and environmental changes at the site.

Figure 2. Map of Yucatan Peninsula. Location of La Joyanca and other sites mentioned in the text.

1.2.2 Modern site

Two types of modern deer samples were used in this study. The first one includes deer that were randomly recovered across Georgia (wild). The second group of samples consist of a group of deer that lived at the Whitehall Deer Research Facility at the University of Georgia

(UGA) in Athens, Georgia during the years 1984-1994 (non-wild). Both groups of samples were provided by the Deer Laboratory at UGA. The climate in Georgia is subtropical humid. The coolest months are December through February, when minimum temperatures average around 5-

7 10 °C (NOAA, 2017). The warmest months are July and August when maximum temperatures average around 32° C. Average precipitation for the year is 1320 mm. Precipitation is evenly distributed throughout the year. Average annual precipitation is influenced by the proximity to moisture sources (Gulf of Mexico and Atlantic Ocean) and the influence of topography, such as orographic lifting and rain shadows (dry areas on lee sides of mountains). The positioning of the

Bermuda High causes warm and moist summers with frequent thunderstorm activity. When this high pressure systems builds to the west, hot and dry weather occurs, although humidity stays high. When the system goes south drought conditions develop (Ingram, 2013). The climatic record for Athens extends from 1944 to 2016 and the closest weather station is 10 km from the

Whitehall Research Facility.

Chapter summary

The two research objectives presented here are essential for understanding the usefulness of white-tailed deer as a paleoenvironmental proxy in tropical areas. The first one address whether rainfall and vegetation seasonality are reflected in the tooth enamel from deer from La

Joyanca and how diachronic changes in these parameters between 1100-1000 BP and 1100-900

BP are recorded. The second aim explores the relationship between rainfall seasonality, diet, and vegetation in tooth enamel and bone collagen of deer from Georgia. These research questions elaborate upon a major theme: the use of paleoenvironmental proxies at the local scale in tropical areas. The research addresses this topic using samples from teeth enamel from archaeological deer from La Joyanca and modern deer from Georgia. Chapter 2 contextualizes the research

8 question through a review of the literature on the environmental setting and cultural history of the Yucatan Peninsula.

9 CHAPTER 2

LITERATURE REVIEW

This chapter describes the context of the research and is organized in four sections. The first section briefly summarizes the previous paleoenvironmental studies in the Yucatan

Peninsula at the regional, local and site scales. The second and third sections review the basics of stable isotope analysis in tooth enamel and bone collagen in herbivores, respectively. The fourth section discusses relevant information of white-tailed deer ecology, including migration and diet habits, which are essential for interpreting the data derived from isotopic analyses.

2.1 Paleoenvironmental reconstructions

The Maya area has been the target of several paleoenvironmental studies at different spatial scales. Here, I provide a summary of the studies at the regional and local levels and finally, the information available for La Joyanca (Figure 3).

2.1.1 Regional scale

The Yucatan Peninsula has been a focus of intensive paleoclimate and paleoenvironmental research, with numerous, multi-proxy paleoclimate studies during recent decades. Speleothems (cave stalagmites), and sediments from marine, lake and wetland contexts have been used to infer past climate conditions at the regional scale (Douglas et al., 2016;

Iannone, 2014). In particular, the two most used approaches for

10 reconstructing past climate in the Maya area use continental archives from the region such as speleothems and lake sediment cores.

Figure 3. Map of the Maya lowlands showing the locations of sites discussed in the text.(SJCh: San Jose Chulchaca; Xca: X’caamal; Tz: Tzabnah; LCo: Lago Coba; LPL: Lago Punta Laguna; LCh: Laguna Chichancanab; LP: LT: Laguna Tuspan; LPA: Laguna Puerto Arturo; LPI: Lago Peten Itza; Fl: Flores; Mc: Macal; JY: Joyanca; PN: Piedras Negras; LP: Lago Salpeten)

Geochemical and palynological evidence from lake sediment cores from central Peten indicate that the transition from Pleistocene savanna to Holocene forests began in the region around 11000 BP (Mueller et al., 2009). Evidence of humid conditions in the early to mid-

11 Holocene has been found in the Caribbean region (Hodell et al., 1991; Islebe et al., 1996). Wetter conditions within the mid-Holocene precede the general, progressive drying trend that began about 4000 years ago (Mueller et al., 2009). Global and pan-Caribbean mesocycles of climatic fluctuation were characterized by periods of cooler and drier and of warmer and moister conditions, lasting hundreds of years (Brenner, 2002). Short-term climatic cycles also affected the precipitation patterns in the region, most notably a 208-year cycle of solar energy pulses during the mid-Holocene (Hodell et al., 2001).

Haug and colleagues (2003) analyzed the titanium content of a marine sediment core from the Cariaco basin, located on the northern coast of Venezuela. The position of the northern coast of Venezuela relative to the movement of the Intertropical Convergence Zone (ITCZ) being similar to the Yucatan's, climatic inferences are possible. These authors found that the

Classic collapse took place during a period of global dryness, punctuated with pluri-annual episodes of intense droughts, around years 1140, 1110 and 1040 BP, with similar signals of drier climate at the time of Pre-Classic abandonment (1750 BP).

In summary, studies from the Yucatan Peninsula and circum-Caribbean show significant

Holocene climate variability (Curtis et al., 1996; Haug et al., 2001, 2003, Hodell et al., 1991,

1995, 2001; Hodell, Brenner, & Curtis, 2005; Kennett et al., 2012; Lane et al., 2009; Wahl et al.,

2014), but few climate reconstructions come from the southern Maya lowlands. Unfortunately, there are no paleoclimate data for the western Maya lowlands and paleoenvironmental data for this region are scant. Weather patterns are highly localized in the Maya area and extrapolating from climate proxies from other parts of the Maya world may be not accurate (Yaeger et al.,

2008). Table 1 summarizes the results from the studies that deal with the Terminal Classic Period in the Maya lowlands. 12 Table 1. Evidence of dry periods in the Maya lowlands during the Terminal Classic

Area Site Description Source Paixban No evidence Wahl et al.(2016) Holmul Anomaly with drier conditions between 1270 – 1040 BP Wahl et al. (2013) Drying trend in the Terminal Classic. The variability in Salpeten Rosenmeier et al.(2002) Southern Maya δ18O values is much smaller than at northern lowlands lowlands Peten Itza No evidence Curtis et al.(1998) Puerto Arturo Weakly evidenced Wahl et al.(2014) Macal Droughts at 1170, 1040, 876 BP Webster (2007) Eight severe droughts, 1144, 1121, 1108, 1093, 1055, Tzabnah Medina-Elizalde et al. (2010) 1041, 1029 and 1015 BP Hodell et al. (1995), Hodell et al Chichancanab Droughts at 1180 – 1080 and 1030 – 850 BP (2005) Lower lake level and drier climate between 1416 – 1357 Curtis et al. (1996), Hodell, Punta Laguna Northern Maya BP, 1200 – 900 BP and mid-fifteenth century Curtis et al (2005) lowlands Generally wetter conditions prevailed until ca. 3750 BP San Jose Chulcaca Leyden (1996) when aridity increase and continue Coba Not drought but drier climate Leyden et al. (1998) No evidence for sustained droughts during the Late and X'caamal Terminal Classic periods. From about 600 –1200 BP, a Hodell, Curtis et al. (2005) slight trend toward increasingly wet conditions.

13 2.1.2 Local scale

A few studies at the local level have contributed to increasing the data associated with archaeological sites. For instance, Repussard et al. (2014)studied the oxygen isotopic signature in

18 bone phosphate (δ Op) of white-tailed deer bones as a local paleoenvironmental proxy for rainfall and humidity levels at the Piedras Negras site. The isotope data showed a possible episode of drought during 1100 – 900 BP, a finding that is in accordance with the dry phase detected by regional studies (Curtis et al., 1998; Rosenmeier et al., 2002). The authors interpreted this finding as a probable contributing factor to hastening the fall of Piedras Negras due to internal sociopolitical causes. Despite the contribution to the understanding of the local signatures at the site, this study did not include teeth as part of the sample, although this proxy could complement the data obtained from bones. The results of other studies on white–tailed deer stable isotopes are summarized in Table 2 and Table 3.

On the other hand, the Maya have been identified as agents of high impact environmental change, particularly in vegetation and land cover. The lines of evidence about how much the

Maya changed their environment include geomorphological, archaeological, paleoclimatological, and paleoecological records (Beach, 2016; Beach et al., 2006, 2009).

From the Yucatan Peninsula, several palaeoecological studies are available that use fossil pollen (Carrillo-Bastos et al., 2010; Islebe et al., 1996; Leyden, 1996; Leyden et al., 1998;

Torrescano and Islebe, 2006). Deforestation has been recorded in different locations around the

Peninsula. In fact, most palynological studies in the Maya Lowlands suggest that human land use, as opposed to climate change, was the dominant driver of vegetation cover change during the late Holocene (Dunning et al., 2014; Turner and Sabloff, 2012). These studies in the Yucatan

Peninsula show attenuated forest taxa values during the period of prehispanic Maya settlement 14 followed by a distinct increase in the early Postclassic. The initial onset of forest taxa decline ranges from ~5600 to 3000 BP; many records exhibit an initial or accelerated decline between

4000 and 3000 BP (Dunning et al., 1998; Islebe et al., 1996; Leyden, 1996, 2002; Pohl et al.,

1996; Rosenmeier et al., 2002; Wahl et al., 2006).

In addition, carbon isotopic signatures (δ13C) can be used to infer changes in the source of organic carbon in lake sediments and soil (Wahl et al., 2013; Wahl et al., 2016). The ancient

Maya cleared large areas of deciduous and evergreen tropical forest (that use the C3 photosynthetic pathway), which led to a large increase in the abundance of grasses and other disturbance taxa (that use the C4 photosynthetic pathway) (Carrillo-Bastos et al., 2012;

Rosenmeier et al., 2002; Turner and Sabloff, 2012). Furthermore, soil organic carbon associated with maize, a C4 grass in the Maya lowlands, exhibits strong 13C enrichment (Douglas et al.,

2012; Sachse et al., 2012).

Another recently developed technique for paleoenvironmental analysis involves the

13 analysis of carbon isotope composition of plant waxes (δ Cwax). This composition is strongly determined by the bulk of plant tissue. In the Maya lowlands, where the dominant natural

13 vegetation is C3 angiosperm forest, the δ Cwax has similarly been applied as a robust indicator of the relative abundance of C3 and C4 plants in other tropical locations (Douglas et al., 2015). This proxy has brought new insights in the land use near Lake Salpeten. During the Classic period, the predominant land use in this lake catchment shifted from low-population-density swidden agriculture, which promoted grasses, to higher-population-density residential land use that promoted the growth of C3 disturbance flora including Asteraceae and Ambrosia.

Table 2. Summary of studies reporting stable isotope values of white-tailed deer bone apatite(VPDB)

Cultural Site Name Mean SD n Mean SD (1σ) n Source 18 13 complex δ Oapatite (1σ) δ Capatite Ontario SW Ontario, multiple sites -10.5 0.8 4 Booth et al. (2012) Maya Lamanal 27.7 0.5 14 -10.2 1.0 14 Repussard (2009) Maya Multiple sites, classic period -10.4 0.8 16 Gerry (1997) Maya Motul de San Jose 30.1 0.7 10 -10.5 1.0 10 Repussard (2009) Maya Piedras Negras 30.0 0.9 67 -10.6 1.0 67 Repussard (2009)

Table 3. Studies reporting stable isotope values of white-tailed deer bone collagen (VPDB)

Cultural Site Name Mean SD n Mean SD n Source 13 15 complex δ Ccol (1σ) δ Ncol (1σ) Maya Aguateca -20.5 1.4 12 Emery et al. (2000) Maya Arroyo de Piedra -20.7 0.8 10 Emery et al. (2000) Maya Bayak -20.5 0.3 5 Emery et al. (2000) Maya Colha -21.1 0.8 16 5.0 1.7 16 White et al. (2001) Maya Copan (Late Classic) -20.0 1.6 20 4.9 1.4 20 White et al. (2004) Maya Copan -20.4 1.6 5 3.8 1.3 Whittington and Reed (2006) Maya Cuello -20.5 0.9 5 5.8 1.3 6 van der Merwe et al. (2002) Maya Dos Pilas -20.5 0.3 16 Emery et al. (2000) Maya Largartero -18.2 5.4 8 5.4 0.9 8 White et al. (2004) Maya Lamanai -21.8 0.3 2 4.5 0.3 2 Repussard (2009) Maya Multiple sites -21.1 0.9 46 4.4 1.3 44 Gerry (1997) Maya Pacbitum -19.2 3.9 5 8.1 4.1 4 White et al. (1993) Maya Punta de Chirmino -20.8 0.9 7 Emery et al. (2000) Maya Tamarindito -20.5 0.7 3 Emery et al. (2000) Maya Tikal -19.7 2.8 9 5.0 1.2 9 White et al. (2004) Mississipian St. Catherine’s Island, GA -21.6 0.8 26 4.7 1.1 26 Bergh (2012)

δ13C analysis of fossil tooth enamel is well suited for detecting the presence or absence of

C4 grasses and the proportions of C3 and C4 plants in the diets of archaeological remains (Lee-

Thorp et al., 2007). Zooarchaeological remains have also been used as supporting evidence to understand human impact in the Maya area. Studies based on stable isotope analyses of zooarchaeological remains show that maize or other C4 species were components of deer and peccary diets, and that a patchwork of forests, fields, and successional plants surrounded Maya sites (Emery et al., 2008; Somerville et al., 2013).

