Past Hydroclimate and Vegetation Variation in Romania Inferred from Isotopic Geochemistry and Pollen of Cave Bat Guano

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Past hydroclimate and vegetation variation in Romania inferred from isotopic geochemistry and pollen of cave bat guano

Daniel Martin Cleary University of South Florida, [email protected]

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Scholar Commons Citation Cleary, Daniel Martin, "Past hydroclimate and vegetation variation in Romania inferred from isotopic geochemistry and pollen of cave bat guano" (2019). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/8349

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Past hydroclimate and vegetation variation in Romania inferred from isotopic geochemistry and

pollen of cave bat guano.

Daniel Martin Cleary

A dissertation submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy with a concentration in Paleoclimatology School of Geosciences College of Arts and Sciences University of South Florida

Major Professor: Bogdan P. Onac, Ph.D. Jonathan G. Wynn, Ph.D. Ioan Tanțău, Ph.D. Phillip van Beynen, Ph.D. H. Len Vacher, Ph.D.

Date of Approval: May 28, 2019

Keywords: nitrogen and carbon stable isotopes, radiocarbon, guano, climate, Romania,

DEDICATION

This dissertation is dedicated to my family that has supported and encouraged me throughout my graduate studies. I also dedicate this work to my uncle Tim Cleary who has always been there for me. Lastly to Bogdan P. Onac, whose guidance I will forever be thankful for.

ACKNOWLEDGMENTS

This research was supported through Romanian CNCS grant PN-II-ID-PCE 2011-3-0588 to

Bogdan P. Onac. I thank Dr. Tudor Tămaș and Alexandra Giurgiu who helped during coring and initial sampling activities associated with the Măgurici core. I acknowledge the administration of Porțile de Fier Natural Park, Mehedinți Plateau Geopark, and the Romanian Speleological

Heritage Commission for granting permission to recover guano profiles from Gura Ponicovei and Topolnița caves, respectively. The assistance of Roxana Grindean with her comments and suggestions during the development of my pollen records is greatly appreciated. Special thanks to Dr. Ioan Coroiu for identifying the bats in both Gura Ponicovei and Topolnița caves and Drs.

Oana A. Dumitru and Ioan Povară for field assistance. I am also deeply grateful to Bogdan P.

Onac and Jonathan G. Wynn for their thoughtful reviews that helped improve the Pollen, δ15N and δ13C guano-derived record of late Holocene vegetation and climate in the southern

Carpathians, Romania manuscript.

TABLE OF CONTENTS

LIST OF TABLES ...... iii

LIST OF FIGURES ...... iv

ABSTRACT ...... v

INTRODUCTION ...... vii

CHAPTER 1: Evidence of long-term NAO influence on East-Central Europe winter precipitation from a guano-derived δ15N record ...... 1

CHAPTER 2: A guano-derived δ13C and δ15N record of climate since the Medieval Warm Period in north-west Romania ...... 2

CHAPTER 3: Pollen, δ15N and δ13C guano-derived record of late Holocene vegetation and climate in the southern Carpathians, Romania ...... 3 AUTHORS ...... 3 ABSTRACT ...... 3 INTRODUCTION ...... 4 STUDY AREA...... 6 METHODS ...... 7 Radiocarbon ...... 7 Isotopic analysis ...... 7 Pollen Preparation ...... 8 RESULTS ...... 9 Radiocarbon ...... 9 Carbon and nitrogen isotopes: Gura Ponicovei ...... 9 Carbon and nitrogen isotopes: Topolnița ...... 10 Palynological and microcharcoal analysis Gura Ponicovei and Topolnița ...... 10 Elemental Data ...... 13 DISCUSSION ...... 13 Climatic information inferred from δ13C and δ15N ...... 14 δ15N winter precipitation reconstruction ...... 16 δ13C summer precipitation reconstruction ...... 21 Vegetation dynamics and the link with climate conditions and human impact ... 24 CONCLUSIONS ...... 28 ACKNOWLEDGEMENTS ...... 29 LITERATURE CITED ...... 29 FIGURES ...... 42

i TABLES ...... 48

APPENDIX A: Evidence of long-term NAO influence on East-Central Europe winter precipitation from a guano-derived δ15N record (Scientific Reports) ...... 49

APPENDIX B: Copyright permission from Nature for use of this article in the dissertation ...... 58

APPENDIX C: A guano-derived δ13C and δ15N record of climate since the Medieval Warm Period in north-west Romania (Journal of Quaternary Science) ...... 59

APPENDIX D: Copyright permission from John Wiley and Sons for use of this article in dissertation ...... 72

LIST OF TABLES

Table 1: Pollen, δ15N and δ13C guano-derived record of late Holocene vegetation and climate in the southern Carpathians, Romania ...... 49

iii

LIST OF FIGURES

Figure 1: Map of Romania with circles indicating locations referenced in text: Gura Ponicovei Cave (1; this study), Domogled National Park (2; Levanič et al., 2012), Topolnița Cave (3; this study), Drobeta-Turnu Severin (4; meteorological station), Zidită Cave (5; Forray et al., 2015), Măgurici Cave (6; Cleary et al., 2017, 2018), Tăul Muced Lake (7; Feurdean et al., 2015a), Bucharest (8) ...... 43

Figure 2: Age-depth models for Gura Ponicovei and Topolnița guano cores ...... 43

Figure 3: δ15N (red), δ13C (black), and Suess corrected δ13C (maroon) values of Gura Ponicovei guano; B) δ15N (blue), δ13C (black), and Suess corrected δ13C (purple) values of Topolnița guano ...... 44

Figure 4: Pollen diagram from Gura Ponicovei Cave guano ...... 44

Figure 5: Pollen diagram from Topolnița Cave guano...... 45

Figure 6: A) %C (red) and %N (black) values from Gura Ponicovei core; B) %C (red) and %N (black) values from Topolnița core ...... 45

Figure 7: A) Correlation analysis of δ15N and δ13C of Gura Ponicovei; B) Correlation analysis of Topolnița δ15N and Drobeta-Turnu Severin autumn/winter precipitation; C) Drobeta-Turnu Severin autumn/winter precipitation (black), Topolnița δ15N values (dotted blue) and with 3-year lag (solid blue) ...... 46

Figure 8: A) Comparison of δ15N values from Gura Ponicovei guano, B) δ15N values from Măgurici guano, C) δ15N values from Topolnița guano; D) 3-year running mean of reconstructed winter precipitation in Europe, E) autumn/winter precipitation Cloșani Cave, F) NAO winter index. Vertical bars indicate similar dry/wet phases ...... 47

Figure 9: A) δ13C values from Gura Ponicovei guano, B) δ13C from Topolnița guano, C) tree ring record of SPI from Domogled National Park D) Tree ring spring/ summer precipitation from southern Czech Republic, E) 3-year running mean of European summer precipitation, and F) 3-year running mean of reconstructed European temperature anomaly. Vertical bars indicate similar dry/wet phases ...... 48

ABSTRACT

While an abundance of paleo-records related to hydroclimate and vegetation exist in

East-Central Europe, currently there is a scarcity of reconstructions that have the resolution to effectively capture the past 2000 years. A more complete understanding of this interval is important as it includes significant climatic events such as the Medieval Warm Period, Little Ice

Age, and the post-industrial revolution human induced climate change. A solution to increasing our understanding of these events is the use of cave bat guano, a relatively underutilized source of climatic information.

Cave bat guano piles commonly have near annual deposition and in Europe can often be found to accumulate for over 1000 years. Additionally, as bats are primarily ingesting herbivorous insects, geochemical and palynological records sourced from guano can be linked to the local environment that lies within proximity to the cave. Variation of nitrogen and carbon stable isotopes can potentially occur in response to fluctuations in certain climatic parameters, such as temperature and water availability. These signals are then transferred from plant foliage to insect to bat and ultimately preserved in guano. Multiple factors also allow for the deposition of pollen grains in guano piles. Insectivorous bats can ingest pollen grains attached to insects, wind dispersed pollen can latch on to foraging bat’s fur later dislodging onto the guano pile, or cave ventilation. Therefore, through isotopic and palynological analysis of bat guano high- resolution late-Holocene records of climate and vegetation can be produced.

Guano cores were obtained from three caves along a north to southwest transect in

Romania for analysis of carbon and nitrogen isotopes and pollen. In the Măgurici record

v (northwestern Romania) a 2‰ increase in δ13C, 1.5‰ decrease in δ15N, and the presence of thermophilous species between AD 881 and 1240 indicate a warm and dry Medieval Warm

Period occurred in the northwestern region of the country. This event is followed by the Little

Ice Age (~AD 1240 – 1850), wherein a decrease from 13.9 to 8.5‰ in δ15N values and a 2.3‰ increase in δ13C values of Topolnița guano (southern Romania) suggest a wetter summer and winter climate.

It was also demonstrated that the North Atlantic Oscillation (NOA) is the primary control of winter precipitation in at each study location. In AD 1970 a 4‰ decrease in δ15N values was found to correspond with a notable transition to a positive phase of the NOA (drier conditions).

Additional positive phases of the NAO were found to be recorded in the Măgurici δ15N series between AD 1820 and AD 1855. In contrast, a 2‰ rise in δ15N values from AD 1940 until AD

1970 occurs contemporaneously with an increased frequency of negative NAO phases (wetter conditions).

Pollen extracted from aliquots of guano were investigated to reconstruct past environments within proximity of the caves. In doing so it was possible to discern whether fluctuations in primary forest taxa occurred in response to climatic and/or human impact. It was found that the development of patchy forests consisting of Tilia, Quercus, and Carpinus betulus following the Little Ice Age around Topolnița and Gura Ponicovei caves is likely associated with the lower water availability at this time. Additionally, the significant contribution of ruderal pollen indicators since AD 1985 is suggesting an increase of agricultural practices around

Topolnița Cave.

INTRODUCTION

For this dissertation, three bat guano cores were recovered from caves in northwestern and southwestern Romania with the intention of reconstructing and better understanding the climatic and vegetal history since the Medieval Warm Period. Study sites were chosen in an effort to determine if there is a clear distinction between the western/northern of Romania and the southwest in terms of climatic influences. Fluctuations in temperature and precipitation in southwest Romania are often controlled by the Mediterranean thermal regime (mild winters), whereas the northern and western regions experience a more temperate-continental climate heavily impacted by the North Atlantic circulation systems.

One of the primary controls of winter precipitation in Europe is the North Atlantic

Oscillation, a system for which very few paleo-records exist. Paper 1 represents a nitrogen isotope analysis completed on a guano core obtained from Măgurici Cave located in northwestern Romania. In this study it is demonstrated that variation in δ15N values from guano can be related to the relative openness of the local nitrogen pool. It was found that the associated nitrogen cycle was primarily controlled by winter precipitation amount. This is explained by the impact of winter hydroclimate on leaching processes that control the amount of plant-available nitrogen produced via mineralization prior to the growing season. Positive phases of the North

Atlantic Oscillation (drier conditions) corresponds with lower of δ15N values, whereas negative phases (wetter conditions) occur contemporaneously with increasing of δ15N. This is most recognizable during the 1970s (Fig. 1) where a shift from negative to positive phase occurred.

vii

Figure 1: (a) δ15N of MC guano from 1850 to 2012(see Paper 1 for details and figure references). (b) The difference in the Sea Level Pressure between 1940 and 1970 (pre-shift) and

the period 1980–2010 (post-shift) (c) As in (b) but for winter precipitation.

As winter precipitation in East-Central Europe is influenced by the North Atlantic Oscillation, past phases of this climatic system were then reconstructed since AD 1650 using the δ15N values of guano. These findings were important in further establishing the utility of δ15N values as a paleoclimatic proxy.

Building off the work presented in Paper 1, Paper 2 details the remainder of the 285 cm core from Măgurici Cave that was analyzed for δ13C, δ15N, and pollen. As guano deposition occurred from AD 881 until 1240 and from AD 1651 to 2013, interpretation of isotopic and palynological data were used to characterize the climate of the Medieval Warm Period and Little

Ice Age (Fig. 2). This effort is important as the timing, duration, and amplitude of these events have been found to vary substantially across the European continent. Additionally, climatic and

viii environmental records of the Medieval Warm Period and Little Ice Age are particularly scarce in

East-Central Europe, and especially within Romania.

Figure 2: The δ13C (blue) and δ15N (inverted axis; red) values from AD 881 to 2012 for Măgurici

15 Cave guano. δ N data are from Paper 1.

Unlike the δ15N-record of winter precipitation developed in Paper 1, this study showed that the variation in δ13C values are related to the effect of summer water availability on plant water use efficiency. This allowed for the production of a near annual reconstruction of summer hydroclimate. Key findings included the determination of a drier Medieval Warm period and progressively wetter conditions following the termination of the Little Ice Age.

Lastly, δ13C, δ15N, and pollen from two cores recovered from Topolnița and Gura

Ponicovei caves located in SW Romania were utilized in Paper 3 to interpret past climatic and environmental changes. With the location of the caves in the southwestern region of Romania, there was potential for a Mediterranean climate influence. Guano deposition began ~AD 1694 at

Topolnița and AD 1537 at Gura Ponicovei. Similar to Paper 2, δ13C values are interpreted to correspond with summer precipitation, whereas δ15N values are inferred, like in Paper 1, to record past phases of the North Atlantic Oscillation through its control on winter precipitation.

ix Differing from the previous two studies, this work placed more emphasis on the palynological records. The inclusion of pollen allowed to understand whether the forest dynamics were influenced by climate. Long term trends in the reconstructed seasonal hydroclimate were found to have some influence on variations in primary forest taxa identified in the pollen records.

However, particularly with the Topolnița record, human impact appears to had a significant effect on vegetation surrounding the cave.

CHAPTER 1: EVIDENCE OF LONG-TERM NAO INFLUENCE ON EAST-CENTRAL

EUROPE WINTER PRECIPITATION FROM A GUANO-DERIVED δ15N RECORD

Note to Reader:

This chapter has been previously published: Cleary, D.M., Wynn, J.G., Ionita, M., Forray, F.L.,

Onac, B.P. - Evidence of long-term NAO influence on East-Central Europe winter precipitation from a guano-derived δ15N record. Scientific Reports, 7: 14095. See Appendix A for the PDF of the published document and Appendix B for permission from the publisher.

CHAPTER 2: A GUANO-DERIVED δ13C AND δ15N RECORD OF CLIMATE SINCE

THE MEDIEVAL WARM PERIOD IN NORTH-WEST ROMANIA

Note to Reader:

This chapter has been previously published: Cleary, D.M., Onac, B.P., Tanțău, I., Forray, F.L.,

Wynn, J.G., Ionita, M., Tămaș, T. - A guano-derived δ13C and δ15N record of climate since the

Medieval Warm Period in north-west Romania. Journal of Quaternary Science, 33(6): 677-688.

See Appendix C for the PDF of the published document and Appendix D for permission from the publisher.

CHAPTER 3: POLLEN, δ15N AND δ13C GUANO-DERIVED RECORD OF LATE

HOLOCENE VEGETATION AND CLIMATE IN THE SOUTHERN CARPATHIANS,

ROMANIA

Authors

Daniel M. Cleary, Angelica Feurdean, Ioan Tanțău, Ferenc L. Forray

Abstract

Two cores of bat guano were recovered from Topolnița and Gura Ponicovei Caves in the southwest region of Romania and analyzed for δ15N, δ13C and pollen. Deposition of guano began in

AD 1537 at Gura Ponicovei and AD 1694 at Topolnița, allowing for the development and interpretation of climate and vegetation records since the Little Ice Age. δ15N values were found to record phases of the North Atlantic Oscillation through the effect of winter precipitation on the local nitrogen cycle. Alternatively, δ13C values are interpreted to correspond with variation in summer precipitation. Both records suggest progressively drier conditions during the termination of the LIA in the region at AD 1870. The transition to landscape openness, with patchy forests of

Tilia, Carpinus betulus, and Quercus during this time could be related to the lower water availability at both sites. Wetter conditions inferred from a 3‰ increase in δ15N values between

AD 1879 and AD 1970 occurred contemporaneously with a similar trend in European winter precipitation and increased frequency of positive NAO phases during this interval. The δ13C values between AD 1885 and AD 1968 suggest a trend towards a wetter summer climate as well.

3 Although there is at times evidence of forest dynamics being controlled by climate, vegetation appears to be more closely associated with human impact, particularly around Topolnița Cave.

With contributions of ruderal pollen indicators from AD 1985 – present is likely related to agricultural practices that were absent around Gura Ponicovei.

Introduction

Recently, cave bat guano records have become more frequently utilized in reconstructing climate. Carbon and nitrogen isotopes records in guano have been served as a proxy for C3 vs C4 plant types (Mizutani et al., 1992a, b; Bird et al., 2007; Wurster et al., 2007; Choa et al., 2016), seasonal precipitation (Royer et al., 2015; Forray et al., 2015; Onac et al., 2015; Cleary et al.,

2016, 2018), migration of the Inter-Tropical Convergence Zone (Royer et al., 2017), and phases of the North Atlantic Oscillation (Cleary et al., 2017). These records most commonly offer a view into the variability of climate over the past 2000 years. Bat guano is particularly useful when considering this interval of time as other resources of climate information (stalagmites, peat cores, etc.) lack the near annual resolution often expressed in large guano deposits.

However, while guano-derived δ15N and δ13C provide an extensive amount of climatic information, pollen records (Carrión et al., 2006; Batina and Reese, 2011; Geantă et al., 2012;

Forray et al., 2015) have the added benefit of describing the associated environment. Pollen enters guano piles in three possible ways; 1) through initial ingestion by bats and later defecation; 2) via deposition through cave air circulation if the cave passages permit (Leroy and

Simms 2006); and 3) attached to the bat’s fur, from where grains can ultimately fall off onto the guano (Maher, 2006). Therefore, pollen grains preserved in guano deposits connect bats to the vegetation growing in the cave proximity (Navarro et al., 2001). Although these mechanisms may result in lower pollen concentrations in guano deposits by comparison to lake and peat

4 settings, guano offers the opportunity to record plant species that rely on insects more than wind for pollination. Particularly in Romania, palynological studies have demonstrated that bat guano can successfully provide insight into past environments (Geantă et al., 2012; Forray et al., 2015;

Cleary et al., 2018). However, only a few studies overall have attempted to use both guano core pollen and δ13C/δ15N records in guano to place the isotope derived climatic information within the context of the reconstructed vegetation composition (Forray et al., 2015; Cleary et al., 2018).

A vast majority of paleoclimate and paleoenvironmental records of Romania have been developed in the western Carpathian Mountains (Feurdean and Willis, 2008; Feurdean et al.,

2009; Cleary et al., 2016), northern Carpathians (Popa and Kern 2009; Tanțău et al., 2011,

2014a; Feurdean et al., 2016; Cleary et al., 2018) and the eastern Carpathians (Tanțău et al.,

2003, 2009, 2014b). A particularly important gap to fill in terms of paleoenvironmental records lies within the southwestern region of Romania. This area has a unique setting due to the potential influence of Mediterranean climate and the associated occurrence of many submediterranean plants (Cristea, 1993; Doniță et al., 2005). The pollen records that do exist for this area of the country often come from high elevation peat and lakes that lack the resolution to properly consider the past 2000 years (Fărcaș et al., 1999; Rösch and Fischer, 2000; Magyari et al., 2018). Additionally, although climate records exist (Constantin et al., 2007; Drăgușin et al.,

2014), very few provide consideration of climate variability in the region since the Medieval

Warm Period (Onac et al., 2014; Warken et al., 2018).

In this study, two guano cores obtained from Gura Ponicovei and Topolnița caves (Fig. 1) were analyzed for δ15N and δ13C and pollen with the aim to develop a paleoclimatic and paleoenvironmental reconstruction since the Medieval Warm Period. Through this approach we determine past vegetational dynamics and independently asses the role of climate on these

5 dynamics. Comparison with instrumental records nearby Gura Ponicovei and Topolnița caves are used to explore any existing relationship between the isotopic values and modern climate and therefore constrain the interpretation of paleoclimate signal extracted from the two cores.

Study Area

Gura Ponicovei Cave (Fig. 1.1) is located in the southwestern region of the Southern

Carpathians, within the Porțile de Fier (Iron Gate) National Park. This 1,666 m long cave system

(Goran, 1982) lies within the Danube Gorge just north of the Danube River at an elevation of 60 m above sea level (a.s.l.). Located within the upper part of the cave, approximately 267 m from the upper entrance is a small (55 cm) accumulation of guano that was deposited by bats on the floor of the Big Room.

An estimated 5 bat colonies of 1,000 individuals of Rhinolophus euryale are present within the

Big Room. In addition to Rh. euryale, Murariu et al. (2004) described populations of Miniopterus schreibersii, Myotis myotis, M. capaccinii within the cave. All species are insectivorous with the exception of M. capaccinii that eats small fish (Levin et al., 2006) from the superficial layer of lakes. The foraging range of these bats is a function of the number of bats in the cave, however they can forage up to 32 km from the cave entrance (Egert-Berg et al., 2018). Gura Ponicovei is surrounded by dense forests containing Carpinus betulus, C. orientalis, Tilia, and Quercus.

Present herbaceous taxa include Poaceae, Chenopodiaceae, Scrophulariaceae, Plantago lanceolata, Filipendula, and Asteroideae. Aquatic species, such as Typha are also common at lower elevations near the Danube.

Topolnița Cave (Fig. 1.3) is situated within the same southern section of the Carpathians, but on the Mehedinți Plateau. Relative to Gura Ponicovei, this cave system is positioned at a slightly higher elevation (~400 m a.s.l.), and has a total length of 22 km (Goran and Povară, 2019). The

6 guano heap is located in the Bat’s Gallery, approximately 100-150 m from the nearest entrance.

The colony above the guano deposit is populated primarily by Rh. euryale, Myotis daubentonii, and M. capaccinii, all primarily insectivorous species (Coroiu, 2019).

Due to the fact that both caves are located in the south-western region of the Southern

Carpathians, the surroundings can potentially be influenced by Mediterranean climate. The seasonal climate includes temperatures averaging 20-22 °C during the hotter months and a mean of 1-3 °C during the winter (Murariu et al., 2004). Records from Târgu Jiu (203 m a.s.l.) and

Drobeta-Turnu Severin (77 m a.s.l.) meteorological stations indicate that precipitation is around

~120-140 mm during the summer (Warken et al., 2018). In contrast, during the winter months this value is much lower (~ <40 mm).

Methods

Radiocarbon

An accelerator mass spectrometer at the Poznan Radiocarbon Laboratory (Poland) was used to measure the 14C age of bulk guano (Topolnița 7 samples and Gura Ponicovei 5 samples). The calibrated years are expressed in years AD and the associated errors are 1 year for modern samples and 25-30 years for pre-modern samples.

Isotopic analysis

The guano deposits were sampled by a manually operated Russian corer (e.g. Forray et al.,

2015). The cores were subsequently sampled at a 2-cm resolution for Gura Ponicovei and a 1-cm resolution for Topolnița. The reason for a lower sampling resolution in Gura Ponicovei was the lack of visible changes in the sedimentology of the core. In contrast, the guano in the Topolnița core was rather well laminated, thus sampled at higher resolution. Guano samples were first homogenized and treated with HCl to remove inorganic carbon (e.g. Onac et al., 2014).

7 Subsequently, 1–2 mg were weighed from each aliquot and then measured for δ13C, δ15N, %C and %N using a Costech ECS4010 Elemental Analyzer coupled with a Delta V Advantage

Isotope Ratio Mass Spectrometer (Thermo Fischer Scientific) at the University of South Florida

Stable Isotope Laboratory. A protein standard B2155 (δ13C: -27.09 ‰, δ15N: 5.94 ‰, %C: 46.5,

%N: 13.3) and a glutamic acid (internal standard: δ13C: -16.49 ‰, δ15N: -6.26 ‰, %C: 41.37,

%N: 9.54) were used during analysis. B2155 and IAEA-C7 were used to calibrate the δ13C value of the glutamic acid. Estimation of the precision of analysis (δ15N: 0.08 ‰; δ13C: 0.04 ‰) is based on replicate internal standards used during each analysis. Suess correction was applied for all δ13C data after AD 1850 (Forray et al., 2015) to correct carbon isotope “contamination” due to fossil fuel burning.

Pollen preparation

All 23 samples of the Gura Ponicovei core were analyzed for pollen at 2-cm intervals. Analysis of the Topolnița core was completed at 4-cm intervals between 0 and 56 cm and at 2-cm intervals between 56 and 94 cm, as the accumulation rate differs at the top and bottom of the core. Due to the samples being entirely organic material, a simplified procedure was used to isolate pollen grains. One cm3 of each sample was first treated with HCl to remove carbonates and dissolve the Lycopodium, and then NaOH (10%) to remove humic acids and organic matter.

Samples were sieved through a 250 µm mesh to remove coarse organic material, and preserved in glycerin. Pollen was counted under microscope (40 X) until a sum of ca. 200 grains was reached. The frequencies of pollen for each taxon were calculated as percentages of the total sum of arboreal pollen (AP) and non-arboreal pollen (NAP). The nomenclature for vascular plants follows Flora Europaea (Tutin et al., 1964–1980). Microscopic charcoal particles were counted

8 along pollen to reconstruct the fire history. The results are presented as a percentage pollen diagram reconstructed in the Tilia software version 2.0.41 (Grimm, 1991).

Results

Radiocarbon

All radiocarbon ages used for radiocarbon dating are in stratigraphic order for both Topolnița and

Gura Ponicovei (Table 1) indicating no contamination of bulk guano samples.

Modern samples found to have high radiocarbon activity were calibrated using CaliBomb

(Reimer and Reimer, 2009) with calibration dataset IntCal13 (Reimer et al., 2013) and North

Hemisphere Zone 1 (NHZI) bomb curve extension (Hua, 2013). The remainder of the 14C ages were calibrated with the Clam code using IntCal13 (Reimer et al., 2013). Ages were interpolated between dated samples every 1 cm. The resulting age-depth models were produced using a linear interpolation between all 14C ages using Clam 2.2 code (Blaauw, 2010) running in R software (R

Development Core Team, 2013).

The age depth model indicates that Gura Ponicovei guano sequence began to accumulate at AD

1537, whereas at Topolnița first deposition occurred beginning with AD 1694. At both sites the guano accumulations were continuous up to present day with no hiatus.

Carbon and nitrogen isotopes: Gura Ponicovei

Low %N and %C prevented isotopic analysis of the lower 5 samples (46-55 cm; AD 1228-1490) of the Gura Ponicovei core. Therefore, the δ13C and δ15N record begins at AD 1537. The δ13C values remain fairly stable between AD 1850 and 1925, varying between -25.7 and -26.3‰ (Fig.

3A). In contrast, δ15N values express a decreasing trend beginning at AD 1537 (7.4‰) reaching a low point at AD 1810 (6.5‰). Both δ13C and δ15N briefly increase at AD 1810 before a substantial decrease beginning at AD 1989. A greater concentration of data occurs between AD

9 1989 and AD 1997 with large apparent variation within this 10-year interval. Starting at AD

1989 (δ15N: 6.9 ‰, δ13C: -26.3 ‰) both δ15N and δ13C time-series trend towards lower values

(δ15N: 4.9 ‰, δ13C: -27.3 ‰).

Carbon and nitrogen isotopes: Topolnița

Between AD 1850 and 1786, δ13C values decrease by 2.8‰, a trend that terminates at the lowest value in the series (-28.2‰; Fig. 3B). The interval, AD 1786-1794 is characterized by a rapid

2.3‰ shift to higher values. Instead, δ15N values become progressively lower until AD 1876, decreasing from 13.9 to 8.5‰. At AD 1876 there is a transition towards higher values in both the carbon and nitrogen isotopic composition of guano (Fig. 3B). The δ15N values continue to increase for the remainder of the series reaching 15.5‰ at AD 2014. There is a brief departure from this trend at AD 2008 wherein δ15N decreases to 5.9‰. The δ13C of guano increases to -

23.5‰ at AD 1985, after which decreases until AD 2008 by 1.4‰.

Palynological and microcharcoal analysis Gura Ponicovei and Topolnița

Pollen counts were too low (<150) in the lower 2 samples of the Gura Ponicovei record to be considered and are therefore removed from analysis. Based on cluster analysis of the pollen records, Local Pollen Assemblage Zones (LPAZ) for Gura Ponicovei (5) and Topolnița (5) were produced. Transitions between zones are indicative of significant changes in the pollen assemblages and therefore vegetation dynamics.

Gura Ponicovei

AD 1395 – 1490 (LPAZ 1)

The pollen record at the oldest interval of the Gura Ponicovei sequence indicates an environment characterized by open forest dominated by Quercus, Tilia, Carpinus betulus, and Fagus sylvatica

(Fig. 4). Herbaceous species occured in low percentages relative to arboreal pollen for most of

10 this interval. Notable constituents of the herbaceous assemblage include Poaceae, Geraniaceae,

Verbascum, Caryophyllaceae, and Scrophulariaceae. Additionally, there were small frequencies of the aquatic species Potamogeton.

AD 1490 – 1810 (LPAZ 2)

This zone is characterized by a gradual decline in Fagus sylvatica to values below 10%.

Quercus, Tilia, Carpinus betulus continued to dominate forests in the area, however, Corylus avellana and Fraxinus excelsior also had a notable contribution. There was increased diversity and abundance of herbaceous taxa during this interval. Apiaceae, Chenopodiaceae, Poaceae,

Scrophulariaceae, Asteraceae and Artemisia displayed the highest percentages.

AD 1810 – AD 1990 (LPAZ 3)

The pollen spectra of this zone demonstrated a sharp reduction in the tree pollen percentages.

Carpinus betulus and Tilia remain the primary forest constituents while the percentages of

Quercus and Fagus sylvatica declined. The abundance of herbaceous taxa increased markedly particularly for Chenopodiaceae, Scrophulariacea, Verbascum, Artemisia, Poaceae, and

Asteraceae. Typha percentages increased in the late 1970’s.

AD 1990 – 1996 (LPAZ 4)

During this interval the frequency of arboreal pollen increased and consisted of Carpinus betulus, C. orientalis, Quercus, Tilia, Salix, Fraxinus excelsior, and Fraxinus ornus. Within herbaceous taxa, percentages of Poaceae pollen increased, contrasting a declined abundance of

Scrophulariaceae and Verbascum. Significant expansion of Ranuculaceae at AD 1992 along with the presence of Plantago lanceolata, Artemisia, Asteraceae, and Chenopodiaceae could indicate increased land disturbance.

AD 1996 – Present (LPAZ 5)

11 Compared to the previous zone, there was a reduction of Quercus, Corylus avellana, and

Fraxinus excelsior, and increased frequencies for Tilia, Salix, and Ulmus. The herbaceous assemblage was characterized by decreased herb diversity particularly taxa associated with land use, Polygonaceae, Rubiaceae, Plantago lanceolata, Artemisia, and Asteroideae.