Although carbon isotopes are not direct indicators of climate conditions, they can provide vegetation data that can be correlated to climatic events. For example, using deer diet as a proxy for the prevalence of agricultural fields, Emery et al. (2000) concluded that there is little evidence for overuse or abuse of the natural environment through agricultural activities in the

Peten area after studying δ13C in bone collagen of deer. Sediment cores from this area support this conclusion (Rosenmeier et al., 2002). In a later study, Emery and Kennedy Thornton (2008) analyzed samples from six drainage basins across the regions of most significant settlement during the Classic period. The results indicate that the ancient Maya land-use practices and local environments varied at a local and site level across time and space. In two regions (the Peten

Lakes and the Petexbatún drainage) maize consumption rose in the Late Classic period, but at the site of Tikal, it appears to have dropped at this time. Oxygen isotope values indicate a correlation between decreasing maize consumption and periods of drought at the Motul de San José polity sites in the Peten Lakes region. Generally speaking, this finding corresponds to paleoenvironmental data obtained from sediment cores (Hodell et al., 2007).

2.1.3 La Joyanca

A record of land use at Laguna Tuspan (near La Joyanca) shows evidence for four main episodes of accelerated erosion between 3000 BP and 1280 BP with the largest occurring by

1280 BP. From 2050 to 1750 BP, there is a marked decrease in agricultural activities, with no signal of the latter by 1850 BP, at the end of the Preclassic (Galop et al., 2004). Later, activities resumed and by 1550-1450 BP they reached a climax when all signals consistently indicate extensive land use near the lake. Then, after 1410 BP (or 1550 BP according to the most recent evidence), a marked decrease in activities is again noted in the same surroundings, while evidence of deforestation and soil erosion indicates continuous land use farther away from the lake. Lastly, by 1150 BP reforestation begins and the erosion signal stops, an interruption in activities that seems to be generalized spatially, suggesting that the regional population left or drastically decreased. Later on, hints of agricultural activities (some degree of deforestation and maize pollen) are dated to 800-750 BP.

Laguna Paixban and lakes Peten Itza and Salpeten show the nearest paleoclimate reconstructions to La Joyanca (Figure 1). None of them present evidence of droughts during the

Terminal Classic, except for Salpeten, which suggests a drying trend during this period of time

(Rosenmeier et al., 2002).

2.2 Stable isotope analysis of tooth enamel

Oxygen (δ18O) and carbon (δ13C) isotope signatures from bones and teeth of herbivores are useful for the study of local climate patterns in the past as well as human-environment relationships (Balasse, 2002; Hedges et al., 2006; Pilaar Birch, 2013). Bones and teeth provide two important advantages when compared with other proxy data. First, they provide information

on the animal species itself. This increases the scope and precision of paleoenvironmental interpretation. Second, they can potentially provide paleoclimatic reconstructions that are local to archaeological sites and directly linked to the periods of human activity (Stevens et al., 2011). In temperate zones, their use has increased due to the limited scope and number of terrestrial archives from which paleoenvironmental records can be obtained. However, their use needs further testing in tropical areas, as the number of studies using them is limited and their conclusions need a deeper analysis (Emery et al., 2008; Repussard et al., 2014).

Teeth provide additional advantages compared to bones. Tooth enamel grows by accretion and preserves incremental laminae that form at a variety of timescales (daily to annual)

(Hillson, 2005). Therefore, a tooth contains a complete record of the phase of rapid growth early in an animal's life (Hedges et al., 2006). It can show a series of individual values that encompasses the time of mineralization of the respective tooth (which can cover several months to a few years depending on species). This allows observing seasonal climate variation that reflects the isotopic composition of the local precipitations over a specific time period (Fricke et al., 1996). Also, tooth enamel is more resistant to chemical and physical alteration because it has a lower organic matter content and a higher degree of crystallinity compared to bone.

Nevertheless, it has not been tested as a paleoenvironmental proxy in the Maya area. Previous studies on tooth enamel in herbivores, such as red deer teeth in Europe, have reported successful results when applied to paleoenvironmental reconstructions (Balasse, 2002; Drucker et al., 2008;

Stevens et al., 2011). Studies of modern horse, yak, goat, bovid, and red deer teeth have allowed an assessment of the degree to which seasonality might be inferred from the intra-tooth oxygen isotopic variation in ancient specimens (Balasse, 2003; Fricke et al., 1996; Zazzo et al., 2006).

19 There is an outstanding opportunity to focus and expand on the limited information on seasonal trends in climate at the local level in northwestern Peten by using the isotopic analyses of the teeth of white-tailed deer from La Joyanca. This will contribute, first, to the understanding of local patterns of past climate change; second, to the expansion of the available local paleoenvironmental proxies in tropical areas and third, to the construction of climate simulations and projections in this area. Local paleoclimate and land cover data will add to the comprehension of the relation of climate events (specifically dry periods) and agricultural activities in the site. In this light, the use of δ13C and δ18O in herbivore teeth is a relevant source of information as they offer data related to a specific site and chronological period and are less likely to be physically and chemically altered compared to bone collagen and apatite.

Here, I review some of the basic principles for stable isotope analysis of oxygen and carbon and their use in archaeological studies.

2.2.1 Oxygen

The δ18O of precipitation is controlled by the ambient temperature and amount of rainfall

(Dansgaard, 1964; Gat, 1980; Rozanski et al., 1993). Lachniet and Patterson (2009)

18 demonstrated that for northern Central America the isotopic signature of rainfall (δ ORain) is

18 ultimately influenced by the amount. The δ ORain decreases with increasing amount of rainfall

(Dansgaard, 1964). Previous studies in herbivores show that seasonal climate patterns are recorded in the isotopic signatures captured by the sequential sampling of the enamel in herbivore teeth (Fricke et al., 1996; Kohn et al., 1998; Lee-Thorp et al., 2007; Nelson, 2005;

Stevens et al., 2011). The samples from enamel can show a series of individual values representing the isotopic composition of local precipitation. Tooth mineral bioapatite carbonate

20 and phosphate precipitate in isotopic equilibrium with body water (Fricke et al., 1996;

Longinelli, 1984; Luz et al., 1984). The major source of oxygen in the body water of white-tailed deer (and ultimately bioapatite phosphate and carbonate bounded oxygen) is the water contained in the leaves ingested by the animal, which comes from precipitation (Cormie et al., 1994; Luz et al., 1990; Villareal Espino-Barros et al., 2005). These leaves come from plants that absorb rainwater directly without depending on groundwater. The overall processes of enrichment of leaf water in δ18O are mainly driven by the humidity gradient between the inside of the leaf and the surrounding air (Repussard et al., 2014). Hence, δ18O signatures reflect changes in the amount of rainfall and humidity levels in the environment frequented by deer. This means that the isotopic signature is only affected by the loss of water via evapotranspiration processes

(Cormie et al., 1994; Luz et al., 1990). In arid habitats, this process can cause an enriched δ18O signal which may not reflect the actual paleoenvironmental variables, particularly in species that survive independently of surface water by obtaining their body water from leaves, such as white- tailed deer. However, white-tailed deer from areas with ≥ 70 % humidity (such as La Joyanca and Georgia) are not affected by this process and can track δ18O precipitation values (Hallin et al., 2012).

The conclusions of this study are considered to be valid given that, previous studies suggest 1000-900 BP, associated with the Medieval Climate Anomaly, was wet (Carrillo-Bastos et al., 2013; Medina-Elizalde et al., 2010). Some studies have identified dry periods during the previous period of 1100-1000 BP, (Table 1) mainly in the northern lowlands. This is not conclusive from the southern lowlands, where La Joyanca is located.

2.2.2 Carbon

The photosynthetic pathway (C3, C4, or CAM) used by the plant species largely determines the relationship between the stable carbon isotopes 13C and 12C, and the relative degree to which these isotopes are incorporated as CO2 into plant tissues (O’Leary, 1981).

Tropical forest ecosystems that are undisturbed by humans are dominated by C3 plants; any increase in C4 plant abundance in these ecosystems is typically linked to swidden agricultural activities and will be proportional in scale to agriculture within the watershed. However, higher- population-density residential land use promotes the growth of C3 disturbance flora, including

Asteraceae and Ambrosia (Brown, 1999; Sage et al., 1999). Carbon isotopes in herbivore skeletal tissues can be used to track variations in the availability of C4 plants (such as maize) across the landscape and over time (Harrison et al., 2003; Krueger et al., 1984). The herbivore tissues in turn have carbon isotope signatures that reflect the main food plants available (Hobson et al.,

1986). Relative 13C/12C ratios in deer bone collagen accurately indicate the proportional dietary incorporation of C4 plants (including maize) to C3 plants (leaves browsed by deer), and can be used to quantify the changing availability of the two plant types to herbivores.

The δ13C value is strictly dependent on the carbon isotopic composition of deer's diet.

Deer subsist almost entirely on C3 plants. In this, the apatite δ13C values of the analyzed deer bones are expected to be ~ -13 ‰. More positive values are indicative of the inclusion of C4 plants in the diet (Koch, 1998).

2.2.3 Tooth mineralization

The samples from enamel can show a series of individual values representing the isotopic composition of local precipitation in a range that varies between months and years. This depends

on the time of mineralization of the teeth after birth. Analyses of white-tailed deer from deciduous forest and rainforest indicate that the second molar (M2) erupts between five and nine months; and the third molar (M3), at one year of age (Brokx, 1972). These results agree with most studies on dental tooth development carried out in deer from high latitudes (DeYoung,

1989; Lockard, 1972; Severinghaus, 1949). Morris (2015) showed using samples of white-tailed deer from Ontario, Canada, that white-tailed deer’s second molars erupt between six and 13 months and the third molars between 15 and 18 months. The mineralization of this species’ second molar enamel starts before two months and is completed by five or six months, and that third molar enamel mineralizes between five and ten months.

Although there are no studies on tooth mineralization from white-tailed deer from tropical locations, growth patterns of deer generally are impacted most dramatically by habitat quality which can vary widely across its range. However, tooth eruption patterns do not seem to differ substantially (Miller, 2016). While there is geographic variability in the development stages and behavior of white-tailed deer across North America (Stewart et al., 2011), these including high

(Ontario) and low latitude (Yucatan Peninsula) areas, growth and dental eruption patterns for the

Yucatan would not be much different than other subtropical regions such as South Texas and

South Florida – and these do not differ from many other temperate regions. Isotopes values of teeth of white-tailed deer primarily reflect ingested leaf water at the very local level (Repussard et al., 2014). Given the lack of tooth mineralization studies for white-tailed deer in tropical areas and the similarity in tooth eruption patterns with deer from temperate areas, this study assumes that the deer from La Joyanca followed the same mineralization patterns as the ones studied by

Morris (2015). Therefore, molar enamel in M2 and M3 is expected to record the variations in

rainfall between two and ten months after the deer’s birth. For instance, assuming a deer born in

May, the M2 and M3 would record the time between July and February.

2.3 Stable isotope analysis of bone collagen

Isotopic analyses of carbon and nitrogen in bone are also well-established markers of dietary practices, and have been successfully applied to zooarchaeological remains for decades

(Ambrose, 1993; Schwarcz, 1991). Usually, bones are more common than teeth in the archaeological record. Bone is composed of a mineral apatite phase (hydroxyapatite) and an organic collagen phase.

2.3.1 Carbon

Carbon isotope ratios from the apatite phase of bone provide different information on the consumer's diet than do carbon isotope ratios from the organic phase of bone (collagen) (Krueger et al., 1984; Lee-Thorp, 2000). The carbon-isotope composition of bone collagen predominately reflect the protein portion of the diet, while apatite reflects the whole diet, i.e., protein, lipid and carbohydrates (Ambrose et al., 1993; Kellner et al., 2007).

A commonly considered upper end point for a C3 only diet in ungulates is –21.4 ‰ in

13 collagen (Cormie et al., 1994; van der Merwe, 1982). Deer with δ Ccol values lower than this could also have been feeding in more deeply wooded areas i.e., a canopy effect (Bonafini et al.,

13 2013). Most studies of deer show δ Ccol ranges from –22.5 to –19.5 ‰ (Bergh, 2012; Emery et al., 2000; White et al., 2001, 2004) which are generally interpreted as reflecting C3 dominant diets, with the exception of some deer who had consumed small amounts of maize probably obtained by browsing at the edge of agricultural fields.

Because of this macronutrient partitioning between tissue fractions, the difference

13 13 13 between δ Csc and δ Ccol (Δ Csc–col) or the carbonate-collagen spacing, can be used as an approximate indicator of degree of carnivory. In herbivores, there is an estimated +5 ‰ increase from diet to collagen, and approximately +12 ‰ increase from diet to structural carbonate

13 resulting in Δ Csc–col mean spacing of ~ +7 ‰ (Clementz et al., 2009; Krueger et al., 1984).

2.3.2 Nitrogen

Nitrogen isotopic compositions of bone and teeth are used in paleodiet studies primarily to differentiate consumption of terrestrial versus marine or aquatic food sources and to identify the trophic position of an organism. The nitrogen isotope composition (δ15N) of animal collagen reflects the source of nitrogen at the base of the food web, e.g. nitrogen fixing plants or fertilized plants. The δ15N values of plants will vary by their environmental context (i.e., soil conditions and climate) and how they incorporate nitrogen. For example, legumes, which fix atmospheric nitrogen, tend to have very low δ15N values (Schoeninger et al., 1982).

In summary, this research will contribute to the expansion of the available local, seasonal, and diachronic paleoenvironmental proxies in tropical areas and to the constriction of climate simulations and projections in this area. Local paleoclimate and land cover data will add to the comprehension of the relation of climate events (specifically dry periods) and agricultural activities in the site. In this light, the use of δ13C and δ18O in herbivore teeth is a relevant source of information as they offer data related to a specific site and chronological period and is less likely to be physically and chemically altered compared to bone collagen and apatite.

2.4 White-tailed deer ecology

Deer are opportunistic in their feeding habits. Although they have preferences, the choice of plants they feed on depends largely on what is available. Throughout their range, deer prefer fleshy fruits but they also relish the leaves and new growth on a variety of woody vines, shrubs and trees. White-tailed deer reject the fibrous wild C4 plants but browse on maize, a modified and non-fibrous C4 crop plant, which forms a significant part of their diet everywhere it is available. Higher maize production in areas accessible to modern white-tailed deer results in documented increases in the inclusion of maize in their diet (Cormie et al., 1994). When maize is not available, they subsist primarily on low-roughage leafy forbs or C3 dicots (Jacobson, 1994).

Clover, vetch, tick trefoil and other legumes are important sources of protein. Instead, deer prefer high-protein and energy rich plants that are highly digestible, like shrub foliage, leafy plants, and acorns over more fibrous grasses (Verme et al., 1984). The plants within the forest are mostly fibrous and deer will resort to them only when compelled by starvation. Deer rummage along forest edges where they are more likely to encounter leafy fodder. Averaging across regions and seasons, white-tailed deer diets consist of 46 % shrub foliage, 24 % forbs, 11 % mast, 8 % grass,

4 % crops, 2 % cactus, 2 % fungus and 3 % other. Grass consumed by deer is typically young and succulent, with high dry matter digestibility (54 -73 %) and at least 14 % crude protein

(Hewitt, 2011). Specific studies in the Yucatan area found that the deer selected mainly trees

(76.5 %), with a smaller proportion of forbs (19.4 %), herbaceous (5.8 %) and a minimum of graminae (0.3 %) (Plata et al., 2009; Ramıreź et al., 2004).

Although their diet may be diverse, white-tailed deer rely on just a few plant species to satisfy their nutrition requirements. Some of these are Acalypha langiana, Croton sp.,

Cardiospermum halicacabum, Coursetia caribaea, Abutilon sp., Ayenia micranta and Spondias 26

purpurea (Arceo et al., 2005). Other common species are Bauhinia divaricata during the entire year. Eugenia sp.and Leucaena leucocephala are common during the rainy and dry season, respectively. The high frequency of herbs during the rainy season is widely recorded in tropical, temperate and arid ecosystems (Arceo et al., 2005; DiMare, 1994). Similarly, the use of shrubs during dry periods is also recorded.

Lowland and montane tropical rainforests that are undisturbed by humans are almost exclusively dominated by C3 plants (Brown, 1999). However, agriculture and associated disturbances can increase the proportion of C4 plants by introducing C4 cultigens and allowing for the spread of native C4 herbs that are rare under closed-canopy conditions (Sage et al., 1999).

In lowland Mesoamerica, other C4 plants include tropical pasture, savannah grasses and species such as Chenopodium album and Amaranthus hybridus. These seem largely absent from the

Maya diet, as they have not been recovered from macrobotanical samples in the area (Emery et al., 2000). Studies of the native grass of the northeastern Mexico have shown low crude protein and digestibility values (between 8 – 12 % and 37 and 49 %), respectively (Lozano, 2007;

Ramı́rez et al., 2004). These characteristics make wild savanna grasses hard to digest for the non- ruminant deer. The CAM plants of the Peten area are represented only by a few tropical succulents (bromeliads and pineapples) and cacti, few of which are important in tropical herbivore diets (Chisholm, 1989). Therefore, the dietary proportions of regular leafy C3 plants and maize in archaeological deer diets can be expected to reflect the availability of C4 plants

(mostly maize) during the lifetime of the deer. The higher the proportion of C4 plants in deer diet, the more positive δ13C values in their tissues. The sizes of deer’s home range are highly variable and based on available resources: deer in areas across higher-quality resources (water, cover, food) that are abundant and well distributed tend to have smaller home ranges than deer 27

that occupy less productive areas (Marchinton et al., 1984). Male white-tailed deer tend to have larger home ranges than females, given that they need more space to meet their nutritional demands. Males also tend to increase their home ranges during the mating season. The average home-range areas for deer in Florida varies between 344 ha for females and 710 ha for males

(Miller et al., 1993). Measurements in tropical deciduous forests indicate 215 ha as the home range and an average between 20 and 57 ha as the activity area (Bello et al., 2004).

Migration behavior in white-tailed deer evolved mainly to mediate changes in forage quality and availability and variable climate or seasonal changes in temperate zones. The available data from northeast United States show migration distances between 7 - 20 km (Stewart et al., 2011). White-tailed deer maximize their energy in the dry season by avoiding traveling. In the rainy season, deer increase their area of activity due to the greater quantity and quality of food, which allows them to choose plants of greater nutritional value.

The reproduction patterns of white-tailed deer are less seasonal in tropical areas than in temperate zones (Weber, 2014). While in Mexico fawns are normally born during the entire year, births have been reported in April and May in Costa Rica (McCoy et al., 1985). Given the climate patterns of the Yucatan Peninsula and the periods of tooth mineralization, it is expected that the upper crown of the second molar forms at the beginning of the rainy season. The lower section of the crown of the second molar and the upper crown of the third molar reflect the rainiest months of September and October. Finally, lower section of the crown of the third molar appears to form during the transition to the dry season (October to February).

Chapter summary

Several studies have provided results on past climate events at the Yucatan Peninsula at different spatial and temporal scales. This research does not support the presence of drought 28

events across the Classic and Terminal Classic. In fact, analyses of bone phosphate from deer show no evidence of this severe dry events. Most of the research has used lake sediments as proxies, which provide relevant information on the catchment area of lakes, but not for the archaeological sites. Studies of tooth enamel from herbivores in temperate zones suggest that this could be an effective proxy for rainfall and temperature seasonality at the local scale.

Additionally, analysis of bone collagen provides insights on the vegetation cover through analysis of the diet of the animal. Finally, an analysis of the behavior, migration and diet habits of white-tailed deer indicates these animals are suitable to use as an environmental proxy.

29 CHAPTER 3

METHODOLOGY

This study investigates the potential of white-tailed deer as a paleoenvironmental proxy using two study groups. The first group includes zooarchaeological remains from La Joyanca from two time periods (1100-1000 BP and 1000-900 BP). The second group (modern white- tailed deer) is used to evaluate the relationship between stable isotopes in enamel and bone from this species and isotopic signatures in rainfall.

This chapter is presented in three sections. The first section describes the materials used in the analysis. The second section describes the laboratory analyses. The final section describes the stable isotope methods used to investigate how archaeological and modern deer remains reflect rainfall seasonality and diachronic changes in precipitation and vegetation.

3.1 Standards

Two additional carbonate standards, one archaeological and one modern, were also included to ensure sample fidelity and to rule out contamination during the sample preparation process. The laboratory standards originated from an archaeological cow from Turkey, and a modern cow tooth from Georgia, United States. These include considerable natural variation in their matrix, and consist of only 3% carbonate. All standards (twenty-five archaeological and sixteen modern) were treated using 0.1 M HOAc (acetic acid). In addition, five of the

30 archaeological standards were treated first with NaOCl (in addition to the HoAc) in order to assess the effect of this pretreatment on the samples.

3.2 Materials

3.2.1 Archaeological deer

A total of ten teeth of adult white-tailed deer (>1.5 years in age) excavated from the archaeological site of La Joyanca, northwestern Peten, Guatemala were analyzed for oxygen

(δ18O) and carbon (δ13C) isotopic compositions in carbonate tooth enamel. The osteological material is associated with two time periods: 1100 – 1000 BP and 1000 – 900 BP. Four teeth

(three individuals) correspond to the first period and to the same stratigraphic context. Six teeth

(three individuals) correspond to the second time period and different contexts (Table 4).

The samples are part of the Florida Museum of Natural History (FLMNH) collection.

Bones were identified to taxon, element, side and age by Dr. Kitty Emery (project zooarchaeologist for La Joyanca) using the comparative collections of the FLMNH

(Environmental Archaeology Program). All the material is from assemblages well dated by stratigraphic and ceramic analysis by archaeologists associated with the project, directed by Dr.

Charlotte Arnauld.

The identifications were also confirmed using the comparative collection in the

Zooarchaeology Laboratory, Georgia Museum of Natural History, University of Georgia

(GMNH-UGA). The identification of wear stages were assigned according to Payne (1973). The wear stages confirmed the age and indicate the suitableness of the teeth to be analyzed.

Table 4. Contextual information for archaeological teeth selected for isotopic analysis Individual Crown height Sample Date Tooth Wear stage Context number (mm) JY1 RM2 D 8.3 Abandonment, 6 2 structure 5D1, layer 3 JY2 RM3 D 8.3

JY3 LM2 E 9.1 Midden, structure 5 2 6F11, layer 1 JY4 LM3 E 8.8

JY5 LM2 D 8.6 Abandonment, 4 2 structure 5D1, layer 3 JY6 LM3 C 9.1 Midden, structure JY7 3 1 RM3 B 9.6 6F11, layer 3 JY8 RM3 E 9.4 Midden, structure 2 1 6F11, layer 3 JY9 RM2 D 7.5 Midden, structure JY10 1 1 RM2 D 7.7 6F11, layer 3 Date: 1: 1100-1000 BP; 2: 1000-900 BP

3.2.2 Modern deer

In addition, twenty-five teeth from modern deer provided by the Deer Laboratory at the

University of Georgia were also analyzed for oxygen (δ18O) and carbon (δ13C) isotopic compositions in carbonate tooth enamel. Thirteen are from wild deer (seven individuals) randomly recovered (unknown dates) from all around Georgia, United States (Table 5). The other twelve (six individuals) come from deer kept at the Deer Laboratory between 1980 and

1994 with known dates of birth and death (non-wild deer,

Table 6). Their diet consisted of Omolene 300 (commercially available feed). In addition, the mandibles from the seven non-wild deer individuals and five from wild deer (individuals 1-5) were also analyzed for carbon and oxygen stable isotope of bone collagen.

Table 5. Contextual information for modern, wild teeth selected for isotopic analysis

Individual Sample Tooth Wear stage Crown height (mm) number MD1 RM3 9.8 1 E MD2 RM2 9.4 MD3 2 RM3 E 9.0 MD4 LM3 11.5 3 E MD5 LM2 12.2 MD8 LM3 9.8 4 D MD9 LM2 10.1 MD10 LM3 11.1 5 E MD11 LM2 10.8 MD12 LM3 8.7 6 E MD13 LM2 9.2 MD14 RM3 7.7 7 E MD15 RM2 8.6

Table 6. Contextual information for modern, non-wild teeth selected for isotopic analysis

Individual Crown Date of Sample Tooth Wear stage Date of birth number height (mm) death MD2 LM3 11.1 A D Spring 1991 8/22/1994 MD22 LM2 10.0 MD3 RM3 9.3 B E Spring 1989 10/14/1994 MD33 RM2 7.7 MD4 LM3 8.5 C E Spring 1986 9/24/1991 MD44 LM2 8.4 MD5 LM3 8.8 D D Spring 1987 1/7/1992 MD55 LM2 8.4 MD6 LM3 8.8 Spring 1987 1/7/1992 E E MD66 LM2 8.3 Spring 1986 2/15/1991 MD7 RM3 7.3 F H 5/28/1984 9/15/1994 MD77 RM2 7.4

33 3.3 Laboratory analyses

Stable isotope analysis of tooth enamel carbonate was carried out at the Quaternary

Isotope Paleoecology Laboratory (QUIP), Center for Applied Isotope Studies (CAIS), University of Georgia. Once sampling and chemical preparation of the specimens was completed, the ratios of stable carbon and oxygen isotopes of the faunal remains were measured by isotope–ratio mass spectrometry (IRMS).

Tooth enamel processing involved the removal of superficial organic matter and then microsampling with a drill. Teeth were drilled from the crown to the cervix of the tooth using a

0.5 mm diamond-tipped drill bit in order to take enamel samples along each crown, in a sequence perpendicular to the growth axis of the tooth. Each sample consisted of a horizontal band (∼1 mm wide and 3–5 mm long) through the depth of the enamel (< 1 mm). The sampled enamel powder of the samples and the selected standards were treated with sodium hypochlorite ∼ 3 % for 24 hours to remove organic matter and then with acetic acid 0.1 M for 4 hours to remove exogenous carbonate (Balasse, 2002). Bioapatite samples weighing approximately 1 mg were reacted with 100 % phosphoric acid at 70°C in individual vessels in an automated cryogenic distillation system interfaced with a MAT253 isotope ratio mass spectrometer. Results are reported with reference to the international standard Vienna Pee Dee Belemnite (VPDB) calibrated through the NBS19 standard and the precision is better than ± 0.13 ‰ for 18O/16O and

± 0.06 ‰ for 12C/13C.

Collagen samples were prepared by demineralizing fragmented 5 g bone chunks in 0.5 M

HCl for several days. The acid was changed every two days until the sample floated and was soft. Then, the sample was filtered using an EZEE filter. The remaining supernatant liquor was gelatinized by heating it in pH 3.0 water at 75 °C for 48 hours. The dissolved collagen was 34

freeze-dried. Subsamples of collagen powders were weighed into tin capsules at CAIS. Collagen samples were analyzed on a Costech Elemental Analyzer coupled to a Finnigan Delta IV Plus

IRMS at CAIS.