Topolnița

AD 1643 – AD 1790 (LPAZ 1)

The beginning of the Topolnița sequence was characterized by forests composed of Quercus,

Fagus sylvatica, Betula, Tilia, and with smaller occurrences of Carpinus betulus (Fig. 5). There was a high diversity and abundance of herbaceous taxa during this interval, primarily, Poaceae,

Asteraceae, Urticaceae, Plantago lanceolata, Artemisia, Cichorioideae, Centaureae, and

Rubiaceae, which suggests land disturbance and development of pastures/meadows in the area.

AD 1790 – AD 1957 (LPAZ 2)

Herbaceous pollen types remained high relative to arboreal pollen throughout most of the 1800’s.

A few instances of forest expansion notable for Fagus sylvatica and Corylus avellana, occurred at AD 1794, AD 1830, and AD 1922. A similar configuration of herbaceous taxa to Gura

Ponicovei LPAZ 3 existed with higher percentages of Asteraceae, Artemisia, Poaceae,

Ranuculaceae, and Cannabis-type. This and the smaller contributions of Euphorbia,

Polygonaceae, Rosaceae, and Fabaceae suggest that plant diversity remained high around

Topolnița Cave.

AD 1957 – AD 1984 (LPAZ 3)

This zone is marked by an expansion of Fagus sylvatica (30%) with additional increased percentages of Salix and Corylus avellana. The pollen percentages of Carpinus betulus, Quercus, and Fraxinus excelsior remained below 10%. There was a corresponding notable reduction in

12 herbaceous taxa in this zone, particularly for Poaceae and Artemisia, whilst others i.e.,

Brasicaceae, Chenopodiaceae, Campanulaceae, and Verbascum increased at this time.

AD 1984 – AD 1991 (LPAZ 4)

This zone is characterized by the decline of Fagus sylvatica at the expense of Carpinus betulus,

Quercus, Tilia, and Betula. Primary constituents of the herbaceous taxa are Poaceae,

Chenopodiaceae, Ranunculaceae, Scrophulariaceae, and Verbascum, however, the diversity of herbaceous pollen is much lower than the previous zones.

AD 1991 – present (LPAZ 5)

The Topolnița region appears to be impacted by human activity since AD 1991 with increased contributions from Artemisia, Urticaceae, Rosaceae, Plantago lanceolata, Ambrosia, and

Cerealia.

Elemental Data

The %C and %N values for the Gura Ponicovei core, notably mirror each other. Fresh surface samples had values of 6.4 (%N) and 25.6 (%C) (Fig. 6A). Both values are lower than fresh samples from other guano studies at Măgurici Cave (%N: 11.3; %C: 36.4) and Zidită Cave (%N:

10.7; %C: 40.5 %), but are relatively stable around each respective surface value for the upper 13 cm of the core. Between 15 and 25 cm there is a distinct decreasing trend in both %N and %C.

At 25 cm %N reaches a minimum value of 3.4 %, whereas %C approaches 14.9%. After this point both series appear to fluctuate around this point.

For the Topolnița core, the lowest values of %C and %N are found in the upper 7 cm (%N: 6.7;

%C: 23.2; Fig. 6B). Both values for the remainder of the core remain relatively stable between 7 and 75 cm with average values of 10.65% (%N) and 35.3 % (%C) over this interval.

Discussion

13 Climatic information inferred from δ13C and δ15N

As all δ13C values are between -23 and -28‰, variability within the time-series can be associated with processes that impact the fractionation associated with the C3 pathway. When a climatic

13 parameter is influencing the δ C values in C3 plants, this signal can potentially be transferred from plant to insect to bat, and ultimately preserved in guano (Forray et al., 2015). While there is a potential ~1 ‰ enrichment that can occur between ingestion of plant and incorporation by the insect into chitin (Potapov et al., 2014), most mammals will have a whole body δ13C value similar to that of their diets (DeNiro and Epstein, 1978). There is a small fractionation that occurs between transfer of bat whole-body and deposition of guano (DeNiro and Epstein, 1978), however the fractionation factor is relatively small with a constant offset to diet. Therefore, if demonstrated that a climate signal is recorded in the δ13C values of Gura Ponicovei and

Topolnița bulk guano, a paleoclimate record based on the carbon isotopic composition can be produced.

13 A majority of δ C records have attributed carbon isotopic discrimination within the C3 pathway to the variation within plant water-use efficiency (WUE) (Farquhar et al., 1982; Bodin et al.,

2013). When there is a scarcity of water or aridity is high, vascular plants with the capability to reduce stomatal conductance are afforded a competitive advantage. Their ability to resist water loss results in less 13C-discrimination and δ13C-plant biomass more enriched in 13C (higher δ13C values). Alternatively, when water availability is not limiting, no competitive advantage occurs.

The higher stomatal conductance and lower WUE under these conditions results in more 13C- discrimination and therein lower δ13C-plant biomass values.

However, δ13C values are not a simple function of mean annual precipitation as factors such as annual water balance, evapotranspiration, seasonality, drainage conditions, etc. can also impact

14 WUE. In addition, carbon isotope discrimination within C3 plants cannot always be associated with WUE (Tang et al., 2014). Variation within this photosynthetic pathway can also be attributed to changes in the concentration of atmospheric CO2 (Silva et al., 2013) as well as light- use and canopy recycling of CO2 (Lambers et al., 2008). Multiple environmental factors, such as temperature, salinity, and/or irradiance can also potentially influence plant-WUE irrespective of precipitation values (Farquhar et al., 1982). With respect to temperature, the effect of warm conditions can potentially outweigh the impact of rainfall received via its influence on evaporative demand during the growing season (Seibt et al., 2008). For example, Xu et al. (2015) found mean annual temperature to have such an influence on δ13C values in alpine areas.

Additionally, Cleary et al. (2018) found evidence of local soil temperature having more impact on water availability than precipitation received locally between AD 1938 and AD 2012.

Therefore, although water availability can often be directly linked with δ13C values, the potential multitude of additional environmental factors can create a less straight forward interpretation.

Factors controlling the δ15N variability in guano are similarly complex. There are a multitude of nitrogen (N) transformations and transfers each with its own associated enrichment ranges that can occur within the soil reservoir (Högberg, 1997). Due to the fact that the significance of these processes cannot be determined based solely on the reservoir δ15N value, paleoclimate studies have largely avoided utilizing nitrogen isotopic records (Royer et al., 2015). However recent work has begun to circumvent these issues by interpreting δ15N values as an integrator of the N- cycle (Cleary et al., 2016, 2017; Wurster et al., 2017). The N pool mixing and any N gains/losses occurring within the soil reservoir can potentially contribute to the δ15N of the system

(soil/biomass). This allows for the interpretation of the δ15N value of a system as it relates to the relative openness of the N-cycle (Robinson et al., 2001). A closed N-cycle occurs when N is a

15 limiting nutrient and therefore more tightly conserved, producing a decrease in δ15N values at each step of the N-cycle (soil, plant foliage, consumers) (Robinson et al., 2001; Szpak, 2014).

Alternatively, when the N-cycle is more open due to abundant or sufficient N, there is an associated increase in δ15N values.

Past monitoring studies have found relationships between the N-cycle and mean annual precipitation (MAP: Austin and Vitousek, 1998; Handley et al., 1999; Amundson et al., 2003).

Szpak (2014) also found the degree of anthropogenic influence to also have a potential control on relative openness. This can occur through the removal of N from the system (deforestation, burning) or the addition of synthetic fertilizer or manure. Paleo-studies have been able to use

δ15N values to reconstruct winter precipitation via the effect of leaching during winter months

(Cleary et al., 2016, 2017) and anthropogenic influence through palynological (Cleary et al.,

2016) and historical records (Wurster et al., 2017).

The utility δ15N as a paleoclimate proxy is dependent upon evidence of a lack of denitrification and ammonia volatilization within the guano pile (McFarlane et al., 1995; Bird et al., 2007;

Wurster et al., 2010). These microbial processes with high enrichment factors reduce the %N in the guano, ultimately altering the initial δ15N values. The %N values of the Topolnița core remain high throughout the entirety of the core, indicating δ15N values are likely to closely match the initial values upon deposition (Fig. 6B). While the %N decreases substantially towards the lower 20 cm of the Gura Ponicovei core (Fig. 6A), these values co-vary with %C. As the microbial processes responsible for N loss in a guano pile are irrespective of those that could remove carbon, we interpret this as evidence that the drop in %N is not due to loss of N following deposition. Therein both δ15N time series are appropriate for interpretation.

δ15N winter precipitation reconstruction

16 Statistical analysis of δ13C and δ15N values of Gura Ponicovei core indicates a correlation between the two isotopes (R2 =0.65; p-value <0.001; Fig. 7). This suggests a similar control of both δ13C and δ15N within the foraging range of the bats. Therefore, it is inferred that as water availability via plant WUE is responsible for the variation in δ13C values, water availability also controls the relative openness of the local N-cycle. This is consistent with results of the other

Romanian guano studies in the northern and central parts of the country (Cleary et al., 2016,

2017) are hereafter interpreted as a proxy for precipitation. Due to the small sample number used for δ15N analysis from Gura Ponicovei between AD 1925 and present, a statistical analysis of nitrogen values and instrumental winter precipitation is not feasible. However, there is sufficient

δ15N data from the Topolnița core. The best correlation (R2 = 0.45; Fig. 7B) was found between

δ15N values and winter/autumn precipitation. Notably, the application of a 3-year lag to the δ15N series (Fig. 7C) was necessary for this result. This lag is believed to be viable considering the errors associated with calibrated ages. In contrast with previous studies (Cleary et al., 2016,

2017), there is a lack of statistical correlation between the δ15N and δ13C values in the Topolnița core. However, this can be explained by a lack of contribution from summer precipitation

(represented by δ13C values) to water availability during the winter months. Therefore, δ15N from both Gura Ponicovei and Topolnița cores are interpreted as a proxy for water availability during the autumn/winter seasons.

The Gura Ponicovei guano record was continuously accumulating through the Little Ice Age termination (LIA; ~AD 1250-1850). This climatic interval was characterized by cool and dry conditions in most parts of Romania (Popa and Kern, 2009; Feurdean et al., 2011 a, b; Haliuc et al., 2017), although milder conditions have been recognized in some areas (Forray et al., 2015).

Interestingly, past work in southwestern Romania found the LIA to begin around AD 1230 based

17 on the deterioration of weather during the Medieval Warm Period/LIA transition (Onac et al.,

2014). However, guano accumulation in Gura Ponicovei begins at AD 1228 (Fig 2A). The trend towards more negative δ13C values and therefore dry climate conditions at Gura Ponicovei (Fig.

8) supports the determination of the LIA onset around AD 1230’s. This could also reflect a deterioration of climate during the termination of the Medieval Warm Period, and not necessarily a precise initiation of the LIA at AD 1230.

Warken et al. (2018) found the driest phase of the LIA in the nearby Cloșani Cave to be between

AD 1675 and AD 1715 (Fig. 8E). At Gura Ponicovei and Topolnița, δ15N values appear to indicate a trend towards drier winters beginning at AD 1500 and terminating at AD 1975. This milder gradual transition out of the LIA resembles that interpreted in the Apuseni Mountains

(Forray et al., 2015). The wettest event recorded in Zidită Cave occurs near contemporaneously with a similar wet event found at Topolnița (Fig. 8). There are no such pulses towards wetter phases in the Gura Ponicovei record. This could be explained by the elevation differences of the caves. Topolnița Cave is positioned higher in the mountains and the surrounding environment is more subject to climate variation. Gura Ponicovei Cave positioned at low elevation is less likely to provide information on finer scale changes in climate, instead is archiving long term variation.

This could also be simply attributed to insufficient data to capture short term variations in winter precipitation.

Past work suggests that negative and positive winter precipitation anomalies in Eastern Europe can often be attributed to East Atlantic/Western Russia system and the North Atlantic Oscillation

(NAO; Bojariu and Paliu, 2001; Perșoiu et al., 2017). Indeed, the NAO is one of the primary influences on climate variability in the Northern Hemisphere (Hurrell et al., 2013). Positive phases of the NAO occur in response to a stronger Azores High and deeper Icelandic low (drier

18 winters in southern Europe). When NAO is in its negative phase, storm tracts migrate southwards which results in increased winter precipitation in the southern and eastern parts of

Europe. Previous studies acknowledge that interferences with Mediterranean circulation can potentially affect the NAO signal in hydroclimate records (Constantin et al., 2007; Roberts et al.,

2012). However, Warken et al. (2018) did find connectivity between North Atlantic forcing and autumn/winter precipitation in SW Romania on centennial to millennial timescales, but a weaker relationship at annual to decadal timescales. Higher resolution records of Topolnița and Gura

Ponicovei guano could potentially better capture the signal.

Most notably the drying trend indicated by a 3.2‰ decrease in the δ15N of Topolnița guano

(Fig. 8C; AD 1824 – AD 1841) occurs near contemporaneously with a progression towards more positive phases of instrumental record of the NAO (AD 1820 - AD 1855; Fig 8F). Additionally, there is a subsequent transition to a more open N-cycle (higher δ15N values) at AD 1879 that corresponds with a trend towards wetter winter conditions in Europe (AD 1882 - AD 1970;

Fig. 8D). Interestingly the initiation of this event appears to precede an interval of more frequent negative phases of the NAO (AD 1923 - AD 1970). However, the Warken et al. (2018) appears to capture this shift towards increased winter precipitation more consistent with the instrumental record of the NAO (Warken et al., 2018; Fig 8E, F). The notable strong positive phase after the mid-1970’s is well recognized with decreasing winter precipitation in Romania (Tomozeiu et al.,

2005; Cleary et al., 2017). Consistent with these findings, the 1.8 ‰ decrease in δ15N values

(Topolnița) beginning at AD 1973 also suggests a drier climate.

Unlike the previous interval, AD 1980 - AD 2014 is characterized by conflicting trends in terms of the associated precipitation. A higher frequency of negative phases of the NAO occurred during this interval, however δ15N values from Gura Ponicovei suggest a pronounced decrease in

19 winter precipitation (Fig. 8A). This is likely due to the fact that southern Romania is particularly vulnerable to drought relative to other areas of the country (Barbu and Popa, 2004). The most prolonged hydrological drought in this region was recorded between AD 1980 and 1995 (Stefan et al., 2004). In agreement, a closed N-cycle and prolonged dry phase is inferred by decreasing trend in δ15N values of Gura Ponicovei guano (Fig. 8A). The 1.7‰ decrease that occurs in the

Gura Ponicovei record at AD 1991 overlaps with a similar decrease (3.1‰) inferred from

Topolnița guano. Similar to the Măgurici record of the NAO (Cleary et al., 2017), δ15N values remain low at Gura Ponicovei after this event (Fig. 8A, B). This interval of ~AD 1970-present is characterized by more a pronounced positive phase of the NAO, agreeing with the sustained dry conditions indicated by the Gura Ponicovei and Măgurici records. Alternatively, increasing

Topolnița δ15N values suggest progressively wetter conditions between AD 1991 and 2014

(Fig. 8C). This appears unrelated to the increased anthropogenic influence in proximity around

Topolnița in the 1900’s (see below). Expansion of human indicator plants suggest deforestation and landscape openness that can result in a more closed N-cycle (lower δ15N values; Cleary et al., 2016; Wurster et al., 2017). An explanation could be associated with an influence of the East

Atlantic/West Russia pattern of climate variability that trends towards more negative values

(wetter conditions) between AD 1960 – present. The absence of a similar trend in the Gura

Ponicovei record could be explained by the lack of data between AD 1960 and AD 2014

(Fig. 8A).