Stable carbon (δ13C ‰ per mil deviation of the ratio of 13C:12C relative to VPDB and stable nitrogen (δ15N ‰ per mil deviation of 15N:14N relative to AIR) isotopic composition measurements were made where repeated measurements of the standards were ± 0.05 ‰ for δ13C and ± 0.01 ‰ for δ15N.

3.4 Data analysis

Seasonality in archaeological and modern tooth enamel. As detailed in section 2.2.3, the assumptions underlying this study include that teeth form at a relatively constant rate; they record two to ten months of the animal’s life; rainfall is the main factor effecting meteoric δ18O water values; the δ18O signal is primarily derived from drinking water, and that meteoric δ18O water precipitates into equilibrium with tooth enamel carbonate.

Following these assumptions, the tooth of an animal that stayed in the same general habitat year-round should record and exhibit up to a full year’s seasonal rainfall signature, i.e. display a sinusoidal curve with a range of variation depending on the degree of rainfall seasonality. A model for predicting influences on the patterns of observed δ18O values in modern sheep show that four parameters were most likely to influence the shape of the curve (Balasse et al., 2012). This included amplitude (A) of the observed δ18O; the period sampled (the number of seasons which the sampled tooth recorded, denoted as “X”); the delay of tooth formation from birth, x0 (influencing where the curve meets the y-intercept); and finally, the change of the mean,

“M”, as when ambient annual temperature for one year may be higher than the last. For the

purpose of this study, the influence of the last two parameters is insignificant. If we assume that deer teeth generally mineralize at the rates described in chapter 2, and that no significant wear has occurred, we can expect that the effect of measured amplitude on the curve is the main factor controlling shape in the results described below. From this information, we can evaluate seasonal rainfall fluctuations recorded in the tooth enamel of individuals (Balasse et al., 2012).

Diachronic changes in archaeological tooth enamel. To evaluate the changes in rainfall across periods (between 1100 – 1000 and 1000 – 900 BP), I compared the maximum, minimum oxygen isotopic values obtained for each period. Then, I calculated an average oxygen isotopic composition for each period using the average values for individual teeth and evaluate whether there was a significant difference with a t test where the data were normally distributed.

Ultimately, this allows tracking diachronic changes in rainfall and humidity. This gives information about significant changes in precipitation patterns (if any) and therefore in possible dry intervals during these periods.

This study uses a series of equations that describe the relationship between deer bone and isotopic values of precipitation (see below). Luz et al.(1990) proposed an equation that describes the dependence of the oxygen isotopic values of white-tailed deer bone phosphate substrates on relative humidity and the oxygen isotopic value of rainfall (Equation 1). This equation allows the

18 18 18 comparison of the δ O of white-tailed deer bone phosphate (δ Op) with δ O of rainfall and relative humidity (RH). The Global Network of Isotopes in Precipitation (GNIP) is a convenient data source for seasonal fluctuation in δ18O of rain. The only data set from GNIP collected near the Maya region is from Veracruz (Mexico). This gives temperature, vapor pressure, and precipitation, as well as monthly δ18O of rain from 1962 to 1989 (n= 130). This dataset provides historical information on maximum and minimum values of rainfall that can be compared to the 36

ones obtained using Equation 1. In fact, Repussard et al. (2014) showed that monthly averages of

δ18O of rain are a function of monthly average precipitation amount (pp) at Veracruz (Equation

2).

Equation 1 18 18 2 δ OP = 34.63 + 0.650 δ Orain – 0.171 RH (n = 44; r = 0.95)

Equation 2

18 2 δ Orain = -0.0121 pp – 0.6915 (n = 12; r = 0.79)

Equation 3

18 18 2 δ OP = 0.98 δ OC – 0.85 (n = 31; r = 0.98)

Equation 4

18 18 δ OV-SMOW = 1.03091 (δ OVPDB) + 30.91

Qualitative comparisons can be made between estimated precipitation δ18O and measured tooth enamel δ18O. The Online Isotope in Precipitation Calculator (OIPC Version 2.2: http://www.waterisotopes.org/) was used to obtain model-generated mean monthly δ18O precipitation for La Joyanca (Bowen et al., 2003). This method has been previously used to calculate local precipitation δ18O for comparison with tooth oxygen isotope signatures (Daux et al., 2008).

The results of this study were also compared to those of Repussard et al. (2014), who calculated a series of expected deer bone δ18O values using modern relative humidity and rainfall values of Flores, Peten, Guatemala and converted them to oxygen isotope carbonate values. The location of Flores, on the southern shore of Lake Peten-Itza, is relevant to this study for two reasons. First, it is situated at an altitude of 130 m, in the same climatic area of La Joyanca, ~70 37

km east of La Joyanca, on the path of the prevailing easterly trade winds that are not blocked by any orographic feature (Williams, 1976). Second, detailed climatic records of monthly averages of temperature, relative humidity and precipitation amounts are available on a monthly basis from 1990 to 2010 at Flores (INSIVUMEH, 2016). Even though it is expected that this town receives higher rain amounts than sites located further away from the lake (due to the direct

18 recondensation of evaporated lake water), this project focuses on the relative differences δ ORain values (and ultimately deer bone δ18O values) between dry and wet periods at a given site

(Repussard et al., 2014).

I compared my results from teeth samples with these present-day data. However, this comparison was made with caution as (1) bones and teeth are matrices with different mineralization processes and (2) there is an offset between the oxygen isotopic signatures in bone phosphate (δ18Op), which were used by Repussard et al. (2014), and the oxygen isotopic signatures in bone carbonate (δ18Oc), which I analyzed in this study. For this purpose, Equation 3 was used. Although error is propagated by using each equation, previous studies suggest that only the results of Equation 1 produce a significant error (Pryor et al., 2014) that varies between

1.6 ‰ and 2.8 ‰ according to the sample size.

To address the changes in C4-C3 vegetation across periods, I compared the maximum and minimum carbon isotopic values obtained for each of them. Then, I calculated an average carbon isotopic composition for each time period using the values for individual teeth. To determine if there is any significant difference between the average values in the carbon isotopic values between periods, I ran a t test. Ultimately, this allows tracking diachronic changes in the consumption of C4 (maize) by white-tailed deer. This study also compared the carbon isotopic

signatures from teeth to the ones obtained by Emery and Kennedy Thornton (2008) for bones in the Peten area.

Relationship between δ18O values of precipitation and δ18O of tooth enamel carbonate.

The δ18O values of enamel tooth mineralization of modern deer from Georgia were also analyzed to address if modern white-tailed deer reflect local temperature or precipitation patterns.

Precipitation oxygen isoscapes in the eastern United States are largely a function of latitude and continentality (Rozanski et al., 1993; Vachon et al., 2010). The continental effect is most pronounced in winter due to sharper temperature contrasts and is greatly subdued in summer due to more homogenous temperatures and greater evapotranspiration. Monthly‐resolved precipitation δ18O values are primarily controlled by air temperature for the eastern United

States, with the highest correlations and slopes observed inland and northward. However, this relationship between precipitation δ18O and air temperature is very weak or non‐existent in southeastern states, perhaps due to greater local sensitivity to changing conditions in the nearby oceanic moisture source (Vachon et al., 2010).

Using the United States Network for Isotopes in Precipitation (USNIP) database, Akers

(2016) calculated weight‐averaged weekly values of δ18O and average surface temperature for the USNIP station in Georgia and grouped them by month and season. The information derived from this analysis will be compared to the one produced in the present work. Detailed climatic records of monthly averages of temperature and precipitation amounts are available on a monthly basis from 1981 to 2016 at the USNIP station in Georgia (NOAA, 2017). These values are used to compare the δ18O values I obtained from modern white-tailed deer.

Relationship between diet, vegetation, δ18O and δ13C. Data on the deer diet inform about the surrounding vegetation (C3 and C4) that was available as food. 39

Chapter summary

It is worth noting that this analysis supposes an analogy of modern conditions to the past.

Some studies suggest the pitfalls in extrapolating long-term “isotope effects” from short-term precipitation datasets, in particular, those in which precipitation amount is calculated (Eastoe et al., 2016). Furthermore, the empirical relations between oxygen isotopes and local temperature and precipitations as derived from seasonal cycles do not apply to time scales other than modern ones. Hence, one should be cautious in using present isotope-climate relationships for long-term paleoclimate reconstructions (Xinyu et al., 2016). In the Yucatan Peninsula, oxygen isotopes may be also correlated to different factors according to different time scales.

The main goal of this research is to understand the relationships between white-tailed deer teeth enamel and bones, climate, environment and societies at the local scale. First, to evaluate whether hard tissues from white-tailed deer reflect rainfall seasonality, the results obtained from the carbon and oxygen stable isotopes analyses of tooth enamel from archaeological deer remains from La Joyanca are compared to the modern records of rainfall and vegetation in the area to understand whether there is a correlation between both groups of signatures. Second, the isotopic analyses provide information on the diachronic changes in rainfall amount between two archaeological periods at La Joyanca (1100-1000 BP and 1000-900

BP). Finally, isotopic analyses in teeth enamel and bone collagen from modern white-tailed deer are used to further evaluate the relationship between rainfall, vegetation and diet in wild and non- wild white-tailed deer. This will aid in the interpretation of the results derived from archaeological remains.

40 CHAPTER 4

RESULTS

This chapter presents the results in three main sections that correspond to those outlined in Chapter 3. First, I present the results derived from the standards; second, the archaeological deer (seasonal and diachronic changes); and finally, the data derived from modern deer, which includes from both wild and non-wild individuals isotopic analyses of tooth enamel and bone collagen.

4.1 Standards

Isotopic composition was measured on a IRMS. The precision was better than ± 0.06 ‰ for 13C/12C and better than ± 0.13 ‰ for 18O/16O based on machine standards. Figure 4 shows the results of all standards for all runs of samples presented in this thesis. Six outliers in the standard samples were likely caused by either variation in physical particle size or electrical fault.

Contamination is considered unlikely, since at least two of each standard were included in each run and the outliers never occurred in the same run. In each of the runs with one outlier, data was critically evaluated and still considered valid for inclusion, as other standards were within the expected range of value.

41 Figure 4. Plot of standard values. Two standard samples of modern cow and four standard samples from archaeological cow were outliers and are attributed to machine fault (circled).

4.2 Archaeological deer

Ten teeth from archaeological deer from the periods 1100-1000 BP and 1000-900 BP were analyzed.

Table 7 shows the minimum, maximum, mean, range and standard deviation for the ten teeth analyzed.

4.2.1 Seasonality

1100-1000 BP

Four teeth (two second molars and two third molars) for this period were sampled, from a total of three individuals (Figure 6). Individual 2 is represented by teeth samples JY8 and JY9.

JY9, its second molar, shows a minor decrease in δ18O values from the beginning of the crown, followed by an increase towards higher values through the end of the tooth. The range of intra-

tooth variation of δ13C and δ18O is very small (0.4 ‰ and 0.8 ‰, respectively). The third molar

(JY8) presents a larger range in δ18O values (1.5 ‰) and a trend towards increasing and undulating values. There is not a marked intra-tooth or inter-tooth variation in δ13C values in this individual, while its δ18O record did not provide evidence of major changes as well.

The second molar (JY10) of individual 3 shows a consistent decrease in δ18O values and it presents the highest intra-tooth variation of all the teeth that were analyzed (3.1 ‰). It also records the maximum δ18O value (1.5 ‰) of all the archaeological deer teeth. Additionally, it presents increasing δ13C values through the crown with a large variation in values of ‰.

Individual 1 (JY7) presents an increasing trend in δ18O, including a large range of variation (2.5

‰) and the minimum recorded value (-3.4 ‰). However, the δ13C signals do not show a clear pattern or tendency to increase or decrease. The variation in modelled monthly precipitation δ18O in La Joyanca (derived using the Online Isotope in Precipitation Calculator) is also shown for comparison of the pattern of annual variation (Figure 5).

1000 – 900 BP

Six teeth (three second molars and three third molars) from this time period were analyzed, for a total of three individuals (Figure 7). JY5 had to be excluded from the analysis because it did not yield a large enough sample. JY1 (M2 of individual 4) shows a slight decrease in δ18O values at the middle of the crown, followed by a slight increase. The range of variation is small (0.5 ‰). Its δ13C values show a minimum range of variation (0.3 ‰) and it is not possible to infer any trend from it. The third molar of this individual (JY2) presents increasing values, with a moderate range (1.6 ‰). This agrees with the tendency shown by the third molars in the former period. The δ13C values of JY2 present a regular, small variation (0.2 ‰).

Table 7. Minimum, maximum, range, and mean values of δ18O and δ13C from white-tailed deer teeth for 1100-1000 BP and 1000-900 BP. All δ values are in per mil VPDB.

δ13C δ 18O Crown Toot Sample Height Minimum Maximum Range Mean Std Minimum Maximum Range Mean Std h (mm) 1000-900 BP JY1 RM2 8.31 -14.7 -14.3 0.3 -14.5 0.1 -2.1 -1.7 0.5 -1.8 0.2 JY2 RM3 8.33 -15.0 -14.8 0.2 -14.9 0.1 -1.7 -0.2 1.6 -1.1 0.7 JY3 LM2 9.13 -14.3 -13.3 1.0 -13.8 0.4 -3.4 -1.4 2.0 -2.3 0.9 JY4 LM3 8.77 -13.8 -12.6 1.2 -13.3 0.6 -3.1 0.0 3.1 -1.7 1.6 JY6 LM3 9.14 -13.6 -12.2 1.4 -13.2 0.7 -5.7 -3.1 2.6 -3.9 1.2 1100-1000 BP JY7 RM3 9.62 -13.3 -12.8 0.4 -13.1 0.2 -3.4 0.0 2.5 -0.8 1.1 JY8 RM3 9.4 -15.3 -14.4 0.9 -15.0 0.3 -2.7 -1.2 1.5 -2.0 0.6 JY9 RM2 7.54 -15.1 -14.7 0.4 -14.9 0.2 -1.7 -0.8 0.8 -1.4 0.3 JY10 RM2 7.67 -16.4 -15.1 1.3 -15.8 0.5 -1.6 1.5 3.1 -0.3 1.3 1 R=right; L=Left; Std= standard deviation (1 σ)

The second molar of individual 5 (JY3) presents decreasing δ18O values across its crown, with a moderate range of 2.0 ‰. The δ13C values increase through the crown, but with a slight variation (1.0 ‰). The third molar of this individual (JY4) shows large ranges of variation towards higher values in both δ18O and δ13C.

Finally, JY6 suggests particularly low δ18O values for individual 6, which was determined to be an outlier. It presents the lowest value recorded for enamel seriation of archaeological deer (-5.7 ‰). This same tooth shows the highest value of δ13C of the sample set

(-12.2 ‰).

-1

‰) ‰) -2

-3

SMOW ( SMOW -

V La Joyanca

O -4

18 Veracruz δ -5

-6

-7 Aug Sept Oct Nov Dec Jan Feb Mar Apr May Jun Jul Month

Figure 5. Variation in modelled monthly precipitation δ18O in La Joyanca (derived using the Online Isotope in Precipitation Calculator (OIPC Version 2.2: http://www.waterisotopes.org/)

Individual 3

Individual 2

. Individual 1

Figure 6. A. Intra-tooth oxygen and carbon isotope ratios of the second and third molars from three white-tailed deer for the period 1100-1000 BP.

Individual 6

Individual 5

Individual 4

Figure 7. Intra-tooth oxygen and carbon isotope ratios of the second and third molars from three white-tailed deer for the period 1000-900 BP.

4.2.2 Diachronic changes

The oxygen and carbon isotopic values were normally distributed according to the

Shapiro-Wilk normality test. The box and dot plot shows a few temporal patterns (Figure 8).

First, there is a significant (p = 0.03) decrease in mean δ18O of 0.9 ‰ from 1100-1000 BP (mean

δ18O = -1.2 ‰; n =25, 3 individuals) to the 1000-900 BP period (mean δ18O = -2.1 ‰; n = 20, 3 individuals).

There is a significant increase (p = 0.02) of 0.65 ‰ in mean δ13C values from 1100-1000

BP (mean δ13C = -14.6 ‰, n=21, 3 individuals) to 1000-900 BP (mean δ13C = -13.9 ‰, n=24, 3 individuals). There is a wider range distribution of δ13C values in the former period.

The δ18O values for individuals in the period 1100-1000 BP have ranges of up to 4.8 ‰ between minimum and maximum values (n=4). In the latter period, the ranges of δ18O values observed in each individual increase to approximately 5.7 ‰ difference between maximum and minimum values (n=6).

4.3 Modern deer

4.3.1 Tooth enamel

4.3.1.1 Wild deer

Thirteen teeth from wild deer with unknown dates of birth and death (seven individuals) were analyzed (

Table 8, Figure 9). The observed δ18O values in precipitation are shown for comparison

(Figure 11). The second molars seem to represent a trend, which begins with high δ18O values that start to fall, then rise again through the middle of the crown before decreasing at the end of the crown. The δ18O values fluctuate between -1.3 ‰ and -4.6 ‰. The second molar of individual 4 presents the highest range of δ18O values, with an abrupt increase from low to high 48 values through the middle of the crown (-1.3 ‰ to -3.7 ‰). Although the second molar of individual 6 follows this trend, it shows low δ18O values that are considered outliers (between -

6.2 ‰ and -5.4 ‰).

Figure 8. Boxplot of medians and distribution of δ18O and δ13C values in white-tailed deer through time

The δ18O signals of the third molars of individuals 1, 4, and 7 (Figure 9) show a decreasing trend at the beginning of the crown and then a trend towards increasing values.

Individual 5 shows the highest range of values (-4.8 ‰ and -2.3 ‰). Although it follows this

trend, the third molar of individual 7 shows an attenuated range of values (0.6 ‰). Individual 5 follows this trend although at the beginning of the crown it follows an increasing trend.

The third molars of individuals 2 and 3 represent exceptions to this trend. In the first case, the δ18O tend to increase across the crown. Conversely, individual 3 begins with increasing values and from the middle of the crown it shows decreasing values. All other third molars present increasing values towards the end of the crown. The third molar of individual 6 maintains the low values shown in its second molar. However, the increase-decrease-increase trend in the third molars of individuals 1,4,5 and 7 is also seen in individual 6.

Overall, the δ18O values of third molars present a higher range of values (-4.8 ‰) compared to the second molars (-3.4 ‰). Second molars also present a slightly lower mean (-3.1

‰) compared to the third molars (-2.8 ‰).

The δ13C values fluctuate between -17.1 ‰ and -14.2 ‰, excluding both the second and third molars of individual 6 which shows particularly low values between -5.8 ‰ and -4.0 ‰.

The range of intra-tooth variation is small compared (1.0 ‰) to the ranges in δ18O. The second molars of individuals 1 and 4 show first an increasing trend in the upper section of the crown and then decreasing values at the end of the crown. The second molar of individuals 5 and 7 present an opposite trend. Beginning with increasing values and then decreasing.

The third molars of individuals 1, 2 and 5 show an increasing trend through the crown.

Individual 3 follows this trend but it shows a tendency to decrease at the end. Individual 4 does not show a clear trend towards high or low values. Finally, individual 7 presents the highest values from all third molars.

Table 8. Minimum, maximum, range, and mean values of δ18O and δ13C from wild white-tailed deer teeth. All δ values are in per mil VPDB.

δ13C δ 18O Crown Sample Tooth Height Minimum Maximum Range Mean Minimum Maximum Range Mean (mm) MD1 RM3 9.77 -16.7 -16.3 0.4 -16.5 -3.6 -2.3 1.3 -2.8 MD2 RM2 9.41 -16.7 -15.9 0.8 -16.3 -3.5 -1.6 1.9 -2.4 MD3 LM3 9 -15.8 -15.4 0.5 -15.7 -1.7 -0.5 1.2 -1.2 MD4 LM3 11.47 -16.5 -16.0 0.5 -16.2 -2.4 -1.1 1.3 -1.8 MD8 LM3 9.81 -16.0 -15.5 0.5 -15.8 -4.3 -2.3 2.0 -3.0 MD9 LM2 10.11 -15.4 -14.2 1.2 -14.8 -3.7 -1.3 2.4 -2.9 MD10 LM3 11.1 -17.1 -16.7 0.5 -17.0 -4.8 -2.3 2.5 -3.9 MD11 LM2 10.77 -16.9 -16.5 0.4 -16.6 -4.6 -2.7 1.9 -3.6 MD12 LM3 8.7 -5.8 -5.4 0.4 -5.7 -5.8 -4.1 1.7 -4.9 MD13 LM2 9.25 -5.1 -4.0 1.1 -4.6 -6.2 -5.1 1.1 -5.6 MD14 RM3 7.74 -14.8 -14.4 0.4 -14.6 -2.5 -1.9 0.6 -2.1 MD15 RM2 8.62 -14.9 -14.6 0.3 -14.7 -4.1 -2.4 1.7 -3.4

51 Individual 1 Individual 2

Individual 3 Individual 4

Individual 5 Individual 6

Figure 9. Intra-tooth oxygen and carbon isotope ratios of the second and third molars from modern wild, white-tailed deer.

Figure 10. Intra-tooth oxygen and carbon isotope ratios of the second and third molars from modern wild, white-tailed deer (Individual 7).

0 -1 -2 ‰) -3

-4

SMOW ( SMOW -

V -5

O 18

δ -6 -7 -8

Month

Figure 11. Variation in modelled monthly precipitation δ18O in Georgia (modified from Akers (2016))

4.3.1.2 Non-wild deer

18 Substantial intra-individual δ Ocarbonate variation was observed over the height of the deer second and third molars (

Table 9, Figure 12). Non-wild deer present the lowest range of variation in δ18O values of all teeth analyzed (3.1 ‰ for the entire group and 1.8 ‰ for intra-tooth variation). Individuals A, D and E show a similar pattern of variation in both the second and third molars, with a decreasing trend towards the middle of the crown and then an increasing trend to the end of it. Third molars show a higher range of variation compared to second molars. Individual A presents increasing δ18O values in both molars. Individual B shows a decreasing trend through the crown.

The δ13C signals show small ranges of variations on all the teeth analyzed. The third molar of individual E is the only one that presents a trend towards decreasing values.

Serial sections of enamel suggest an entirely C4-based diet for the first year of life of the six non-wild deer. All six deer maintained uniform enamel mean δ13C values between -7.9 ‰ and

-4.9 ‰ indicative of a C4 diet. In addition, the range of intra-tooth variation is low, with the highest range being 1.2%.

There is also a significant difference (p < 0.001) between the δ18O values of non-wild and wild deer, with the former showing lower values. As these deer were captive, the reason for the low values could be the source of their drinking water, which was provided in a stall at the Deer

Laboratory facilities. On the other hand, the wild deer obtained water from plant leaves and this value is more similar to the one recorded by the archaeological deer (

Figure 13). Table 10 summarizes the results obtained after the analysis of tooth enamel from archeological and modern deer.

Table 9. Minimum, maximum, range, and mean values of δ18O and δ13C from non-wild white-tailed deer teeth. All δ values are in per mil VPDB.

δ13C δ 18O Crown Sample Tooth Height Minimum Maximum Range Mean Minimum Maximum Range Mean (mm) MD2 LM3 11.08 -7.1 -6.6 0.5 -6.9 -5.7 -4.9 0.8 -5.5 MD22 LM2 9.97 -7.9 -7.1 0.7 -7.6 -5.7 -4.7 1.0 -5.4 MD3 RM3 9.35 -6.1 -5.7 0.5 -5.9 -5.3 -3.4 1.8 -4.3 MD33 RM2 7.67 -7.4 -6.7 0.7 -6.9 -5.9 -4.8 1.1 -5.3 MD4 LM3 8.46 -6.3 -5.8 0.5 -6.2 -6.0 -5.0 1.0 -5.6 MD44 LM2 8.38 -7.5 -6.4 1.1 -6.8 -6.1 -5.4 0.7 -5.6 MD5 LM3 8.76 -6.6 -6.3 0.3 -6.5 -5.9 -5.2 0.8 -5.6 MD55 LM2 8.39 -7.5 -6.4 1.1 -6.9 -6.0 -5.5 0.6 -5.6 MD6 LM3 8.75 -6.4 -5.8 0.6 -6.0 -5.3 -4.1 1.2 -4.8 MD66 LM2 8.34 -5.6 -4.9 0.7 -5.3 -6.0 -4.9 1.1 -5.3 MD7 RM3 7.28 -6.7 -6.6 0.1 -6.1 -5.3 -5.1 0.2 -5.3 MD77 RM2 7.45 -6.8 -5.8 1.0 -6.2 -6.5 -5.3 1.2 -5.8 1 R=right; L=Left; Std= standard deviation

Individual A Individual B Individual C

Individual D Individual E Individual F

Figure 12. Intra-tooth oxygen and carbon isotope ratios of the second and third molars from modern, non-wild white-tailed deer

Figure 13. Boxplot of medians and distribution of δ18O and δ13C values in non-wild and wild white- tailed deer (non-wild: n=50; 1000-900 BP: n=59).

4.3.2 Bone collagen

Collagen was deemed well-preserved on the basis of carbon content (%C), nitrogen content (%N) and atomic carbon-nitrogen (C/N) ratios (Ambrose, 1991) (Table 11).

13 15 The δ Ccol and δ Ncol values for bulk bone collagen are plotted in Figure 14. The mean

δ13C and δ15N values in collagen are shown in Table 12. A closer examination of deer by type reveals no significant difference for wild deer and non-wild deer for nitrogen isotopic values.

However, there is a significant difference (p < 0.001) in the carbon isotopic values.

The mean values of the enamel carbon isotopic values of the thirteen deer whose carbonate was analyzed in addition to collagen are shown in Table 12. Figure 15 superimposed over regression lines that illustrate three different protein diets with C3 and C4 energy endpoints, described in greater detail elsewhere (Kellner et al., 2007). This confirms the composition of the deer diet described before.