In summary, it appears that δ15N of guano from Gura Ponicovei and Topolnița track winter precipitation associated with the NAO through its influence on the local N-cycle. However, consistent with Warken et al. (2018), this study finds a non-persistent relationship between signals of the NAO in these records on annual timescales. Although 50 to 100-year trends agree

20 well, there is less frequent correspondence with 5-10 year variability. This is likely due to the smaller data size in comparison to the Măgurici record (Cleary et al., 2018). Therefore, the potential exists for a higher resolution record acquired from the region to provide a more complete record of NAO influence on winter precipitation in SW Romania.

δ13C summer precipitation reconstruction

No statistically significant correlation was found between δ13C values and instrumental records of temperature, soil temperature, and atmospheric CO2 concentration between AD 1960 and

2014. However, Topolnița guano δ13C values (Fig. 9B) were found to correspond well with the

Levanič et al. (2012) summer standardized precipitation index (SPI) record (<50 km from study site; Fig. 9C). An apparent strong relationship exists between the two records, with lower δ13C values occurring contemporaneously with drought conditions indicated by negative values of the

SPI. Interestingly this is paradoxical to the generally accepted effect of water availability on plant-WUE and the associated carbon isotope fractionation. As mentioned above, typically

13 13 higher δ C values of C3 are associated with drought conditions and samples depleted in C are interpreted to occur as a result of increased water availability (Lambers et al., 2008; Bodin et al.,

2013). Although this type of relationship has been demonstrated to exist in peat mosses

(Skrzypek et al., 2007), they function entirely different than vascular plants.

When considering temperature, the warmer European climate (Fig. 9F) between AD 1950 and

2014 as well as at AD 1782 does correspond with more negative values in δ13C. However, unique relative to other reconstructions of drought indices, an SPI record inherently is based solely on monthly precipitation data (McKee et al., 1993). There is no dissimilarity between the

SPI and guano-δ13C values during this period of similar trends with temperature. Therefore, while there is likely some influence of temperature on precipitation, the most consistent and

21 strong relationship exists between δ13C values and the local SPI (Fig. 9E). This suggests that although carbon isotopic fractionation associated with WUE is counter to the previously established process, the local water balance is the source of variation in δ13C values.

A primary control of precipitation on WUE rather than other environmental factors could be explained by the fact that the southern Romania is the most vulnerable to drought relative to other regions of the country (Levanič et al., 2012). Therefore, water availability would likely be the primary limiting ecological factor for plant growth in the region. Additional evidence to this point is provided by the good agreement between the summer SPI and δ13C records of SW

Romania and reconstructed summer precipitation in Europe (Pauling et al., 2006; Fig. 9E).

Although we cannot offer an explanation as to why drought conditions would result in low WUE in plants, for the reasons listed, there is sufficient evidence of a precipitation control on the carbon isotopic composition of guano. This allows us to interpret the δ13C values in terms of past water availability.

Although the Levanič et al. (2012) SPI record provides a detailed record of dry and wet years, guano δ13C values can offer information regarding trends in precipitation. Gura Ponicovei δ13C values indicate that climate in the region was becoming progressively wetter from AD 1537 until

AD 1982 (Fig. 9A). However, an interval of decreased precipitation does occur between AD

1689 and AD 1781 in the Topolnița record (Fig. 9B). This event appears to coincide with four extreme dry years (AD 1725, 1748, 1782, and 1784) in Domogled (Levanič et al., 2012; Fig.

9C). Lower water availability indicated by higher δ13C values within this interval also occur contemporaneously with less precipitation in the Czech Republic (Fig. 9D) and overall European summer precipitation (Fig. 9E). Additionally, each record features a brief wet pulse that occurs within this interval at AD 1726. Spring precipitation in Europe was generally low throughout this

22 interval (Pauling et al., 2006) providing additional evidence that summer climate likely has more impact on plant water use efficiency around Topolnița cave. The SPI indicates that this area of

SW Romania during the nineteenth century lacked extreme wet and dry years. Similarly, there is little variation in δ13C values of Topolnița guano between AD 1791 and AD 1878 (Fig. 9B).

Summer precipitation in Europe during this interval also stabilized relative to prior and subsequent years with mean annual rainfall fluctuating between 150 and 232 mm (Fig. 9E).

Additional evidence of this connection can be found in the gradual trend towards wetter conditions between AD 1795 and AD 1841. The higher summer water availability also occurred in the Metaliferi Mountains (Forray et al., 2015), on the Someș Plateau (Cleary et al., 2018), and in the Tăul Muced (Feurdean et al., 2015a).

As indicated by the precipitation data, the aforementioned interval of relative stability in terms of

European summer water availability, is followed by 83 years of progressively wetter conditions data (AD 1885 – AD 1968). A corresponding increase in δ13C values over roughly the same interval (AD 1878 – AD 1978) suggests that summer precipitation also increased in SW

Romania (Fig. 9B). A trend is not apparent in the SPI until a drought occurs in AD 1946, where after a higher frequency of extreme wet events occur (AD 1969, 1970, 1975, and 1982; Fig. 9C).

This increase in wet events also corresponds with the highest precipitation received at both

Topolnița and Gura Ponicovei caves (Fig. 9A, B). Interestingly, other European records of precipitation (Fig. 9F, G) indicate a drier climate over the same interval, in the Swiss Alps (van der Knaap et al., 2011), Northern Britain (Charman et al., 2006) and in the eastern Mediterranean

(Touchan et al., 2005). However, the study by Briffa et al. (2009) of summer moisture availability found that dry and cool summer conditions were less widespread across Europe in the latter half of the nineteenth century. This is supported by geographically extensive European

23 records of precipitation (Pauling et al., 2006; Büntgen et al., 2011) and more localized records of

Romania (Feurdean et al., 2015a) that indicate a wetter climate over the same interval.

The initiation of this wet period appears to correspond with the termination of the LIA, a prolonged cold phase that affected many parts of Europe (Mann et al., 2009). The amplitude, duration, and climate associated with the LIA event (~AD 1240-1850) were variable at the regional level (Mann, 2002). At other sites in Romania the end of the LIA is usually recognized by a shift to drier conditions (Forray et al., 2015; Cleary et al., 2018). The contrasting results from Topolnița and Gura Ponicovei caves data could be attributed partly to the precipitation pattern altered by topography (Gura Ponicovei Cave less obstructed by mountains than

Topolnița). The demise of the LIA in Romanian records occurred at varying times. Studies in the northwestern region (Cleary et al., 2018) and Metaliferi Mountains (Forray et al., 2015; Forray et al., 2019) found abrupt terminations at AD 1890 and AD 1870, respectively. Alternatively, other records in the Călimani Mountains (Popa and Kern, 2009) suggests the end of the LIA occurred earlier in the nineteenth century around AD 1840. In agreement with the former, a shift towards wetter conditions in this study, places the demise of the LIA at ~AD 1870 (Fig. 9B).

This wet event is subsequently followed by a sharp change towards drier conditions. This shift appears to be inconsistent across Romania with wetter conditions existing in western (Forray et al., 2015) and northern (Popa and Kern, 2009) regions of the country. However, a decreased discharge of the Danube (Rîmbu et al., 2002) and droughts in AD 1987 and AD 2000 in

Domogled (Levanič et al., 2012) provide evidence of a drier SW Romania during this period.

This decreasing trend in δ13C values of Topolnița and Gura Ponicovei guano corresponds with a progressively drier and warmer climate in Europe (Pauling et al., 2006; Büntgen et al., 2011).

Vegetation dynamics and the link with climate conditions and human impact

24 AD 1395-1643: Open mixed deciduous forest

Our pollen record from Gura Ponicovei Cave near the Danube River, show abundant concurrence of thermophilous species such as Quercus and Tilia but also of Fagus sylvatica. As

Tilia is an entomophilous species and bats are ingesting insects, insect pollinated species are better represented in guano-derived pollen records than in those from peat or lakes. The presence of Fagus sylvatica, a species that typically thrives in cool and moist summer and mild winters

(Peterken and Mountford, 1996) suggests warm winters and moist summers between approximately AD 1395 and 1550 in the region. This agrees well with the stable wet winter and summer precipitation in Europe during this interval (Fig. 8D, 7E; Pauling et al., 2006).

AD 1643-1957: Increased landscape openness

Pollen records from both caves indicate a reduction in the forest cover beginning at AD 1643 around Topolnița Cave and AD 1810 at Gura Ponicovei. In agreement with modern elevational distribution of the forest belts, the patchy forests were dominated by Tilia and Carpinus betulus admixed with Corylus avellana and Quercus at our low elevation cave (Gura Ponicovei) and by

Fagus sylvatica, Tilia, Carpinus betulus at Topolnița, our cave located closer to the Fagus forest belt (Fig. 1). Topolnița Cave also features a stronger anthropogenic influence. The reduction in percentages of mesophilous species, and in particular of Fagus sylvatica, could be associated with a lower water availability, an interpretation also supported by the δ15N and δ13C values indicative of drier winter and summer seasons during the LIA (AD 1537 and AD 1781). As noted by Barbu and Popa (2004), southwestern Romania is particularly vulnerable to drought.

Therefore, it appears that water availability rather than temperature is a primary control of vegetation dynamics around Topolnița and Gura Ponicovei caves. Peak dry conditions in the region between AD 1775 and 1800 likely led to the near complete disappearance of Fagus

25 sylvatica from the Gura Ponicovei area, whilst more drought resistant species such as Tilia and

Quercus were able to persist. The persistence of greater amount of Fagus sylvatica at Topolnița could indicate that the area was less susceptible to drought due to the higher elevation. On the other hand, the decreased tree cover and the parallel increase in anthropogenic pollen indicators expressed by our records partially overlaps with the pollen and map-based reconstruction of grasslands and agricultural land expansion in Transylvanian Depression between AD 1700 and

1850 (Feurdean et al., 2015b). This trend may suggest that tree cover decline may have also been the response to human impact not only to changing climate conditions.

AD 1957-1985: Open mixed deciduous foothill forest belt

Unique to the Topolnița record, a transition to a 28-year expansion of Fagus sylvatica dominated forests with contributions from Corylus avellana, Fraxinus excelsior and Alnus occurs contemporaneously with a reduction in the diversity and abundance of herbaceous taxa. Notably there is also an increased proportion of Typha, likely related to the construction of the Iron Gate I dam in AD 1971 (Orghidan and Negrea, 1979). The subsequent localized ~20 to 35m water level rise in the Danube river could have increased the potential of foraging bats to interact with Typha pollen through ingestion of aquatic insects. There is no sign of evident climatic change which can be traced by δ13C and δ15N values in guano samples.

However, the reduction in tree cover was very typical during the Socialist Period in Romania, when agricultural activities (pasture, croplands) intensified (Hartel et al., 2013; Feurdean et al.

2017). This reduction in herbaceous diversity could be attributed to the application of artificial fertilizers, which has been found to contribute to expanding grasses while repressing other herbs

(Akeroyd and Page 2011; Wesche et al., 2012). While increasing agricultural practices had a significant impact on the relative proportions of tree versus herbaceous taxa, climate could

26 explain the proportion of primary forest constituents at this time. Fagus sylvatica, Alnus, and

Fraxinus excelsior fair better with increased precipitation and soil moisture (Peterken and

Mountford, 1996; Dobrowolska et al., 2011). Topolnița guano δ13C and δ15N values are indicative of progressively wetter climate from AD 1885 until AD 1968. In northern Romania,

Popa and Kern (2009) found a brief cold spell in the 1910s, preceding the initiation of the warming trend at ~AD 1980.

AD 1985-present: Forest expansion at Gura Ponicovei and human impact at Topolnița

Forests around Gura Ponicovei consisting of Carpinus betulus, C. orientalis, Quercus, and Tilia are typical of modern vegetation for such a low elevation site (Abdi et al., 2009; Fărcaș et al.,

2006). In the post Socialist period (AD 1989 to present) increased forest cover occurred in most of the Carpathian region as former croplands and pasturelands were abandoned (Griffiths et al.,

2013). However, the pollen record from Gura Ponicovei suggests that agricultural practices were minimal in the region since the 1950’s at this location. Notably is the presence of C. orientalis, a species very indicative of a sub-Mediterranean environment and which thrive under warmer conditions. This type of forest assemblage with increased contribution of thermophilous and drought resistant tree species e.g. Pinus, Salix, and Fraxinus ornus is likely indicative of a warm/dry climate. Indeed, both δ13C and δ15N records feature a contemporaneous shift to drier winters and summers. Lower precipitation (Pauling et al., 2006) and higher temperatures

(Büntgen et al., 2011) appear to have also impacted most of Europe at this time.

Contrarily to the Gura Ponicovei Cave, there is an overall reduction in arboreal taxa and increased contributions ruderal pollen indicators at Topolnița Cave (Fig. 5; 6). There does not appear to be substantial fluctuations in primary tree taxa (Carpinus betulus, C. orientalis, and

Quercus) during this interval that could indicate a lack of deforestation activities. Forests

27 dominated by Quercus, Tilia, and Carpinus betulus are typical of lower elevation in Romania (>

300 m a.s.l.) and it appears that these thermophilous species replace the previously dominant

Fagus sylvatica, in the pollen record. Human impact around the cave is likely attributed to agricultural practices that are not as prevalent around Gura Ponicovei. The microcharcoal spike around AD 1930 can be associated with controlled fire practices with the purpose of land cleaning (Whitlock and Larsen, 2001; Feurdean et al., 2013) that was also recognized by Forray et al. (2015) in the Mada region of the Apuseni Mountains.

Conclusions

The δ15N values of guano from Gura Ponicovei and Topolnița provide a record of winter precipitation in SW Romania. Variability in water availability seems to be associated with the

NAO through its influence on the local N-cycle. It was also determined that δ13C values reflect the relationship between summer precipitation amount and plant WUE. These records indicate that drier winters and summers characterize the conditions of the LIA termination (AD 1870).

Increased presence of thermophilous and drought resistant species in the vegetational assemblage agree with this climatic setting. Water availability appears to have increased in subsequent winter and summer seasons through the 1970’s. Expansion of Fagus sylvatica around Topolnița Cave can likely be attributed to these milder conditions. Gura Ponicovei and Topolnița δ15N and δ13C values indicate that droughts became more frequent in both seasons over the past 30-40 years.

Both δ15N and δ13C reconstructions of precipitation compare more favorably with European records of precipitation than other Romanian data, indicating that climate within the country is more variable and well linked to a greater region than the country itself. Future work in SW

Romania will be needed to better understand the climate disconnect between this region and more interior areas of the country.

28 Acknowledgments

The authors greatly appreciate the assistance of Roxana Grindean for her comments and suggestions during the development of these pollen records. We also thank Bogdan P. Onac and

Jonathan G. Wynn for their insight and comments on this project and early drafts of this manuscript. Funding for this research was provided by Romanian CNCS grant PN-II-ID-PCE

2011-3-0588 to Bogdan P. Onac.

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41 Figures

Figure 1: Map of Romania with circles indicating locations referenced in text: Gura Ponicovei

Cave (1; this study), Domogled National Park (2; Levanič et al., 2012), Topolnița Cave (3; this study), Drobeta-Turnu Severin (4; meteorological station), Zidită Cave (5; Forray et al., 2015),

Măgurici Cave (6; Cleary et al., 2017, 2018), Tăul Muced Lake (7; Feurdean et al., 2015a),

Bucharest (8).

Figure 2: Age-depth models for Gura Ponicovei and Topolnița guano cores.

–24.5

–25.0 (‰) –25.5 C 13 δ –26.0 8.0 –26.5 Ponicovei 7.5

–27.0 7.0

–27.5 6.5 (‰) N 15 6.0 δ

5.5 Ponicovei 5.0 A 4.5 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000

13 (‰) N

11 15 δ a ț

–24 Topolni 7

–25

(‰) 5 C 13

δ –26 a ț

–27 Topolni

–28 B

1650 1700 1750 1800 1850 1900 1950 2000 Age (AD)

Figure 3: A) δ15N (red), δ13C (black), and Suess corrected δ13C (maroon) values of Gura

Ponicovei guano; B) δ15N (blue), δ13C (black), and Suess corrected δ13C (purple) values of

Topolnița guano.

43 s li s a a r li t in io a s a la ic e e s n u t o r � e a a e l s a l n a e f e e a e e p e l a e u ll tu ie ic s e c jo m o p a e e e e c c ty p n a r c n e e r t i e a n a . u a y e a ae a a c a ia - y e o e o b s x r v b o a d a e la p i b la t c e e a e e a ll r m t a e t e a rc m e e o a lv i s e e a a a id m s m d r ae - la d c i c ac e e l y la s - e m a e ea e m a e i s s s s s y ir u a s e d si si re o o o o u o e ae o u e is u i a b a a a u h u cu e a c u e e g c c u c ab u u u s u u s v r a s e u a i i i g g g tr p a e is d c b c o c r in n ce e n p s it ic la ic c a o a ra ia i ch s a n n c lu in in s s e r m n s c l e o ro u r a a a c o x e c n a a n c i o g o a c a o h a t n u r a e a r e n b s a l i i r r y p p u es s u p x e u a s u a u c r b m ta o t t t li n e ac a u e c n u sa ss h a g i a p p b n o e i c m h e s a o ro u u e ix tu ax ax e ia e r r r g i u i ri d c l i ic p a te te n h n n n a e m i s g ip ti n n a p r ly b b m ry o r o r im p m ia ta p p o r il c in in ic l e r r u il c o a a a b ln ln n a e is g it lm r o o s m r e ic la la la h h u p o an il r a a ip r u o o u a a a cr e d e r y a il o y y r e p i Age (AD) Depth P P P Sa B F F Q T A C C C F A A A Ju L H V Ju V U E P P A A A C C P P P T C R A R S F U C R D B E B P R F C C S V O V P H L L P T C D G E M Zone CONISS 2014 0 2 LPAZ 5

1996 4 6

12 LPAZ 4 14

20 1990 22

26 LPAZ 3 28

1810 32

36 LPAZ 2 38

1490 42

44 46 LPAZ 1 1395 48 50 20 20 20 20 20 20 20 20 20 20 1 2 3 4 Total sum of squares

Figure 4: Pollen diagram from Gura Ponicovei Cave guano

e a e e e a id a e o e id c d o a i ri s ro o ip e is t ch l D ) s i a a f. s a A C t in b li li . . la c u e m a a f f fi s e e a e iu s t e b b o r f e a a e p n u n r u u e o o e a e e e c y e a l ie e s s c j . m a p e a e e a e c a -t p n g e a rn r C n a p iu ty c e a a e c a i y e to r e o o o ( e a e la m s d rb la e - a c e e c e la ll r m t a e a a ea rc s e a a a e a m e e o a s l a c ia c a a e u y la u s - e m a e p e c m s s s s s u a s e e e i i r e o o o u o a a s u e i u li a b a n a n h u c e a c u e e g S c a u u u s u u r a s e u a a c s is u c g g g tr p x e i d c b c o ic r in ce e a p h s it ic la ic c a o / a ra ia iu h s a la in n c lu in in s s e r m n s c l e e a ro a a a a a ic o e e c u n a a n if s o g o a c p o p a t n u r a e m a r e n b c u ix x i r r y p p u s u ip x e u la u a u c c r m t r t t t l n c a g e c n u r s h a g i a y o b n o e i c h e s a o ro e l tu x e ia e r r r g ie n ri d c g is c p a a te b te n te n n n a e m ia s n ip ti n n p p r ly b b m r r r o r im p m a ta p p o r il c in ic a e ra ra u il c o a a a b ln u a e is u it lm ri o o o s m r e s la la la h h u p o a il r a a a ra u o o u a a a c e d e r y a ili o y y r e p i Age (AD) Depth P P S B F F Q T A C C C F A A J L H V J V U E P P P A A A C A P P P T C R A R S F U C R C B E B P R F C C S V O V P H L L P T C D G E M Zone CONISS 2014 0

5 LPAZ 5

10 1991 15

20 LPAZ 4 25

1984 35

45 LPAZ 3 50

1957 60

70 LPAZ 2

75 1790 80

85 LPAZ 1 90

1643 95 100 20 20 20 20 40 20 20 2 4 6 8 10 Total sum of squares

Figure 5: Pollen diagram from Topolnița Cave guano.

%C %C 15 20 25 30 20 25 30 35 40 45 0 0 A B 5 5 10 15 10 20 25 15 30 35 20 40 45 25 50

55 (cm) Depth Depth (cm) Depth 30 60 65 35 70 75 40 80 85 45 90 95 50 100 2 3 4 5 6 6 8 10 12 %N %N

Figure 6: A) %C (red) and %N (black) values from Gura Ponicovei core; B) %C (red) and %N

(black) values from Topolnița core.

Figure 7: A) Correlation analysis of δ15N and δ13C of Gura Ponicovei; B) Correlation analysis of

Topolnița δ15N and Drobeta-Turnu Severin autumn/winter precipitation; C) Drobeta-Turnu

Severin autumn/winter precipitation (black), Topolnița δ15N values (dotted blue) and with 3-year lag (solid blue).

45 Figure 8: A) Comparison of δ15N values from Gura Ponicovei guano, B) δ15N values from

Măgurici guano, C) δ15N values from Topolnița guano; D) 3-year running mean of reconstructed winter precipitation in Europe, E) autumn/winter precipitation Cloșani Cave, F) NAO winter index. Vertical bars indicate similar dry/wet phases.

Figure 9: A) δ13C values from Gura Ponicovei guano, B) δ13C from Topolnița guano, C) tree ring record of SPI from Domogled National Park D) Tree ring spring/summer precipitation from southern Czech Republic, E) 3-year running mean of European summer precipitation, and F) 3- year running mean of reconstructed European temperature anomaly. Vertical bars indicate similar dry/wet phases.

47 Tables

Table 1. Radiocarbon ages, calibrated years AD and the dates used in construction of the age depth models for Topolnița and Gura Ponicovei guano cores.

Topolnița 14C Activity Calibrated Age used Sample ID yrs BP Depth (pMC) years (CE) (CE) Surface - 0 2012-2015 2014 TP14 104.88 ± 0.31 - 14 1980-1994 1987 TP30 106.38 ± 0.36 - 30 1978-1992 1985 TP44 111.12 ± 0.36 - 44 1974-1988 1981 TP56 125.91 ± 0.36 - 56 1962-1976 1969 TP71 160 ± 30 71 1719-1788 1795 TP85 200 ± 30 85 1731-1809 1780 TP94 250 ± 30 94 1627-1690 1683

Gura Ponicovei 14C Activity Calibrated Age used Sample ID yrs BP Depth (pMC) years (CE) (CE) Surface - 0 2012-2015 2014 PI2 111.3 ± 0.33 - 2 1993-1999 1996 PI12 138.82 ± 0.37 - 12 1989-1995 1992 PI26 115.33 ± 0.35 - 26 1967-1990 1975 PI42 350 ± 30 42 1530-1608 1537 PI55 810 ± 30 55 1181-1269 1228

APPENDIX A: EVIDENCE OF LONG-TERM NAO INFLUENCE ON EAST-CENTRAL

EUROPE WINTER PRECIPITATION FROM A GUANO-DERIVED δ15N RECORD

Note to Reader:

This chapter has been previously published: Cleary, D.M., Wynn, J.G., Ionita, M., Forray, F.L.,

Onac, B.P. Evidence of long-term NAO influence on East-Central Europe winter precipitation from a guano-derived δ15N record. Scientific Reports 7:14095. See Appendix A for the PDF of the published document and Appendix B to see permission from the publisher.

49 www.nature.com/scientificreports

OPEN Evidence of long-term NAO infuence on East-Central Europe winter precipitation from a guano- Received: 25 April 2017 15 Accepted: 11 October 2017 derived δ N record Published: xx xx xxxx Daniel M. Cleary1, Jonathan G. Wynn1,6, Monica Ionita2,3, Ferenc L. Forray4 & Bogdan P. Onac 1,4,5

Currently there is a scarcity of paleo-records related to the North Atlantic Oscillation (NAO), particularly in East-Central Europe (ECE). Here we report δ15N analysis of guano from a cave in NW Romania with the intent of reconstructing past variation in ECE hydroclimate and examine NAO impacts on winter precipitation. We argue that the δ15N values of guano indicate that the nitrogen cycle is hydrologically controlled and the δ15N values likely refect winter precipitation related to nitrogen mineralization prior to the growing season. Drier conditions indicated by δ15N values at AD 1848–1852 and AD 1880–1930 correspond to the positive phase of the NAO. The increased frequency of negative phases of the NAO between AD 1940–1975 is contemporaneous with higher δ15N values (wetter conditions). A 4‰ decrease in δ15N values at the end of the 1970’s corresponds to a strong reduction in precipitation associated with a shift from negative to positive phase of the NAO. Using the relationship between NAO index and δ15N values in guano for the instrumental period, we reconstructed NAO-like phases back to AD 1650. Our results advocate that δ15N values of guano ofer a proxy of the NAO conditions in the more distant past, helping assess its predictability.

Global atmospheric circulation has a number of preferred patterns of variability, all of which have expressions in surface climate. Regional climates may vary out of phase, due to the action of such teleconnections, which modulate the location and strength of the storm tracks and poleward fuxes of heat, moisture and momentum1–3. Over Europe, the strongest infuence is given by the North Atlantic Oscillation (NAO)4–6. NAO has a substantial efect on the European climate by modulating the position of North Atlantic storm tracks, which in turn control short and long-term changes in precipitation and temperature. When NAO is in its positive phase (e.g., a stronger than normal Azores High and a deeper than normal Icelandic Low), winter storm tracks are defected northward resulting in wet winters over the northern part of Europe (positive correlation between winter NAO index and winter precipitation) and dry winters over the southern and eastern part of Europe (negative correlation between winter NAO index and winter precipitation) (Fig. 1). During the negative phase of NAO, storm tracks are shifed southwards, thus bringing wet and mild winters over the southern and eastern parts of Europe and dry winters over the northern part of Europe. At a regional scale, the Carpathian Mountains have a strong infuence on NAO related precipitation and temperature anomalies in Romania5. Due to orographic efect of the Carpathians, the winter NAO related signal is stronger in the northwestern part of the country7,8 where Măgurici Cave (hereafer MC) is located (see Supplementary Fig. S1). While studies have demonstrated that the NAO is an important factor in winter climate variability in East-Central Europe (ECE)8–10, except a recent study reconstructing winter air temperature and

1School of Geosciences, University of South Florida, 4202 E. Fowler Ave, NES 107, Tampa, FL, 33620, USA. 2Paleoclimate Dynamics Group, Alfred-Wegener-Institute for Polar and Marine Research, Bussestrasse 24, Bremerhaven, D-27570, Germany. 3MARUM, Center for Marine Environmental Sciences, University of Bremen, Bremen, Germany. 4Department of Geology, Babeș-Bolyai University, Kogălniceanu 1, 400084, Cluj-Napoca, Romania. 5Emil Racovită Institute of Speleology, Romanian Academy, Clinicilor 5, Cluj-Napoca, 400006, Romania. 6Present address: National Science Foundation, 4201 Wilson Blvd., Arlington, VA, 22230, USA. Correspondence and requests for materials should be addressed to B.P.O. (email: [email protected])

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Figure 1. Correlation map between (a) winter (DJF) NAO index and winter precipitation (PP), and (b) temperature (TT) derived from the CRU TS3.24.01 data set54. Green contours outline Romania. Dotted areas indicate correlation coefcients signifcant at 95% signifcance level on a standard t-test. Te fgure was produced using Matlab 2014b (http://de.mathworks.com/products/new_products/release2014b.html).

moisture source11, there is a lack of paleo-records of the NAO in ECE. Furthermore, to place recent NAO variability in a long-term context, there is a pressing need to develop longer records. Here we use coupled carbon and nitrogen isotope ratio data (δ13C and δ15N values) of a 286 cm guano core from MC in ECE, in an attempt to characterize the hydroclimate infuence on the N-cycle since the lat- est part of the Little Ice Age (LIA; AD 1650 to 1850) and up to AD 2012. We frst compare these data to the December-January-February (DJF) NAO index that represents the strength of the system during the winter months. Ten we use DJF meteorological records and a ECE reconstructed precipitation series from Pauling et al.12 that directly and indirectly relate to the NAO to further examine the impact of changes in vegetation and hydrology of ECE on the isotopic composition of guano. Guano and Climate Bat guano is primarily composed of loose organic material, such as insect chitin, with a geochemistry characterized by an abundance of transition metals13,14. Although there are numerous organic compounds in guano, chitin is the most abundant in MC deposit. Terefore, measured δ15N values refect chitin derived N, while the contribution of other N sources to bulk guano is likely insignifcant. If bats are not changing their roost sites, guano can accumulate as thick deposits (3 m or more) over long time periods (centuries to thousands of years)13,15. Depending on the morphology and hydrology of each cave system, fooding of underground passages may cause guano deposits to be interbedded with clay and silt layers. Such circumstances ofer additional information with respect to local cave environmental changes while guano was being deposited16,17. Bat guano may provide an ideal record of past vegetation because the δ13C value of plants in the region is transferred from plant to insect to bat and ultimately recorded in guano. Te δ13C values of foliage are distinct between 18 13 the two main photosynthetic pathways (C3 and C4) . Additional variation in δ C values occurs within C3 plants in response to the water use efciency of photosynthesis19. Te preservation of these values within guano provides new and critical information on the changing vegetational assemblage through time20–25. Although more complex, recent studies have demonstrated that δ15N values in bulk guano can be interpreted as an integrator of the nitrogen cycle (N-cycle)26,27. When nitrogen is a limiting nutrient, nitrogen is conserved and as a result less nitrogen is lost and δ15N values decrease (i.e., a relatively closed N-cycle)28,29. A relatively open cycle results in high δ15N values under the opposing conditions. When it can be demonstrated that a climatic infuence controls whether the N-cycle is open or closed, δ15N values can also be used as a paleoclimatic proxy26. Since the NAO has a strong infuence on precipitation and temperature it is possible that the nitrogen isotopic composition of cave bat guano, which has already been shown to refect changes in water availability could provide insight into the infuence of the NAO beyond the historical record. Results and Discussions Te nitrogen isotopic composition of bat guano may be afected by diagenesis afer deposition via processes of ammonia volatilization or denitrifcation13,30. Te conditions under which alteration may have been significant are in part refected in variation of %N along the profle (see Cleary et al.26). Te %N in the MC core (see Supplementary Dataset 1) shows little deviation from near surface values (~11.3%) to the lower most portion of the core (mean = 10.6%). Terefore, the increasing trend in δ15N values between AD 1650 and AD 1980 (see Supplementary Fig. S2) is likely unrelated to diagenesis and can be interpreted as primary variation, considered here as a proxy of the NAO variability. Te subsequent interval of lower δ15N values (7.5 to 9.5‰; AD 1980 to 2012) represents a change in local N-cycle (see discussion below) as opposed to diagenetic processes. It is difcult to elucidate past δ15N values at any stage of the food web (plant-insect-bat-guano), however it is possible to interpret δ15N as an integrator of the N-cycle28. Te isotopic fractionations, N pool mixing, and N gains/losses produce the δ15N value of the system (soil, biomass, consumers). Terefore this resulting value integrates these processes and can be used to interpret the state of the N-cycle. Te fractionations occurring