Table 10. Minimum, maximum, range, and mean values of δ18O and δ13C from archaeological and modern white-tailed deer teeth. All δ values are in per mil VPDB. δ13C δ18O Period/ Me Mi M Ran St Me M M Ran St Variable an n ax ge d an in ax ge d 1100–1000 -14.6 -16.4 -12.8 3.5 1.0 -1.2 -3.4 1.5 4.8 1.1 BP 1000-900 -14.0 -15.0 -12.2 2.8 0.8 -2.1 -5.7 0.04 5.7 1.3 BP Wild -14.1 -17.1 -14.2 2.9 0.8 -3.2 -4.8 -0.5 4.8 1.0 Non-wild -6.5 -7.9 -4.9 2.9 0.7 -5.3 -6.5 -3.4 3.0 0.6

Table 11. Carbon and nitrogen stable isotope collagen values, total nitrogen and carbon and carbon/nitrogen ratio of the deer mandibles analyzed

Sample δ13C (‰) δ15N (‰) Total %C Total %N C/N ID VPDB AIR JD1 -20.0 -0.5 43.8 15.5 3.3 JD2 -18.5 7.9 41.8 14.7 3.3 JD3 -17.3 2.9 43.4 15.2 3.3 JD4 -16.2 5.7 44.2 15.3 3.4 JD5 -22.2 5.3 44.0 15.1 3.4 JD6 -17.0 5.7 43.3 15.0 3.4 JD7 -16.1 3.9 43.9 15.4 3.3 JW1 -23.1 4.8 43.4 15.2 3.3 JW2 -22.0 6.3 43.8 15.3 3.3 JW3 -22.3 4.5 42.5 14.8 3.3 JW4 -13.7 4.6 43.8 15.3 3.3 JW5 -22.5 4.9 42.5 14.7 3.4 JW6 -23.4 5.9 38.3 13.6 3.3

13 15 Table 12. Mean values of δ C and δ N from modern white-tailed deer teeth

13 15 Deer δ CVPDB (‰) δ NAIR (‰) Wild -22.7 4.4 Non-wild -18.2 4.9

59 Figure 14. Stable carbon (δ13C) and nitrogen (δ15N) ratios of Georgia deer jaw collagen.

13 13 Figure 15. Stable carbon isotope data from apatite (δ apt) and collagen (δ Ccol) from Georgia deer mandibles

Chapter summary

The oxygen isotopic signatures of intra-tooth enamel from several archaeological deer from La Joyanca and modern, wild deer from Georgia exhibit a range of variation and a sinusoidal pattern as expected. The carbon isotopic signatures do not seem to follow this trend. A statistical evaluation of the diachronic changes in the oxygen isotopic signature between the two archaeological periods of 1100-1000 BP and 1000-900 BP suggests that there is a significant but minor increase in the rainfall amount during the latter period, but there is not any change in the type of vegetation consumed by deer between the periods. The carbon isotopic signatures from both groups show small ranges of variation, with no sinusoidal pattern and with values that confirm a diet based entirely in C3 plants.

Conversely, the isotopic signatures from non-wild Georgia deer do not record any seasonal trend and suggest a mixed C3 and C4 diet. Finally, the results from the carbon and nitrogen isotopic analyses of bone collagen indicate a C3 diet for the wild deer and a mixed C3-

C4 diet for the non-wild deer.

CHAPTER 5

DISCUSSION

This chapter addresses the application of the results of the research objectives, which focus on understanding the potential of white-tailed deer teeth as paleoenvironmental proxies in the tropics and in particular at the archaeological site of La Joyanca. The results derived from modern deer from Georgia aid the interpretation of isotope values of deer teeth from archaeological sites and illustrate the relationship of isotopic signals in white-tailed deer to environmental variables.

This chapter consists of three sections. The first section evaluates the methodology and precision of this study. The second section describes the results derived from the isotopic analyses in archaeological deer, focusing on the seasonality and the diachronic changes. The third section examines the relationship between δ18O values of precipitation and δ18O of tooth enamel carbonate and the relationship between diet, vegetation, δ18O and δ13C.

5.1 Methodology and precision

There is still no agreement on the effects of removing organic matter from teeth on isotopic values of bone carbonate and phosphate, neither is there agreement regarding the optimal reaction time (Koch, 1997; Zazzo et al., 2006). Previous studies demonstrated that the reaction between NaOCl and bone alters the isotope values of bone, causing a decrease in δ18O and δ13C values (Schöne et al., 2016; Wierzbowski, 2007). However, the modern deer study used

fresh bone (presumed to be unaltered), in which the organic content is higher than in archaeological bones, so the shift induced by NaOCl treatment may reflect the removal of actual contaminants. Conversely, other studies consistently use NaOCl in the pretreatment of their samples (Balasse, 2002; Garvie-Lok et al., 2004; Stevens et al., 2011).

In the present study, the effects of the pretreatment with NaOCl were tested by preparing two different sets of standards. One set was exposed to NaOCl and the other was not. The analyses did not show any significant difference in the values of the two datasets. In addition, each sample was subjected to the same organic matter removal protocol, using the same NaOCl stock solution and the same reaction times. It is thus considered that a possible offset of the isotopic values would have been the same for each sample, allowing a meaningful comparison between the obtained values.

In addition, the interpretations derived from the methodology followed in this study should consider some other factors. First, the major issue when reconstructing past environmental and climatic changes using archaeological material stems from its scarcity. The variable duration of archaeological ceramic phases may contribute to uncertainty in the climatic and environmental representativeness of the data, as well as in comparing records between sites.

The resolution of the cultural sequence of the site, based on seriation, only allows for the association of bones and teeth within a range of 100 years (1100-1000 BP or 1000-900 BP).

While radiocarbon dating might help narrow the temporal relationship to ± 20 years, it is cost prohibitive. The temporal resolution of tooth enamel carbonate is limited to the time of mineralization of each tooth, up to ten months of record. The time frame that is being sampled could be any two to then months in those 100 years. In order to address this issue, one must therefore be sure to analyze a sufficient number of individual deer teeth, to provide a broad range 63

of specimens from across the phase. In this case, though a small sample size, all available specimens were analyzed. At the same time, the inter-individual variability in the timing of growth of the M2 and M3 is also a factor that needs to be taken into account.

5.2 Archaeological deer

5.2.1 Seasonality

Several studies have stated that deer body water reflects ingested water, as discussed previously. According to the model described above, more positive values of δ18O in rainfall are considered as evidence of drier conditions, and more positive δ13C values indicate a higher

18 proportion of C4 plants in the diet. The archaeological enamel δ O values from the six individuals reflect body water composition which changes over approximately a 10-month time period and thus corresponds with seasonal variation in δ18O of meteoric water.

Previous studies have assessed the relationship between rainfall amount and δ18O values.

Particularly, for the Yucatan Peninsula, precipitation will have higher δ18O values in the driest months and lower δ18O values in wettest months (Lachniet et al., 2009). The available climatic dataset from Flores shows that the rainy season takes place from May throughOctober, although the majority of precipitation usually falls in summer (from June to October). The dry season

18 takes places between November and April, while the highest values of δ ORain are seen during the winter (February) (Lachniet et al., 2009).

Over the height of the second and third molars, substantial intra-individual δ18O variation was observed. A broadly sinusoidal pattern in the δ18O values was found across the second and third molars of selected individuals, although some variation is observed between individuals.

Both the second and third molars were available from individuals 2, 4, 5 and 6 and were

expected to provide at least ten months of record. Samples from these individuals show what would be expected for a deer born in April or May, in which decreasing values are seen through its second molar (where the rainy season starts) and a drying trend towards more positive values between November and February in the third molar.

The interpretation for those individuals with just one tooth (a second or a third molar) is limited given the short time period that is represented within a single tooth. However, individuals

1 and 3 showed increasing and decreasing values, respectively. This follows what would be expected if third and second molars reflect the rainfall pattern of the area. As an exception and outlier, sample JY6 (individual 6) (period 1000-900 BP) shows an anomalous pattern, suggesting the presence of high amounts of rain during November-December. These months are anticipated to be dry according to the modern climate records. This animal could have been born in a different season or during a year with anomalous rainfall amounts.

Some samples, such as JY1 and JY9, show a dampened signal, which prevents conclusions regarding the trend shown by them. It is important to note that this effect can be caused by physiological effects (Kohn et al., 1998), mineralization rates (Balasse, 2002, 2003;

Zazzo et al., 2006) and sampling (Hoppe, 2006). Figure 6 and Figure 7 indicate that the teeth sampled for this project show the lowest δ18O values between June and October, matching the start of the rainy season. Also, the third molars seem to capture the transition from the rainiest months to the dry season (November to February). Individual 1 and Individual 2 show the drying trend that represents this pattern, in which lower values represent rainy conditions and the transition to the dry season is seen in higher values of δ18O.

In addition, the δ18O values are higher in the second molars than in the third molars during 1100 – 1000 BP. A possible explanation for this pattern could be that second molars capture the first three months of the rainy season (which are not the rainiest), while the third molars record part of the end of the rainy season (including the rainiest months of September and

October) and the beginning of the annual dry period. However, during 1000-900 BP, the δ18O values of second and third molars are almost the same. This trend seems to be the result of the latter period being wetter than the former or the individual variation in birth date and tooth

development, as

Figure 8 shows. This conclusion should be taken with caution given the broad range of variation of the δ18O values.

Third molars also show higher ranges of intra-individual δ18O values compared to second molars in both periods. This may be the result of the larger time period that is recorded by third molars, which is reflected in the fact that two of the second molars analyzed (JY1 and JY9) show the smallest ranges of variation (0.5 ‰ and 0.8 ‰). More studies that address the mineralization process of white-tailed deer in tropical areas would clarify this argument.

The results discussed above suggest that serial sampling from white-tailed deer teeth record seasonal variations in rainfall through isotopic signatures that present sinusoidal 66

patterns of variations in the Peten area. This trend has been well-documented in domestic sheep and goat (Balasse, 2002, 2003; Balasse et al., 2012). In red deer, Stevens et al. (2011) recently found a broadly sinusoidal, seasonal pattern that corresponds to the sinusoidal pattern observed in precipitation δ18O over an annual cycle for the non-migratory population on the island of Rum (Scotland).

The ranges of inter (4.8 ‰ – 5.7 ‰) and intra-tooth (0.5 ‰ –3.1 ‰) variation of δ18O values in this study are similar to those found in other studies that involved white-tailed deer bones in the Peten area of around 3-4 ‰ (Repussard, 2009) and Scotland (Stevens et al., 2011).

By applying Equation 4 to the mean δ18O values in the present study, it is possible to transform them to V-SMOW and then compare them to the ones reported by Repussard (2009), whose study was based on bone bioapatite phosphate and stated values between 27.5 ‰ and 30.3 ‰.

When converted to V-SMOW values, the present study finds a range of values between 26.9 ‰–

30.6 ‰. The intra-tooth sampling of enamel provides a higher temporal resolution for tracking seasonal variations in rainfall, as opposed to bones, which have a resolution of around 2.5 years.

The damping of most of the δ13C values does not allow inferences about seasonal change in the vegetation in the individuals studied at La Joyanca, as there is no value below 13 ‰.

5.2.2 Diachronic changes

The statistically significant change in mean δ18O values between periods indicates that between 1000-900 BP there were on average slightly wetter climatic conditions than during

1100-1000 BP. Both periods show the same range of δ18O variation, which suggests that neither period presented extreme wet or dry events. These results agree with paleoenvironmental reconstructions at Laguna Paixban and Lake Peten Itza. Indeed, none of them present evidence of

severe arid conditions during the Terminal Classic, with the exception of Salpeten, which suggests a drying trend during this period of time.

The sedentary lifestyle of deer makes it a very local recorder of past conditions. It is inferred that in the Maya Lowlands, the home range of deer is a few km2. It is also expected that the deer consumed by the Maya were hunted locally, so it is considered that the isotopic signal in deer bones and teeth excavated at a given site represents very local conditions. This makes difficult to compare the results from this study to ones using lake sediments or speleothems which represent larger catchment areas. However, the results of this study (which suggests that the period between 1100-1000 BP was drier than the following one) are in agreement with some studies in the southern and northern Maya lowland. In the first region, the study by Wahl et al.

(2013) indicates the presence of drier conditions during 1270–1040 BP. Evidence of drought was also found in 1040 BP (Webster et al., 2007). For the northern lowlands, Medina-Elizalde et al.

(2010) proposed the possibility of short droughts during 1144 and 1015 BP, which corresponds with the first period evaluated in the present study. Other studies suggest the presence of severe dry conditions during the whole timeframe of the Terminal Classic (Curtis et al., 1996; Haug et al., 2003; Hodell et al., 2007). There are more available studies in the northern lowlands and some of them provide contradictory results (Table 1). These discrepancies could possibly be explained by the uniqueness of Yucatan's climatology, as well as the restricted spatial resolution of our method. In any case, the significant difference in δ18O values between periods should be taken with caution as the range of variation within each period is similar value of the difference between periods.

These results are in agreement with the paleoenvironmental reconstructions at La

Joyanca, which suggest that by 1150 BP reforestation began and the regional population left or 68

drastically decreased. Later, hints of agricultural activities (some degree of deforestation and maize pollen) are dated to 800-750 BP (Carozza et al., 2007).

At this time, it is not possible to assess if rainfall δ18O has mostly reﬂected the same indicators (such as precipitation amount) in the past. Future studies would provide insights in the correlation between time period (modern deer vs. archaeological deer) and δ18O values, which could indicate the continuity of body water composition over a long period.

When comparing the precipitation δ18O and tooth enamel δ18O values, it is clear that the individual range of estimated δ18O of drinking water for each of the deer is less than that of the

18 population as a whole, and is also less than that of the modelled δ O in precipitation range. This could be the result of both the sampling procedure or the intra or interannual variation in the oxygen isotopic value of precipitation. There is the possibility that the sampled deer are representing years with anomalous or unusual amounts of rain. The range and absolute values of estimated δ18O of drinking water for the population as a whole is similar to that of the modelled

18 18 δ O in precipitation range and very similar to that of the Veracruz δ O in precipitation. This

18 suggests that the full amplitude of seasonal changes in δ O in precipitation is detectable in the

18 tooth δ O carbonate values of the population, although this cannot be confirmed without direct

18 measurements of δ O in precipitation on a site closer to La Joyanca.

The comparison between the δ18O values derived from deer teeth and the modern δ18O rainfall should be taken with caution. First, the accuracy of the modelled δ18O precipitation is related to the availability of precipitation isotope data from locations around the world. Uneven sampling through time and space can affect the interpolated isotopic results in certain locations.