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during the metabolic processes within bats and insects as nitrogen is transferred from plant to guano remain fxed through time. Since these fractionations follow conservative pathways, variation in the nitrogen isotopic composition of guano can ultimately be related to changes that occur in the soil inorganic nitrogen reservoir from which plants access nitrate and ammonia28,29. Such changes have been connected to the state of the N cycle (open: more nitrogen loss and higher δ15N values; closed: less nitrogen loss and lower δ15N values)29 and attributed to hydrological infuence on the state of the N-cycle31–33. Cleary et al.26 suggested variation in δ15N values of guano (ultimately related to those of foliage) are strongly correlated to instrumental record of winter precipitation. Tis may result from a lag between the preservation of δ15N values in foliage during the growth phase (spring-early summer), which refect soil N conditions from months immediately prior to growth (late fall-winter). In temperate forests, the maximum plant-available N occurs just prior to the onset of the growing season due to limited plant uptake of N that has been produced largely by microbial mineralization in months prior34. Although there may be signifcant wet deposition of N during the spring and summer months, this fux into terrestrial ecosystems is on average lower than that produced via mineralization35,36. Tis pool of plant-available N is soluble; thus during the winter season it is very sensitive to leaching processes, which is in turn driven by winter climatic conditions such as snow melt and the type of winter precipitation (snow vs. rain) when temperatures that straddle the threshold of freezing37,38. Since the state of the N-cycle is controlled by the amount of N in the system, we infer that any change in the N-cycle is in response to the impact of winter hydroclimate on leaching processes. During the subsequent growing season when plants begin to access the soil N-pool, the state of the N-cycle established by the amount of leaching in the winter is then recorded in the new foliage. Increased leaching would refect a more open N-cycle and result in higher δ15N val- ues39 at each level of the food chain and ultimately guano. Terefore, although bats forage in the summer months, nitrogen delivered to the soil via spring/summer precipitation will likely not infuence the δ15N values of guano. Cleary et al.26 compared δ15N values of bat guano to corresponding δ13C values and to meteorological data from a nearby weather station in the Mada region (Metaliferi Mountains, W. Romania), and interpreted that water availability is a primary control of δ15N values of guano. Given the well-documented relationship of δ13C values of leaf tissues to water-use efciency19,40 the carbon isotopic composition of MC guano (−26.5 to −21‰) suggests variation within the C3 pathway that may ultimately be related to changes in water availability. Based on these observations, we test a hypothesis for a hydrological connection between δ13C and δ15N values of guano by examining the correlation between each proxy in the Măgurici Cave guano record. Excluding two outliers, the resulting statistical analysis indicates that the δ15N and δ13C values in the MC record between AD 1800 and 2012 show a negative correlation (p-value = <0.001; R2 = 0.62; n = 105; see Supplementary Fig. S2a). Tis suggests some dependence of the N-cycle of the bats foraging area on water availability, as the latter can be interpreted 13 15 as the primary control on δ C values of C3 vegetation. Te relationship between δ N and water availability is in contrast to other studies32,33,41, however, is consistent with results obtained from modern guano25 and precipitation in NW Romania. Although both δ15N and δ13C values of guano refect hydrological conditions, it is likely that they are record- ing diferent seasonal infuences on water availability. While δ13C values of foliage are related to water stress during photosynthesis (spring/summer), δ15N values of guano are related to the winter precipitation infuence on leaching. Terefore, correlation between δ13C values and δ15N values is ultimately the result of the winter precipitation (control of N-cycle) contributing to the degree of water stress in the spring/summer (control of δ13C). Consequently, if there is an infuence of the NAO on precipitation in ECE, the nitrogen isotopic composition of guano should retain this signal more accurately than δ13C values. In accordance, hereafer we focus our interpre- tations of δ15N values of guano on reconstructing DJF precipitation. One of the most striking features of our δ15N reconstruction is the abrupt decrease (~4‰) at the end of 1970’s (Fig. 2a) representing a shif of the NAO to a strong positive phase afer the mid-1970s. Tis swing corresponds to regional climatic efects such as milder and wetter winters in northern Europe42, a reduced discharge of the Danube River43, and decreasing winter precipitation over Romania44. Tis shif at the end of 1970’s is clearly observed in the sea level pressure (SLP) and precipitation patterns (Fig. 2). Te diference map in the SLP feld between 1940–1970 and 1980–2010 is indicative of a period characterized by an increased frequency of positive NAO phases afer the 1970s (Fig. 2b), which is associated with a strong reduction in winter precipitation over the southern part of Europe (Fig. 2c). Te resulting precipitation anomaly that occurs between northern and southern Europe during the winter months is also recognized in instrumental records across these regions1. Tis regional decrease in winter precipitation that occurs at the end of the 1970’s is demarcated by the most signifcant decrease in δ15N values of MC guano. Given the direct link between precipitation and the current phase of the NAO, we interpret the δ15N values to largely refect a hydrologic component of the regional climate with a signal that can be related to the NAO. Due to the absence of δ15N values for certain years it is difcult to confdently utilize a statistical analysis to compare the record directly to the NAO index. However, there is a correlation (p-value = <0.002; R2 = 0.43) between the frst derivatives of time series of the NAO index and of the δ15N values of MC guano (AD 1981–2012; interval of near annual ages of guano; see Supplementary Fig. S3). Additionally, since AD 1800, lower (higher) δ15N values, which are indicative of drier (wetter) conditions, occur preponderantly during positive (negative) phases of the NAO (Fig. 3). Te occurrence of more negative phases of the NAO (AD 1940–1975) corresponds with progressively wetter conditions expressed in the δ15N record. Likewise, trends towards drier conditions interpreted from δ15N values appear near contemporaneous with a higher frequency of NAO positive phases (AD 1848–1852; AD 1875–1930; and AD 1975–2012). Our interpretation of δ15N values of MC guano as a NAO proxy (Fig. 3a) is supported by comparison to other precipitation proxies that are more directly infuenced by the NAO. Lower δ15N values from MC correspond well with drier conditions indicated by a DJF reconstructed precipitation record for ECE12 (Fig. 3b), a long-term precipitation measurement in southern France45 (Marseille; Fig. 3c), and nearby Budapest and Baia

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Figure 2. (a) δ15N of MC guano from 1850 to 2012 (see text for details). (b) Te diference in the Sea Level Pressure between 1940 and 1970 (pre-shif) and the period 1980–2010 (post-shif) based on data from NCEP/ NCAR Reanalysis55. (c) As in (b) but for winter precipitation derived from CRU TS3.24.01 data set54. Te fgures were produced using (a) pro Fit 7.0 (http://www.quansof.com) and (b,c) Matlab 2014b (http://de.mathworks. com/products/new_products/release2014b.html), respectively. Te digital version was generated using Adobe Illustrator CC17 (https://www.adobe.com/products/illustrator.html).

Mare meteorological stations45 (Fig. 3d,e). All three are located over regions strongly infuenced by NAO (see the blue shaded band in Fig. 1a). Frequently there is contemporaneous overlap between a negative NAO index and wetter conditions indicated by precipitation records and the δ15N values from MC guano (Fig. 3h). Tis agrees with an interpretation of a wetter NW Romania during the negative phase of the NAO. Tus, the state of the N-cycle that is preserved in δ15N values of MC guano appears to be infuenced by DJF precipitation, which in turn is modulated in ECE by the NAO. A few of the general trends in hydroclimate that are expressed by the NAO index, meteorological records, and the nitrogen isotopic composition of MC guano can also be found in other Romanian and southern Europe paleo-records spanning the period AD 1850 to present. δ15N values from the MC core trend towards wet conditions until AD 1800, fuctuates between 1800 and 1975, afer which the climate abruptly became drier until present. Broadly, the δ15N values from Zidită and Măgurici guano frequently show similar trends. Te two records are well-correlated afer AD 1900, when both feature a gradual trend towards drier conditions interrupted by a wetter interval between AD 1940 and 1975 (Fig. 3g,h). δ15N values from Zidită, Măgurici, and δ13C values in a Sphagnum core from the Tăul Muced46 (Rodnei Mountains, N. Romania; see Supplementary Fig. S1), all refect a drying trend afer ~AD 1975 (Fig. 3f–h). However, the MC record appears to correspond more consistently with the NAO index, suggesting that the infuence of the NAO is more prevalent in NW Romania than in other regions of the Carpathians. Given that the signal of the NAO instrumental record is refected in the δ15N values since AD 1850, we extend our interpretation of this proxy to infer past phases of the NAO prior to the instrumental record. Indeed, using the ECE DJF precipitation reconstruction12 and measurements45 (Marseille) as additional evidence, δ15N values suggest that the positive phase of the winter NAO dominated the circulation between AD 1650 and 1800. Over this 150-year interval, there were fve prominent periods of at least 2 years each during which the negative phase was dominant (Fig. 3h). Our core reveals a major depositional hiatus, when guano accumulation ceased and a silty-clay layer was deposited between ~AD 1713 and 1715 (Fig. 3h). Tis period corroborates well with one of the highest reconstructed value of DJF precipitation across ECE12, suggesting unusually wet winter seasons. Concurrent, historical hydroclimate records elsewhere in Europe47 document an increased in fooding frequency at this time. Interestingly, a testate amoeba record from Tăul Muced ombrotrophic bog located ~100 km NE of our cave documents a decline in water table depth over this period46. Furthermore, the sharp decrease of the δ13C values of Sphagnum at the same location (Fig. 3f) was interpreted to indicate prevalence of drier conditions44. Tese site-specifc contrasting precipitation patterns are not surprising since the winter NAO signal is stronger in the intra-Carpathian region8. Following the guano hiatus, the δ15N time series suggests that beginning at ~AD 1720, a gradual transition to recurring positive phases of the NAO occurred, with the atmosphere remaining locked into this mode until

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Figure 3. Comparison of guano δ15N with other hydroclimate proxy records between AD 1650 and 2012: (a) Winter (DJF) NAO index56; DJF precipitation data series from ECE12 (b), Marseille45 (c), Budapest45 (d), and Baia Mare45 (e), δ13C of Sphagnum from Tăul Muced46 (f), δ15N values of guano from Zidită Cave26 (g), and Măgurici Cave (h; this study). Te black smoothed lines in a–e represent the 3-year running mean.

AD 1790. During this 70-year interval, signifcant negative phases of the pattern appeared only three times (AD 1760, 1775, and 1785), all coincident with wettest winters recorded at the Marseille weather station (Fig. 3c,h). From AD 1800 to 1820, we identifed an abrupt transition to more positive nitrogen isotopic values, which suggest increased regional moisture delivery mostly over the winter period. As inferred from our guano δ15N values, this begins a period of ~20 years of mild and wet winters in the ECE relative to the earlier interval, which ends ~AD 1840 when precipitation started to decrease. Te onset of this period of positive winter NAO phase is coeval with a marked phase of ice ablation in the St. Livre Cave (SW Switzerland), thought to represent almost three decades of warm and dry winters48. It also agrees well (but opposite sign) with a composite speleothem annual growth-rate record in NW Scotland, refective of positive winter NAO states49. It is apparent from the discussion above that the changes in the phases of the NAO may partly explain the climate variability over the last part of the LIA. Conclusions A nitrogen isotopic proxy record of guano provides new information regarding the efect of DJF hydroclimate system on the N-cycle and the infuence of the winter NAO on the ECE. Te evidence of a NAO signal contained within the MC guano δ15N series is the temporal strong correlation of the winter precipitation amount in the instrumental record. Tis study demonstrates that future guano research should consider not only precipitation, but also larger scale climatic systems when utilizing the nitrogen isotopic composition of guano. Te use of nitrogen isotopic composition of bat guano it is possible to add to, and improve the historical record of the NAO. As such, our results suggest that the δ15N values of guano can be utilized to reconstruct past phases of the NAO beyond the instrumental record and demonstrates that the δ15N values of guano can ofer a proxy of the NAO in regions where instrumental or historical records are limited. Methods Guano coring and sampling. In October 2012, a Russian peat corer was used to extract a 287 cm core from a guano pile located in the Circular Room of MC (Fig. 1 in Johnston et al.50). Te coring site was adjacent (within 1 m) to the one investigated by Johnston et al.50. Both cores have similar stratigraphy, however, the lower 29 cm of the frst one recovered only clay, whereas ours penetrated a 4-cm thick clay layer at 237 cm revealing an additional 46 cm of guano beneath it. Except for the clay layer, the entire core length was sampled for isotopic analyses and radiocarbon measurements.

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Radiocarbon dating and age-depth models. Twenty aliquots of bulk bat guano from various depths of the Măgurici core were submitted for radiocarbon dating by accelerator mass spectrometry (AMS) at the Poznan Radiocarbon Laboratory (Poland) and returned ages in stratigraphic order. Sample MG-15 was contaminated with young organic matter and thus discarded. Since guano is mainly composed of chitin (>95%), which makes it an excellent material for AMS age determinations51, no sample preparation was needed. Te age-depth models (see Supplementary Fig. S4) are based on a linear interpolation between each14C age in the upper 50 cm of the core, whereas for the rest of the sequence a type 4 smooth spline was applied. Both models were generated using Clam code52. Te reasoning for employing a linear age-depth model for the upper 50 cm is because continuous observations since 1965 confrmed that the size of the bat colony has not changed, and therefore, it is expected that the guano accumulation remained constant. Te default calibration curve utilized by Clam is the northern hemisphere terrestrial curve IntCal13.14C (cc = 1) from Reimer et al.53. Te samples in the top 50 cm of the core are characterized by high radiocarbon activity (130.06 ± 0.4 and 132.46 ± 0.34 pMC) resulting in modern ages (1979–1980 and 1977–1978 cal. years). Guano began to accumulate in the Circular Room at ~AD 881, shortly before the beginning of the Medieval Warm Period (MWP: ~AD 950–1300). One hiatus is inferred from the age depth model between AD 1237–1651, an interval that corresponds to the frst half of the LIA. Te raw14C data are included in Supplementary Dataset 2, and the results of modeling in Supplementary Dataset 3).

Elemental and stable isotope analyses. Contiguous 1-cm bulk guano sub-samples were recovered for isotopic analyses along with a modern sample collected in 2012 to anchor the isotope chronology. Chitin is the dominant organic compound in MC guano, therefore, we considered compound specifc extraction to be unnecessary. Due to the cave climate (see Supplementary Information) it is highly unlikely that any soluble guano-derived N-compound will survive and potentially impact the nitrogen isotopic composition. All samples were prepared for δ15N and δ13C analysis following the procedures described in Forray et al.25 and Cleary et al.26. Out of these samples, 1–2 mg aliquots were weighed and placed in tin cups and then measured for δ15N, δ13C, %N, %C, and C:N. Analysis was completed using a Costech Elemental Analyzer coupled to a Delta V Isotope Ratio Mass Spectrometer hosted in the Stable Isotope Laboratory (School of Geosciences, University of South Florida). A glutamic acid (internal standard; δ15N: −6.28‰; δ13C: −16.50‰; %N: 9.54%; %C: 41.37%) and a protein standard B2155 (δ15N: 5.94‰; δ13C: −26.98‰; %N: 13.32%; %C: 46.5%) were used during analysis. Certifed reference materials, B2155 and IAEA-N1, were used to calibrate the δ15N value for the internal standard. B2155 and IAEA-C7 were used to calibrate δ13C value of the glutamic acid. Estimation of the precision of analysis (δ15N: 0.08‰; δ13C: 0.04‰) was based on replicate internal standards during each run (Supplementary Dataset 1).

Statistical methods. Correlation analysis between δ15N values (mean = 10.4‰; std. dev. = 1.5) and δ13C values (mean = −24.5‰; std. dev. = 0.7) (n = 105) was completed using SPSS. Tis statistical test is appropriate as both data sets were extracted from the MC core with the same sampling and temporal resolution. Resulting p value andR2 (p-value = <0.001; R2 = 0.62) from the 2-tailed test indicate statistical correlation. MATLAB was used to convert unevenly sampled data to evenly sampled δ15N values, whereas EXCEL was used to compute the frst derivatives (in 1-year time steps). Tis step allowed for the examination of year-to-year changes in values. Correlation analysis (n = 26; using SPSS) between the frst derivatives of δ15N time series (mean = 8.4‰; std. dev. = 0.6) and DJF NAO index (mean = 0.5; std. dev. = 1.4) data sets was completed. Tis analysis was performed for years between 1981 and 2012 that included a respective δ15N value. Te decision of testing the derivatives is appropriate due to the fact that the sensitivity of change being a more probable representation of infuence of the NAO then raw values. Te 2-tailed test resulted in a p-value = <0.002 and R2 = 0.43. References 1. Hurrell, J. W., Kushnir, Y., Ottersen, G. & Visbeck, M. An overview of the North Atlantic Oscillation. In The North Atlantic Oscillation: Climatic Signifcance and Environmental Impact. Geophys. Monogr. Ser. 134(eds Hurrell, J. W., Kushnir, Y., Ottersen, G. & Visbeck, M.) 1–35 ISBN: 9781118669037 (2003). 2. Quadrelli, R. & Wallace, J. M. Varied expressions of the Hemispheric circulation observed in association with contrasting polarities of prescribed patterns of variability. Am. Meteo. Soc. 17, 4245–4253, https://doi.org/10.1175/JCLI3196.1 (2004). 3. Trenberth, K. E., Fasullo, J. & Smith, L. Trends and variability in column-integrated atmospheric water vapor. Clim. Dyn. 24, 741–758, https://doi.org/10.1007/s00382-005-0017-4 (2005). 4. Hurrell, J. W. & Van Loon, H. Decadal variations in climate associated with the North Atlantic Oscillation. Climatic Change 36(3), 301–326, https://doi.org/10.1023/A:1005314315270 (1997). 5. Bojariu, R. & Paliu, D. In Detecting and Modelling Regional Climate Change and Associated Impacts (eds Brunet, M. & Lopez, D.) 345–356 (Springer, 2001). 6. Cassou, C., Terray, L. & Phillips, A. S. Tropical Atlantic infuence on European heat waves. J. Climate 18, 2805–2811, https://doi. org/10.1175/JCLI3506.1 (2005). 7. Busuioc, A. & Von Storch, H. Changes in the winter precipitation in Romania and its relation to the large-scale circulation. Tellus 48, 538–552, https://doi.org/10.1034/j.1600-0870.1996.t01-3-00004.x (1996). 8. Bojariu, R. & Giorgi, F. Te North Atlantic Oscillation signal in a regional climate simulation for the European region. Tellus 57A, 641–653 (2005). 9. Bednorz, E. & Wibing, J. Snow depth in eastern Europe in relation to circulation patterns. Ann. Glaciol. 48, 135–149, https://doi. org/10.3189/172756408784700815 (2008). 10. Casanueva, A., Rodríguez-Puebla, C., Frías, M. D. & González-Reviriego, N. Variability of extreme precipitations over Europe and its relationship with teleconnection patterns. Hydrol. Earth Sys. Sci. 18, 709–725, https://doi.org/10.5194/hess-18-709-2014 (2014). 11. Perșoiu, A. et al. Holocene winter climate variability in Central and Eastern Europe. Sci. Rep. 7, 1397, https://doi.org/10.1038/ s41598-017-01397-w (2017). 12. Pauling, A., Luterbacher, J., Casty, C. & Wanner, H. Five hundred years of gridded high-resolution precipitation reconstructions over Europe and the connection to large-scale circulation. Clim. Dyn. 26, 387–405, https://doi.org/10.1007/s00382-005-0090-8 (2006). 13. Bird, M. I. et al. A long record of environmental change from bat guano deposits in Makangit Cave, Palawan, Philippines. Earth Env. Sci. T. Roy. Soc. Edin. 98, 59–69, https://doi.org/10.1017/S1755691007000059 (2007).

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APPENDIX B: COPYRIGHT PERMISSION FROM NATURE TO USE THE ARTICLE

EVIDENCE OF LONG-TERM NAO INFLUENCE ON EAST-CENTRAL EUROPE

WINTER PRECIPITATION FROM A GUANO-DERIVED δ15N RECORD IN

DISSERTATION

APPENDIX C: A GUANO-DERIVED δ13C AND δ15N RECORD OF CLIMATE SINCE

THE MEDIEVAL WARM PERIOD IN NORTH-WEST ROMANIA

Note to Reader:

This chapter has been previously published: Cleary, D.M., Onac, B.P., Tanțău, I., Forray, F.L.,

Wynn, J.G., Ionita, M., Tămaș, T. A guano-derived δ13C and δ15N record of climate since the

Medieval Warm Period in north-west Romania. Journal of Quaternary Science 33(6):677-688.

See Appendix C for the PDF of the published document and Appendix D to see permission from the publisher.

59 JOURNAL OF QUATERNARY SCIENCE (2018) ISSN 0267-8179. DOI: 10.1002/jqs.3044

A guano-derived d13C and d15N record of climate since the Medieval Warm Period in north-west Romania

DANIEL M. CLEARY,1* BOGDAN P. ONAC ,1,2,3 IOAN TANTS AU,! 2 FERENC L. FORRAY,2 JONATHAN G. WYNN,4 MONICA IONITA5 and TUDOR TAMA! S 2 1Karst Research Group, School of Geosciences, University of South Florida, Tampa, FL, USA 2Department of Geology, Babes-BolyaiS University, Cluj-Napoca, Romania 3Emil RacovitSa! Institute of Speleology, Cluj-Napoca, Romania 4National Science Foundation, Alexandria, VA, USA 5Paleoclimate Dynamics Group, Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany Received 14 February 2018; Accepted 25 April 2018

ABSTRACT: A 285-cm core of bat guano was recovered from Magurici! Cave in north-west Romania and analyzed for d13C, d15N and pollen. Guano deposition occurred from AD 881 until 1240 and from AD 1651 to 2013, allowing for the interpretation of summer variations in precipitation and temperature during the Medieval Warm Period (MWP) and the Little Ice Age (LIA). A 2‰ increase in d13C, 1.5‰ decrease in d15N, and the presence of Ulmus, Quercus and Carpinus betulus indicate a warm and dry MWP occurred in the region. The lack of deposition during the beginning of the LIA suggests a possible climate-induced change in prey availability resulting in bats vacating the cave. Variation of d13C values between 25 and 23‰ at AD 1650 (LIA) indicates similar drier À À13 conditions as at the end of MWP. However, a 2‰ decrease in d C values that occurred between AD 1790 and 1900 suggests climate was trending towards wetter conditions at the end of the LIA. From AD 1938 to 2013, d13C values appear to be more inﬂuenced by temperature, indicating that this parameter had a more signiﬁcant effect on carbon discrimination than water availability. Copyright # 2018 John Wiley & Sons, Ltd.

KEYWORDS: bats; paleoclimate; pollen; Romania; stable isotopes.

Introduction The connection between vegetation and guano is such that the carbon and nitrogen isotopic signature is transferred from The Medieval Warm Period (MWP) represents the most recent plant foliage, to insects, to bats, and ultimately preserved in extended warm interval in the North Atlantic region that guano. Pollen can reach guano in one of three ways: (i) by occurred before pre-industrial times (Trouet et al., 2009). pollen ingestion and subsequently defecation by bats, (ii) by In contrast, the Little Ice Age (LIA) was a lengthy period with air circulation/wind transport, and (iii) trapped on bat skin/hair lower temperatures that were experienced mainly in the (Carrion et al., 2006). Northern Hemisphere (Mann et al., 2009). However, the In locations where it can be shown that C4 and CAM plants occurrence, amplitude and duration of these events are absent, and thus most of the vegetation follows the C3 have been found to vary distinctly over different regions. photosynthetic pathway (d13C values typically between 24 Additionally, although paleo-records of these recent major À and 32‰) plant water-use efficiency (WUE) may largely be À 13 climatic events exist for Europe, they are scare within responsible for variation in d C variation of plant biomass. Romania. Therefore, more information is needed to better Plant WUE describes the ratio of net photosynthesis to characterize these climate intervals within this region in terms transpiration (Farquhar et al., 1980; Nobel, 1980). Plants of precipitation, temperature and vegetation dynamics. have some capacity to reduce stomatal conductance and/or Cave bat guano has the potential to be a very useful resource increase photosynthetic capacity, which allows for some for paleoclimate and paleoecological reconstructions. Studies optimization of conditions resulting in higher WUE in using d13Cvalueshaveprovidedinformationrelatedtotheshift conditions of low water availability and/or high CO2 concen- between C3 and C4 plants as well as variation in plant water- trations (Manzoni et al., 2011). In more arid conditions, these use efficiency (Des Marais et al., 1980; Mizutani et al., 1992a,b; plants can minimize water loss (Farquhar et al., 1982) and as Bird et al., 2007; Wurster et al., 2007; Forray et al., 2015; Royer 13 15 a result discriminate less against the heavier C and produce et al., 2017). Guano-derived d Nvalueshavealsobeenused less negative d13C values in foliage. Less water-stressed to reconstruct regional winter precipitation (Cleary et al., 2017) conditions create the opposite effect, wherein little competi- and to assess the anthropogenic and climatic influence on a tive advantage is afforded to plants with high WUE, leading regional nitrogen pool (Cleary et al., 2016). In addition, pollen to higher carbon isotopic discrimination (lower d13C values). preserved in guano has been shown to be a reliable resource for This variation in plant foliage d13C values may be transferred reconstruction of vegetation dynamics (Carrion et al., 2006; to bat guano and can therefore be used to reconstruct the ! Geanta et al., 2012). This type of proxy record is ideal for the hydroclimate within the foraging range of the bat colony study of the MWP and LIA in Romania as deposits have (Forray et al., 2015). ! 13 frequently been found to be as old as AD 800 (Geanta et al., Although d C values of guano have been used more 2012; Onac et al., 2014) with guano accumulation often frequently in paleoclimate studies, recent work has demon- occurring through parts of the LIA (Forray et al., 2015). strated that d15N values can provide information related to changes in precipitation (Cleary et al., 2016, 2017) and ÃCorrespondence: Daniel M. Cleary, as above. E-mail: [email protected] vegetation assemblage (Wurster et al., 2017). Because

60 2 JOURNAL OF QUATERNARY SCIENCE nitrogen pathways are more complex than those of carbon, Speleogenesis proceeded in the fossiliferous limestones nitrogen isotopic composition can alternatively be used as an (Cozla Formation) of Eocene–Oligocene age that outcrop on integrator of the local nitrogen cycle (N-cycle) rather than as the Purcare! t-BoiuS Mare Karst Plateau in the north-east part of a simple tracer (Robinson, 2001). In this context d15N values the SomesS Plateau (Onac and Todoran, 1987; Bucur et al., in guano can be associated with shifts between an open and 1989; Prica,! 2001). closed N-cycle. In environmental conditions under which a The climate of the region can be described as temperate- multi-proxy approach suggests that climatic or environmental continental with mean annual temperatures of 7.4 ˚C in the factors control the amount of nitrogen in the system, higher altitudes (>300 m a.s.l.) and 8.5 ˚C at lower elevations. d15N values of guano can be used as a paleoclimate proxy Annual average precipitation is between 700 and 900 mm in (Cleary et al., 2016). the region (Sandu et al., 2008). The current climatic regime is In this study, a 285-cm-long core was extracted from a ideal for the main taxa present in the forests within proximity guano heap in Magurici! Cave (north-west Romania; Fig. 1) of the cave. Primary constituents include Quercus petrea, with the aim to use carbon and nitrogen stable isotope values Fagus sylvatica, Carpinus betulus, Populus and Salix (Geanta! (d13C and d15N) along with the pollen record to interpret et al., 2012). Agriculture is more prevalent at lower elevations changes in the vegetation assemblage and the temperature with prominent crops, fields and pastures. The most common and hydroclimatic regime of the MWP and LIA. Records of herbaceous pollen families encountered in the area are temperature and precipitation from the instrumental period of Chenopodiaceae, Plantaginaceae, Caryophyllaceae, Poaceae, climate history are used to confirm or reject how these factors Urticaceae and Apiaceae (Ratiu,S 1966; Dragulescu! and influence d13C and d15N values and if they have local or Macalik, 1999). regional significance. The 285-cm guano accumulation is located in the Circular Room (See fig. 1 in Johnston et al., 2010) where a large nursing bat colony is currently present. The underground Background climate conditions in this sector of the cave are characterized Study area by a mean annual temperature of 11.8 ˚C and relative humidity ranging between 85.6 and 98.5% throughout the Magurici! Cave is located in nort-west Romania (Fig. 1) and year (Borda and RacovitSa,! 2000–2001). The stable climatic consists of 543 m of galleries with a total vertical range of conditions documented in the Circular Room are due to the 30 m (Borda et al., 2004; Onac and Tama! s,S 2018). unique cave morphology, wherein a mud bank at the

Figure 1. Map of Romania with circles indicating locations referred in the text: Taul! Muced (1; Feurdean et al., 2011a,b), Magurici! Cave (2); Calimani! Mountains (3; Popa and Kern, 2009), Cluj Napoca (4), Lake Ighiel (5; Haliuc et al., 2017), Zidita! Cave (6; Forray et al., 2015), and Gaura cu Musca! Cave (7; Onac et al., 2014). The locations of Czech Republic (8; Brazdil et al., 2002) and Budapest (9, Kiss et al., 2011) studies, and position of Romania (red outline) within Europe are indicated on the inset.