Second, the model does not currently calculate confidence intervals for the mean monthly δ18O precipitation it generates. To assess further whether the values and seasonal pattern in δ18O 69

precipitation estimated by OIPC are reasonable, published δ18O precipitation data obtained from precipitation collected at Veracruz (IAEA, 2017; Tyler et al., 2007) were obtained. The δ18O precipitation data are available from Veracruz; however, the data from this location cannot be used to assess the quality of the OIPC as they are included in the IAEA data-set on which the

OIPC is based. The annual mean, mean dry season minimum and mean wet season maximum precipitation δ18O at Veracruz are very similar to those predicted for La Joyanca by OIPC, suggesting that both the values and the seasonal pattern in δ18O precipitation inferred by OIPC is likely to be a reasonable estimation of the actual values.

Serial sections of enamel suggest an entirely C3-based diet for the first year of life of archaeological deer. All deer showed enamel δ13C values between -16.4 ‰ and -12.2 ‰ indicative of a C3 diet. In addition, the range of intra-tooth variation is small. This agrees with what is expected of a C3 diet. Values higher than -13 ‰ represent a mixed diet of C3 and C4 plants. However, the interpretation of these values is complicated due to the possible mixed effects of C4 proportion in the diet, the density of forest coverage and the effect of water stress on the isotopic signal. The proportion of C4 plants in an individual’s diet may increase in the case of starvation, likely to occur during more arid conditions, when C3 plants with sufficient moisture would no longer be available. Therefore, high δ13C (higher than -13 ‰) could be interpreted as reflecting drier conditions. The canopy effect could also cause lower δ13C values.

However, La Joyanca is in a zone of dense rainforest and was likely never deforested during its history. Due to the canopy effect, the plants ingested by the deer in the region surrounding La

Joyanca are expected to be relatively 13C depleted. However, the small range of intra-tooth variation in δ13C values does not suggest seasonal changes in the deer analyzed (c.f. Drucker et al., 2008). 70

The δ13C values derived from the present study differ from the ones obtained by other studies in the Yucatan Peninsula. While the mean δ13C values from enamel in this study are between -14.1 ‰ and -14.6 ‰, the range reported by previous studies for bone apatite fluctuate between 10.2 ‰ and 12.4 ‰. This difference could be the result of the deer of La Joyanca consuming an entirely C3 based diet, while deer at other Maya sites had a mixed C3-C4 diet.

While the results concerning δ13C in the present study are more difficult to decipher than the

δ18O, it can be observed that the range and average δ13C remained quite stable between both periods of time, but higher values are observed during the 1000–900 BP. Finally, a weak correlation between the carbon and oxygen isotopic values indicates that these signals do not reflect the same variables (Figure 16).

Figure 16. δ13C against δ18O in the archaeological deer teeth dataset(r2=0.2)

Comparison of precipitation δ18O and tooth enamel δ18O values. Figure 17 compares the OIPC

18 18 modeled δ O in precipitation with the estimated δ O of drinking water of deer. To compare the range of variation of δ18O of drinking water with the annual range of variation in δ18O in precipitation, it is necessary to have both the minimum and maximum rainfall values recorded in

18 the tooth isotope sequences. The minimum δ Oc values are expected to be recorded in the M3 isotope sequences of all the individuals, matching the wet season, whereas the maximum ones are recorded in the M2 isotope sequences, matching the dry season. The whole population δ18O of drinking water values for the period 1100-1000 BP have a range of 7.5 ‰, from a maximum of 2.3 ‰ to a minimum of −5.2 ‰ (V-SMOW). The period between 1000-900 BP have a range of 8.9 ‰, from a maximum of 0.04 ‰ to a minimum of −8.8 ‰ (V-SMOW). These ranges are

18 larger than that observed for the modelled δ O in precipitation (range of 6.1 ‰, rainfall maximum of – 0.3 ‰ and rainfall minimum of – 6.4 ‰ (V-SMOW), and that observed at

Veracruz (range of 5.7 ‰, mean rainfall maximum of -5.2 ‰, mean rainfall minimum of − 0.5

‰ (V-SMOW).

18 Figure 17. Range of calculated drinking water δ OSMOW values based on intra-tooth M2 and M3 enamel carbonate measurements for each of the deer teeth whose isotope sequences cover both 18 maxima and minima rainfall together with the seasonal range of Veracruz precipitation δ OSMOW 18 values and modelled precipitation δ OSMOW values for La Joyanca (modelled using the Online Isotope in Precipitation Calculator (OIPC Version 2.2: http://www.waterisotopes.org/).

5.3 Modern deer

5.3.1 Tooth enamel

5.3.1.1 Wild deer

Oxygen. Most of the individuals sampled for this project seem to represent a trend in which more positive δ18O (August) values start to fall in September and October and then rise again through at the end of the autumn, ending with more positive values in early winter (January and

February). This follows the seasonal trend found for Georgia. When grouped by season, summer presents the highest values. Conversely, March and October show particularly low values.

As stated before, the tooth mineralization process indicates that the second molar could record August through November and the third molar October to March. While the intra-tooth sequences of the second and third molars of individuals 1, 4 and 5 follow the δ18O seasonal pattern proposed for Georgia, individuals 3,6 and 7 do not follow a clear trend. Individual 3

73 shows a trend towards lower values in the third molar record, while individual 6 presents fluctuations between high and low δ18O values through the sequence recorded by the second molar. In addition, these values are particularly low compared to the equivalent ones in other individuals. Finally, individual 7 shows an attenuated signal in the third molar sequence. The large range of values at the top of the crown in the third molar sequence also complicates the interpretation of the δ18O values. Taking into account that these deer were randomly collected and their year of collection is unknown, these differences in the absolute values of δ18O are expected.

In summary, δ18O values from wild deer largely follow the pattern shown by the δ18O seasonal values in water precipitation. However, as the correlation and relationship between rainfall and temperature and δ18O values in precipitation is weak in Georgia, inferences about past climate conditions using deer in this area of the United States are limited.

Carbon. Serial sections of enamel suggest an entirely C3-based diet for the first year of life of the wild deer. All deer maintained uniform enamel mean values between -17.1 and -14.2

‰ indicative of a C3 diet. In addition, the range of intra-tooth variation is low, with the highest range being 1.2 ‰. As individual 6 showed variations between -4 and -5.8 ‰, it was considered as an outlier within this framework. Compared to the previously published literature suggesting warm weather maize consumption, a seasonal pattern of maize consumption is suggested by the

δ13C values of the serial-sectioned enamel data of modern deer, at least during the first year of life. For the modern wild deer in this study, C4 plant consumption matches the summer period.

5.3.1.2 Non-wild deer

Most of the second molars of the analyzed non-wild deer do not follow the pattern that would be expected according to the δ18O values of precipitation. However, most of the third molars seems to follow the trend towards higher values, a trend that is expected for the fall and winter. Deviations from the expected values in δ18O of non-wild deer are likely due to the fact that the sources of their drinking water were ponds or tap water, and not leaves.

Serial sections of enamel suggest a mixed diet of C3 and C4 for the first year of life of the non-wild deer. This is in agreement with the known diet of these deer, who were fed with

Omolene 300, a mixture of whole oats, cracked corn, dehulled soybean meal, cane molasses, wheat middlings, coarse barley, soybean oil, stabilized rice bran. Although these deer were all fed the same diet, they present an inter-tooth range of variation of 2.9 ‰.

5.3.2 Bone collagen

The archaeological bone in this study could not be sampled in order to limit destructive analysis. However, the carbon isotopic analyses of collagen in modern deer confirm the differences between the diet of wild deer and non-wild deer, the latter having significantly more positive δ13C values compared to the former. The δ13C values of bulk bone collagen from wild deer suggest that they consumed mostly C3 plants. This is consistent with previous studies of

13 modern deer from the region where δ Ccol values greater than –20 ‰ were assumed to indicate a

C4 component in the diet (Cormie and Schwarcz 1994). The non-wild deer show significantly more positive values compared to the wild deer values due to the mixture of grains that compose their diet. These results are similar to the values reported for archaeological white-tailed deer on

St. Catherine’s Island, in Georgia (Bergh, 2012). In addition, in an analysis of North American

modern deer remains, Cormie and Schwarz (1994) found that where maize is a major crop, deer bone collagen δ13C values are as high as -17.8 ‰, whereas in areas not growing C4 plant, the

δ13C values ranged down to -23.3 ‰. When corrected for pre-industrial atmospheric values

(Stuiver, 1978), these are equivalent to 16.3 ‰ for deer feeding partially on maize, and 21.8 ‰ for those feeding entirely on C3 plants.

15 The δ Ncol values of wild and non-wild deer are consistent with those expected for an herbivore. The lower modern deer results are similar to those observed by Cormie and Schwarz

(1994) across the United States and Canada. On average, there is less than 3 ‰ variation among wild and non-wild deer from sites spanning 3000 years, suggesting consistency in deer diet

(Figure 14). The nitrogen isotopic signature is also higher in non-wild deer due to the effects of fertilizers in the commercially available feed they consumed.

Figure 15 illustrates that the whole diet and the protein portion of wild deer consisted of

C3 sources. The isotopic signals from non-wild deer confirmed the fact that these deer had a C4 component in their diets.

Because maize leaves can have δ13C values several per mil lower than the grains

(Tieszen, 1991), the relatively low proportion of maize in the diets of modern wild deer may be explained by a preference for leaves that are available during warmer months of the growing season. A previous isotopic laser analysis of individual osteons in deer bone indicates increasing

C4 consumption during warmer, summer months (Larson et al., 2007). The enamel series of δ13C does not support this idea, as there is no pattern suggesting higher maize consumption during the summer.

18 18 Figure 18 and Figure 19 compare the δ O in precipitation with the estimated δ O of drinking water of deer. Equivalent to the archaeological deer, the minimum values are expected to be recorded in the M3 isotope sequences of all the individuals, whereas the maximum ones are recorded in the M2 isotope sequences. The whole population δ18O of drinking water values for the wild deer have a range of 10.41 ‰, from a maximum of 0.7 ‰ to a minimum of approximately. -9.7‰ (V-SMOW). The non-wild deer population has a range of 4.74 ‰, from a maximum of -5.4 ‰ to a minimum of approx. -10.1 ‰ (V-SMOW). These ranges are larger than those observed at the Georgia USNIP Station (range of 4.2 ‰, mean rainfall maximum of -

2.7 ‰, mean rainfall minimum of −6.9 ‰ (V-SMOW)).

The individual range of estimated δ18O of drinking water for each of the deer is less than

18 that of the population as a whole, and is also less than that of the δ O in precipitation range. This could be the result of the intra or interannual variation in the oxygen isotopic value of precipitation. There is the possibility that the sampled deer represent years with anomalous or unusual amounts of rain. The range and absolute values of estimated δ18O of drinking water for

18 the population as a whole is similar to that of the Georgia δ O in precipitation. This suggests that

18 18 the full amplitude of seasonal changes in δ O in precipitation is detectable in the tooth δ O carbonate values of the wild deer population, but not in the non-wild deer.

In summary, the data from wild deer suggest that white-tailed deer teeth enamel record the seasonal changes in rainfall in Georgia. By having a controlled diet, the range of values retrieved by the non-wild deer (2.9 ‰ and 3.0‰ for carbon and oxygen, respectively) suggest that ranges of variation above these numbers imply a significant change in rainfall or/and diet/vegetation. In fact, Table 10, shows that the archaeological and the wild deer show larger ranges of variation compared to the non-wild. This demonstrates the ability of white-tailed deer 79

to record changes in rainfall amount in both geographical areas. Both the archaeological and wild deer show similar ranges of variation to the non-wild deer, but non-wild deer demonstrate that individuals managed by humans may not be viable proxies for environmental conditions.

Chapter summary

Oxygen and carbon isotopic analyses of teeth from white-tailed deer are used to address the relationships among this species, its diet, rainfall and vegetation and its potential as a paleoenvironmental proxy at La Joyanca, Yucatan Peninsula. The major findings as they relate to the research objectives are reviewed below.

The deer from La Joyanca show variability in the oxygen isotopic signatures among intra and inter-tooth samples as well as individuals. This reflects the seasonality in rainfall reported over a 10-month period in modern records of the area. The range of oxygen isotopic values in the archaeological deer fall within the modelled and observed values from La Joyanca and Veracruz, respectively. The carbon isotopic signatures suggest a diet based on C3 plants and do not show any vegetation seasonality. The period 1000-900 BP appears to be wetter compared to 1100-1000 BP. This agrees with the results from previous paleoenvironmental studies in the region.

The oxygen isotopic signatures of modern, wild white-tailed deer reflect the rainfall seasonality recorded in the isotopic signatures of precipitation, while the carbon isotopic signatures suggest a diet entirely based on C3 plants. This differs from the results from non-wild deer, which do not reflect rainfall seasonality and show a mixed diet composed of C3 and C4 plants. While the range of variation of the oxygen isotopic values reflect the observed ones from

Georgia, the non-wild deer results appear to be significantly lower. This confirms the influence of diet when using white-tailed deer as an environmental proxy. The nitrogen and carbon 80

isotopic values from collagen confirmed the results obtained from the carbon isotopes of teeth enamel.

CHAPTER 6

CONCLUSIONS

The research presented here is the first attempt to explore the potential of white-tailed deer teeth as an environmental proxy in tropical areas. The project relies on white-tailed deer because of their importance and ubiquity in archaeological sites in the Maya area, where the main study site of this project was located. This research set out to clarify three points about the use of deer as an environmental proxy: (1) whether stable isotope analysis of tooth enamel records seasonal changes in rainfall and vegetation at the archaeological site of La Joyanca; (2) whether there is any change in rainfall and vegetation between 1100-1000 BP and 1000-900 BP at this site, and (3) to explore the relationships among modern deer, diet, rainfall and temperature.

Seasonality in archaeological deer

The analyses of sequential sub-samples from white-tailed deer second and third molars from this species show that enamel δ18O of carbonate values vary within teeth and that the pattern of intra-tooth isotopic variation broadly corresponds to the sinusoidal pattern observed in precipitation δ18O over a 10-month period. Intra-tooth oxygen isotope signatures of white-tailed deer are a good proxy for tracking rainfall seasonality at the archaeological site of La Joyanca.

The deviations from this pattern could be the result of actual changes in precipitation at that time period, mineralization rates, physiological effects, or the loss of sample due to tooth wear.

82 For the population of archaeological deer as a whole, the range in calculated drinking water δ18O values is similar to that of the local modelled precipitation δ18O at La Joyanca and the measured precipitation δ18O at Veracruz. This supports the use of white-tailed deer teeth as a useful rainfall proxy for tropical areas, which could aid in the development of modelling studies that advance our knowledge of the future of climate in tropical areas. Additionally, this information could help in the understanding of the human-environment relationships in the archaeological record at the local scale. Carbon isotopic values of teeth enamel from archaeological deer do not record any seasonal changes in vegetation through the 10-month period recorded by this proxy, expected in tropical areas.

Diachronic changes

Paleoclimatic studies disagree on the conditions prevailing during the Classic and

Terminal Classic in the Yucatan Peninsula. While some of them suggest a drying trend until

1000 BP, a single pattern cannot be extrapolated from any study and imposed on at the local scale (and at archaeological sites). The data gathered in this study show diachronic changes in the δ18O values of deer teeth enamel between 1100-1000 and 1000-900 BP. Though a small sample, these results agree with previous studies at the local and regional level, which suggest that the latter period was wetter compared to the earlier one. A larger sample of specimens from other archaeological sites would contribute to the strength of this conclusion.

The intra-tooth carbon isotope signatures suggest that the individuals included in this study (except for the modern non-wild deer) consumed an entirely C3 diet during their first ten months of life. The data did not show any signal of C4 consumption or canopy effect.

Modern deer diet and its relationship to rainfall

The data obtained from modern, wild white-tailed deer from Georgia, a subtropical setting, support the use of this species as a proxy for δ18O in precipitation values because the

δ18O values calculated from the tooth isotope signatures are similar to the monthly and seasonal

δ18O values in Georgia. The data from non-wild deer did not follow the seasonal pattern of variation and the δ18O values were significantly more negative than the recorded ones. This confirms the influence of the diet and the significance of water source in the isotopic signature of deer tooth enamel carbonate and bone collagen. The data from non-wild deer provided a control range of variation in δ18O and δ13C values from which to compare the variation in carbon and oxygen isotopic values in archaeological and modern, wild deer.

The stable isotopic values of the mandible bone collagen in wild and non-wild deer confirmed the data obtained with the tooth enamel bioapatite in each individual. These measurements suggest a C3-based diet for wild deer and a mixed of C4 and C3 for the non-wild.

This agrees with what was expected given the diet of the non-wild deer.

Finally, while the interpretation of these data is limited by the number of available samples and precise dating them, more research on the tooth mineralization process of white- tailed deer from tropical areas is required to better understand the role of its tooth enamel as a paleoenvironmental proxy. Additionally, furthering the knowledge about the differences in the signatures recorded in second and third molars is essential in order to take full advantage of the data derived from isotopic analyses of tooth enamel of deer. More research on deer from hunt stands could be valuable when studying wild deer isotopic values because they inform about the location of their home range.

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APPENDIX

Table A- 1. Details of archaeological deer teeth samples

Height of FMNH Internal Length Width crown to be Catalog lab Date Side Element Tooth (tooth) (tooth) (mm) sampled number number (mm) (mm) 1801:2 JY1 1000-900 Right Mandible M2 13.34 9.53 8.31 JY2 BP Right M3 18.04 9.77 8.33 673:1 JY3 Left Mandible M2 13.92 9.4 9.13 JY4 Left Mandible M3 18.21 9.52 8.77 1801:3 JY5 Left M2 12.67 8.21 8.57 JY6 Left M3 17.06 9.01 9.14 2628:4 JY7 1100-1000 Right M3 16.64 7.71 9.62 BP 2628:3 JY8 Right Mandible M3 17.65 9.3 9.4 JY9 Right M2 12.38 9.67 7.54 2628 JY10 Right Tooth M2 14.98 13.72 7.67

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Table A- 2. Details of individual tooth sub-sample isotopic measurements for archaeological deer Distance from Sample Date Tooth crown (mm) δ13C (VPDB) δ18O (VPDB) JY1a M2 0.52 -14.69 -1.69 JY1b M2 2.02 -14.48 -1.67 JY1c M2 3.16 -14.51 -2.12 JY1d M2 4.39 -14.34 -1.84 JY1e M2 5.65 -14.54 -1.66 JY2a M3 0.73 -14.94 -1.74 JY2b M3 2.17 -14.82 -1.60 JY2c M3 3.65 -15.02 -1.22 JY2d M3 4.72 -14.98 -0.68 JY2e M3 6.15 -14.93 -0.18 JY3a M2 0.75 -14.19 -1.18 JY3b M2 2.25 -14.26 -1.45 JY3c 1000-900 BP M2 3.7 -13.73 -1.84 JY3d M2 5.11 -13.75 -2.59 JY3e M2 6.16 -13.47 -3.41 JY3f M2 7.49 -13.30 -3.25 JY4a M3 0.89 -13.67 -3.07 JY4b M3 2.11 -13.79 -3.04 JY4c M3 3.69 -13.65 -2.23 JY4d M3 5.45 -12.57 0.03 JY4e M3 6.51 -12.77 0.04 JY6a M3 1.56 -13.57 -3.56 JY6b M3 3.68 -12.17 -5.68 JY6c M3 4.56 -13.47 -3.13 JY6d M3 5.99 -13.43 -3.40 JY7a M3 0.71 -13.12 -3.36 JY7b M3 1.95 -13.21 -2.42 JY7c M3 3.76 n/a n/a JY7d M3 5.01 -13.28 -0.51 JY7e M3 6.23 -13.20 0.04 JY7f M3 7.6 -12.84 -0.16 JY8a M3 0.47 -14.44 -2.50 JY8b M3 1.64 -14.97 -2.69 JY8c 1100-1000 M3 2.97 -15.00 -1.79 JY8d BP M3 3.9 -15.31 -1.82

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Table A- 2. Details of individual tooth sub-sample isotopic measurements for archaeological deer Distance from Sample Date Tooth crown (mm) δ13C (VPDB) δ18O (VPDB) JY8e M3 4.92 -15.19 -1.23 JY9a M2 0.94 -15.03 -0.83 JY9b M2 2.18 -14.85 -1.47 JY9c M2 3.05 -15.09 -1.66 JY9d M2 4.07 -14.97 -1.66 JY9e M2 5.18 -14.71 -1.32 JY10a M2 0.57 -16.38 1.49 JY10b M2 1.58 -16.00 0.24 JY10c M2 2.61 -15.66 0.07 JY10d M2 3.8 -15.67 -1.51 JY10e M2 5.33 -15.09 -1.59

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Table A- 3. Details of individual tooth sub-sample isotopic measurements for modern, wild deer Distance from crown Sample Tooth δ13C (VPDB) δ18O (VPDB) (mm) MD1a M3 2.46 -16.59 -2.68 MD1b M3 3.61 -16.70 -3.59 MD1c M3 4.52 -16.59 -2.98 MD1d M3 6.09 -16.49 -2.89 MD1e M3 7.19 -16.47 -2.55 MD1f M3 8.37 -16.30 -2.33 MD2a M2 1.94 -16.10 -1.56 MD2b M2 3.63 -15.92 -2.57 MD2c M2 5.07 -16.36 -1.99 MD2d M2 7.04 -16.71 -3.50 MD3a M3 2.28 -15.80 -1.72 MD3b M3 3.86 -15.64 -1.27 MD3c M3 5.08 -15.85 -1.30 MD3d M3 6.77 -15.82 -1.08 MD3e M3 8.05 -15.38 -0.47 MD4a M3 2.1 -16.46 -1.65 MD4b M3 3.97 -16.19 -1.09 MD4c M3 4.93 -16.07 -1.66 MD4d M3 6.85 -15.98 -2.03 MD4e M3 8.14 -16.05 -2.38 MD8a M3 1.93 -15.49 -3.07 MD8b M3 3.41 -15.96 -4.30 MD8C M3 4.73 -15.84 -2.66 MD8d M3 6.34 -15.92 -2.30 MD8e M3 7.26 -15.82 -2.89 MD9a M2 2.08 -14.88 -3.71 Md9b M2 3.13 -14.62 -3.74 MD9d M2 6.6 -14.18 -1.30 MD9e M2 7.83 -15.38 -2.89 MD10a M3 2.92 -17.10 -4.76 MD10b M3 4.11 -16.99 -4.36 MD10c M3 5.71 -17.11 -4.77 MD10d M3 6.99 -17.04 -3.34 MD10e M3 8.78 -16.66 -2.30 MD11a M2 2.21 -16.76 -3.26 Md11b M2 3.66 -16.74 -3.78 MD11c M2 4.87 -16.48 -2.72 MD11d M2 6.27 -16.53 -2.81 108

Table A- 3. Details of individual tooth sub-sample isotopic measurements for modern, wild deer Distance from crown Sample Tooth δ13C (VPDB) δ18O (VPDB) (mm) MD11e M2 7.77 -16.50 -4.20 MD11f M2 8.99 -16.86 -4.65 MD12a M3 1.58 -5.43 -5.28 MD12b M3 2.67 -5.54 -5.34 MD12c M3 4.24 -5.84 -4.69 MD12d M3 5.51 -5.79 -5.15 MD12e M3 6.83 -5.76 -4.11 MD13a M2 1.53 -5.04 -5.39 MD13b M2 2.69 -5.10 -6.21 MD13c M2 3.41 -4.67 -5.48 MD13d M2 5.26 -4.03 -5.80 Md13e M2 6.5 -4.24 -5.12 MD14a M3 1.73 -14.84 -1.94 MD14b M3 3.17 -14.43 -2.48 MD14c M3 4.78 -14.70 -1.90 MD14d M3 5.94 -14.62 -2.16 MD15a M2 1.52 -14.66 -3.68 MD15b M2 3.21 -14.92 -3.58 MD15c M2 4.12 -14.75 -3.31 MD15d M2 5.99 -14.72 -4.09 MD15e M2 7.66 -14.64 -2.38

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Table A- 4. Details of individual tooth sub-sample isotopic measurements for modern, non-wild deer Distance from enamel Sample Tooth δ13C (VPDB) δ18O (VPDB) -root junction (mm) MD2a M3 10.76 -6.75 -5.64 MD2b M3 7.98 -6.62 -5.73 MD2c M3 6.38 -7.13 -5.57 MD2d M3 4.83 -6.94 -5.41 MD2e M3 3.82 -7.02 -4.91 MD22a M2 9.04 -7.86 -4.66 MD22b M2 7.48 -7.43 -5.40 MD22c M2 5.89 -7.15 -5.69 MD22d M2 4.04 -7.85 -5.71 MD22e M2 2.43 -7.49 -5.43 MD3a M3 3.17 -6.13 -5.25 MD3b M3 4.47 -5.78 -4.18 MD3c M3 5.86 -5.97 -4.46 MD3d M3 7.72 -6.04 -4.01 MD3e M3 8.5 -5.67 -3.45 MD33a M2 2.96 -7.38 -5.69 MD33b M2 4.28 -6.78 -5.92 MD33c M2 5.8 -6.95 -4.82 MD33d M2 7.16 -6.72 -5.19 MD33e M2 7.94 -6.82 -5.11 MD4a M3 8.44 -6.26 -5.72 MD4b M3 7.23 -6.13 -5.95 MD4c M3 5.4 -5.79 -4.98 MD4d M3 3.94 -5.95 -5.76 MD44a M2 8.44 -7.48 -5.42 MD44b M2 6.94 -7.20 -5.45 MD44c M2 5.08 -6.89 -5.59 MD44d M2 4.05 -6.43 -6.13 MD5a M3 7.82 -6.42 -5.93 MD5b M3 6.06 -6.33 -5.60 MD5c M3 4.3 -6.59 -5.56 MD5d M3 2.62 -6.42 -5.18 MD55a M2 7.65 -6.44 -6.02 MD55b M2 6 -6.71 -5.50 MD55c M2 5.2 -7.54 -5.64 MD55d M2 3.02 -7.46 -5.45 MD6a M3 8.37 -5.82 -5.29 MD6b M3 5.89 -5.81 -5.06 110

MD6c M3 4.83 -5.89 -4.71 MD6d M3 3.26 -6.44 -4.10 MD66a M2 7.64 -5.59 -4.90 MD66b M2 6.35 -5.51 -5.86

MD66c M2 4.78 -4.93 -5.98 MD66d M2 3.43 -5.24 -5.39 MD7a M3 7.31 -6.64 -5.08 MD7b M3 6.1 -6.60 -5.33 MD7c M3 4.63 -6.73 -5.22 MD77a M2 Md77b M2 5.56 -5.95 -6.15 MD77c M2 4.48 -5.84 -6.50 MD77d M2 2.34 -6.83 -5.28

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Figure A- 1. Photographs of the isotopically analyzed teeth: (a) JY7; (b) JY1 and JY2

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