61 A GUANO-DERIVED d13C AND d15N RECORD 3 entrance of the gallery constricts the passage way and limits a sum of ca. 300–400 pollen grains was reached. The airflow into the deeper parts of the cave. This could explain frequencies of pollen for each taxon were calculated as the continued presence of the bat colony within the Circular percentages of the total sum of arboreal pollen (AP) and non- Room between April and September as the existing climatic arboreal pollen (NAP). The nomenclature for vascular plants regime is ideal for bats. Common species present in Magurici! follows Flora Europaea (Tutin et al., 1964–1980). Microscopic Cave are Myotis myotis, Miniopterus schreibersii and Myotis charcoal particles were also counted to reconstruct the fire blythii (Borda et al., 2004). history. The results are presented as a percentage pollen diagram reconstructed in the Tilia software version 2.0.41 Core description (Grimm, 1991). Guano cored for this work was completed within 50 cm of the core used by Johnston et al. (2010) and Geanta! et al. Results (2012). The 271-cm core from the aforementioned studies has a largely similar lithostratigraphy to the guano analyzed here, %N and %C content of guano which can be described as loose disaggregated insect remains There is a 8% decrease in %C between 0 and 96 cm depth for the upper 236 cm of the core. Visually this younger (36.4–28.1%), after which values stabilize around 34% material appears as dark brown guano. In the previous work, (average %C: 31.5; Fig. 2). In contrast, excluding minor coring was completed until a silty clay sequence was reached fluctuations, %N appears to be stable throughout the (Johnston et al., 2010). However, in this study a silty clay sequence (average %N: 10.6). There are two notable minima layer at 236–241 cm was cored through revealing an addi- in %N that occur at 259 (8.5%) and 280 cm (8.1%), tional 45 cm of guano. Below this detrital layer, guano is suggesting a possible loss of nitrogen at these depths. These more compact and drier than in the upper section of the core. minima are mirrored in the %C plot by values of 24.3 and 21.0%, respectively. Methods d13C and d15N of guano Radiocarbon The entire d13C time series features values that fall between An accelerator mass spectrometer at the Poznan Radiocarbon 26 and 21‰ (Fig. 3), suggesting that plants with a Laboratory (Poland) was used to measure the radiocarbon age À À C photosynthetic pathway have been dominant in the of 20 samples of bulk guano. The ages obtained are in 3 vegetation assemblage since AD 881. During the MWP stratigraphic order, although sample MG-15 was contami- the d13C values are the lowest for the entire core ( 26‰), nated with younger organic material and thus omitted from À before trending towards less negative values at AD 1076. the constructed age–depth model (Cleary et al., 2017). Due This course continues until the beginning of the hiatus at to associated errors with the radiocarbon dating ( 1 year for 13 Æ AD 1240 where d C reaches 24.3‰ (Fig. 3). There are a modern samples and 25–30 years for pre-modern samples), À Æ few slight differences between the two time series as the expressed years are not exact dates but rather d15N values first increase by 1‰ between AD 881 and approximations. 933, whereas d13C values lack variation until AD 970. After this interval values decrease and plateau at 12.4‰ (AD Isotopic and elemental analysis 962–1052) before briefly trending towards more positive After coring, sampling was completed at 1-cm resolution. d15N values at AD 1076. Sample preparation followed the method described in detail Following the lengthy hiatus that starts just after the by Forray et al. (2015). Guano samples of 1–2 mg were beginning of the LIA, deposition resumed at AD 1651. The weighed from each aliquot and then measured for d13C, d13C values following this interval are relatively stable until d15N, %C and %N using a Costech ECS4010 Elemental the end of the LIA (d13C: 24.9 to 22.9‰; mean: À À Analyzer coupled with a Delta V Advantage Isotope Ratio 23.9‰). However, two notable variations occur during À Mass Spectrometer (Thermo Fischer Scientific) at the Univer- this apparent stability. The first is the short increase of 1‰ sity of South Florida Stable Isotope Laboratory. A protein in d13C values between AD 1651 and 1687 that is followed standard B2155 (d13C: 26.98‰, d15N: 5.94‰, %C: 46.5, % by a rapid approach to more negative values at AD 1696 À 13 N: 13.3) and a glutamic acid (internal standard: d C: ( 24.6‰). The second trend visible in this interval is the À 13 16.5‰, d15N: 6.28‰, %C: 41.37, %N: 9.54) were used gradual increase in d Cvaluesfromthisminimumtomore À À 13 during analysis. B2155 and IAEA-C7 were used to calibrate positive values at AD 1785 (d C 23.1‰). This is ¼À 13 the d13C value of the glutamic acid. Estimation of the followed by another peak at AD 1821 (d C 23.3‰) 13 ¼À precision of analysis (d15N: 0.08‰; d13C: 0.04‰) is based on before a 80-year decrease in d C values to 24.5‰ replicate internal standards during each analysis. punctuates the end of the LIA (AD 1850). Next, there is a rapid increase in d13C values until AD 1938 where the Pollen preparation most positive value is achieved (d13C 21.4‰). After this ¼À maximum peak, two sudden drops mark the return to lower Palynological work was previously completed on a different d13C values, which remain below 25‰ until AD 2012. Magurici! guano core (see Geanta! et al., 2012), but did not 15 À The d N values display a gradual increasing trend from capture the lowermost part of the deposit. Samples for pollen AD 1651 until 1850. After this interval, there is a prolonged analysis were taken at 1-cm intervals from this section located stability before a drastic decrease in d15N values at AD between 250 and 285 cm. Due to the samples being entirely 1960 (Cleary et al., 2017). organic material, a simplified procedure was used to isolate 3 pollen grains. One cm of each sample was treated with Pollen and microcharcoal NaOH (30%) solution, centrifuged and washed repeatedly with distilled water. This in effect removed humic acids and Upon completion of palynological analysis, two local organic matter, excluding pollen and spores. Samples were pollen assemblage zones (LPAZs) were defined in the lower then staged on glass thin slides and pollen was counted until 35 cm of guano (Fig. 4). The first occurred during the MWP

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Figure 2. Variation of %C and %N content with depth. Silty/clay layer is represented by a brown bar and dotted lines indicate average values for each series.

(AD 899–1168; LPAZ 1), whereas the second encompasses Apiaceae, Rubiaceae, Ranuculaceae and Scrophulariaceae. a short period (AD 1651–1700; LPAZ 2) within the LIA. In LPAZ 2 there is a decrease in most of the aforemen- Analysis of the two LPAZs suggests that hiatus (AD tioned forest taxa with the exclusion of Fagus sylvatica, 1240–1650) corresponds to signiﬁcant changes in the which appears to expand greatly. There is also a signiﬁcant pollen record, and thus probably also in the vegetation increase in Poaceae during this interval. Other herbaceous cover. The primary forest constituents in LPAZ 1 were plants appear to continue at the approximate same percen- Betula, Alnus, Ulmus, Quercus, Carpinus betulus and Fagus tages across the LPAZs, although Asteraceae and Plantago sylvatica with the prominent herbaceous families being lanceolata become more prevalent.

Figure 3. The d13C (blue) and d15N (inverted axis; red) values from AD 881 to 2012 for Magurici! Cave guano. d15N data are from Cleary et al. (2017).

63 A GUANO-DERIVED d13C AND d15N RECORD 5 Pollen diagram for the lower 35 cm of the guano core. Figure 4.

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Figure 5. Comparison of d13C (A) and d15N (B) values from Magurici! guano to d13C of Gaura cu Musca! guano (C; Onac et al., 2014, 2015), depth to water table at Taul! Muced (D; Feurdean et al., 2015), Ti inﬂux into Lake Ighiel (E; Haliuc et al., 2017), temperature anomaly (F; Popa and Kern, 2009) and the 10-year running mean of reconstructed precipitation totals in Europe (G; Buntgen€ et al., 2011) from AD 800 to 1300 (diamonds on the d15N series indicate samples with low %N).

Although there was an expansion of Fagus sylvatica, the Discussion proportion of tree pollen decreases across LPAZ 2 while Suitability of guano for d15N analysis herbaceous plants increase. The entire period is characterized by low microcharcoal percentages, except for the level from The d15N values of bat guano can be altered after deposi- 258 cm (AD 1675) to 260 cm (AD 1670). tion through denitriﬁcation, which preferentially removes

65 A GUANO-DERIVED d13C AND d15N RECORD 7

14N, resulting in a 15N-enriched residual guano. Due to the precipitation records (Fig. 5C,F,G) that are not visible in the range of the enrichment factor associated with this process Magurici! d13C record (Fig. 5A). The absence of these events in (28–33‰; Robinson, 2001) and the inability to determine this record could be explained by the lower resolution in ages the quantity of N that was denitrified, it is not possible to of the Magurici! core. It is also possible that less rain and higher account for the amount of decomposition that may have summer temperatures occur in the Magurici! Cave region in occurred. Other enriching processes such as ammonia- contrast to the Zidita! Cave, due to its lower elevation and volatilization have been shown to affect only urea and not landscape openness. the nitrogen isotopic composition of bulk guano (McFarlane The presence of Ulmus, Quercus and Carpinus betulus is et al., 1995). The degree to which denitrification may have consistent with this interpretation as they are reliant on altered d15N values can be screened by observing warm and dry conditions (Abdi et al., 2009; Feurdean the variation in %N with depth. Typically, denitrification et al., 2011a,b). Although Fagus sylvatica and Alnus,by increases with age, resulting in a trend towards lower %N contrast, prefer wetter conditions (Peterken and Mountford, values with depth (Bird et al., 2007; Cleary et al., 2016). If 1996) the aforementioned species are typically confined to %N shows little variation and values are near the value for lower elevations and are low pollen producers. Therefore, modern guano accumulation, we may consider the associ- their relatively higher percentages at this mid-elevation site ated guano as acceptable for use in interpretation of d15N suggests that there were probably larger forests of these values as a proxy for paleoclimate, anthropogenic influence taxa at low elevations due to the warmer and drier overall and trophic position (Cleary et al., 2016). climatic regime. The %N in the MC core shows little deviation from near Both this guano pollen record (Fig. 4) and that of Geanta! surface values between 0 and 240 cm depth (Fig. 2). Beyond et al. (2012) indicate that F. sylvatica forests began to expand 240 cm depth, there are intervals of %N values that remain AD 1100. This is a highly competitive species that thrives high, but there are a few instances of anomalously low %N. under wetter conditions and cooler summers (Kutzbach and The associated d15N values for the guano with two low %N Webb, 1993). However, the gradual increase in d13C values values between 250 and 285 cm (Fig. 2) are considered here (decrease in d15N) suggests drier conditions before the hiatus. but their interpretation should be treated with caution. It is likely that the region was progressing towards less water In contrast, the %C values of guano display moderate availability, although the cooler conditions led to a decrease variation throughout the core. However, there are no major in the warm reliant taxa (Ulmus, Quercus and Carpinus diagenetic processes that result in fractionation of carbon betulus). The dominance of F. sylvatica indicates that isotopes in guano, with only a 0.8‰ enrichment between precipitation was still at an amount that allowed for the diet and animal feces (Des Marais et al., 1980). Even when % expansion of this species, although the greater impact on this C is low, the d13C values of the corresponding guano are change in vegetational assemblage is probably due to the interpreted to reflect the original value upon deposition shift to cooler conditions. (Forray et al., 2015). Therefore, because no shifts between C3 13 and C4 plants were expected in the region, the d C values Little Ice Age from the entire series are appropriate for interpretation of past water-use-efficiency. Based on the gap between the interpolated radiocarbon ages at 268 and 269 cm in our age model (Cleary et al., Pre- and Medieval Warm Period 2017), a depositional hiatus is interpreted to occur between AD 1240 and 1651. The absence of guano could be the There is a contrast in trends between the carbon and nitrogen result of (i) a flash flood within Magurici! Cave preventing isotopic values from AD 881 to 940, where d13C values are access to the Circular Room or (ii) a change in climatic relatively stable and d15N values become progressively more conditions just before the onset of the LIA (AD 1250) positive. This is probably related to the fact that the first five wherein the bats left the cave. Climate would seem to be a values of d15N from the base of the core show low %N more viable explanation as there is no evidence of a flood (7.1–9.9%; Fig. 2) that could indicate a possible alteration of (associated silt or clay deposits). Additionally, isotopic the nitrogen isotopic composition. This alteration can occur in (Fig. 5) and palynological results (Fig. 4) also suggest that older samples, when denitrification and/or ammonia volatili- the climate was transitioning towards drier and cooler zation results in the guano becoming 15N-enriched. The conditions at AD 1075 and this trend continued until AD increasing d13C values and decreasing d15N isotopic records 1240. Therefore, the cause of this hiatus (AD 1240–1651) since AD 1075 suggest that the trend in climate was towards may more likely be associated with this change. The gap in drier conditions (Fig. 5A,B). The same event is visible in the this record covers more than half of the LIA, representing a lower d13C values from the Gaura cu Musca! guano record lengthy period with no bats roosting in the Circular Room (Fig. 5C; Onac et al., 2015), deeper water table in Taul! Muced (Johnston et al., 2010). The fact that the hiatus occurs (Fig. 5D; Feurdean et al., 2015), and lower Ti count at Lake contemporaneously with the presumed timing of the LIA in Ighiel (Fig. 5E; Haliuc et al., 2017). However, the initiation of Europe provides further evidence of the climate event in the drying trend appears to begin only around AD 1110 at north-western Romania (Johnston et al., 2010; Geanta! Gaura cu Musca! and Taul! Muced (Fig. 5C,D). Therefore, the et al., 2012). The absence of the bats may be related to the onset of this drying event appears to have occurred later at cooler conditions associated with the early parts of the LIA more southern and eastern locations in Romania. These results (Geanta! et al., 2012), which would have reduced the are consistent with a drier Europe during the MWP, with availability of prey for the bats (Dietz et al., 2006). With precipitation values fluctuating approximately 80 mm around less food present, the bats would probably have vacated Æ 220 mm between AD 1050 until AD 1250 (Fig. 5G; Buntgen€ Magurici! Cave for a region that experienced milder climatic et al., 2011). With mean annual rainfall at 100–250 mm, this conditions over this period. Based on the age of the guano, can be classified as a semi-arid environment (Dean, 2004). bats probably returned to the cave when conditions became There are also a few wet events during this semi-arid period warmer and wetter around AD 1651 (Fig. 3). Although we that occur contemporaneously around AD 1100, AD 1168 cannot infer the climate of the LIA due to the absence of and AD 1142 in the Calimani,! Gaura cu Musca! and European guano for this interval, the d13C values just before the

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Figure 6. Comparison of d13C values from Magurici! guano (A), Ti inﬂux into Lake Ighiel (B; Haliuc et al., 2017), d13C of Zidita! guano (C; Forray et al., 2015), depth to water table at Taul! Muced (D; Feurdean et al., 2015), precipitation values from southern Czech Republic (E; Brazdil et al., 2002), 10-year running mean of reconstructed precipitation totals in Europe (F; Buntgen€ et al., 2011), temperature anomaly for Calimani! Mountains (G; Popa and Kern, 2009), and the temperature record in Budapest, Hungary (H; Kiss et al., 2011) from AD 1600 to AD 2012. Brown bar at AD 1712–1715 represents the occurrence of the silty clay layer. depositional hiatus suggest a trend towards drier conditions. Feurdean et al., 2011a,b). Cool and dry conditions may have The interpreted climate from the Magurici! guano record at AD been less hospitable to bats in this region of Romania in 1651 adds to previous work which found alpine areas of comparison with the Mada region (Fig. 1; locality 6) where Romania to be cooler during the LIA (Popa and Kern, 2009; there was a gradual transition to the LIA (possibly due to

67 A GUANO-DERIVED d13C AND d15N RECORD 9 milder conditions) without the cessation of guano deposition wetter climatic conditions. This agrees with other Romanian (Forray et al., 2015). A possible reason for these contrasting studies that found wetter conditions in the Apuseni Mountains conditions between the Magurici! and Mada regions could be (Forray et al., 2015; Cleary et al., 2016), Taul! Mare-Bardau! due to the northern areas being on the rain shadow side of the peat bog (Cristea et al., 2014), and Taul! Muced (Feurdean Carpathians. et al., 2015). Temperature records from the Calimani! Moun- Following AD 1651, forests were still dominated by Fagus tains and Hungary indicate climate was progressing towards sylvatica in addition to a larger Poaceae component (Fig. 4). warmer conditions (Popa and Kern, 2009; Kiss et al., 2011). This suggests that this vegetational assemblage probably Increased precipitation in Romania is also consistent with the persisted through the LIA and continued to AD 1700. The relatively higher precipitation throughout Europe, which d13C values indicate that the hydroclimate was relatively stable transitioned towards a wetter climate as well (Fig. 6F; between AD 1651 and 1900. However, a brief drying trend Buntgen€ et al., 2011). did occur from AD 1651 to 1687 that was followed by a rapid There is debate over the precise initiation and demise of shift to wetter conditions at AD 1696. Following this interval the LIA in Europe, with many studies concluding differing and until AD 1785, the d13C values progressively became less intervals of time (Matthews and Briffa, 2005). With respect to negative, suggesting that climate was drier for nearly 100 years. Romania, the LIA appears to have covered AD 1370 until This interpretation is consistent with the findings in the Rodna AD 1840 in the Calimani! Mountains (Popa and Kern, Mountains (1360 m a.s.l.) where the water table in a swampy 2009). In the Apuseni Mountains (western Romania), Forray lake was much lower during this time (Feurdean et al., 2015; et al. (2015) found this event to begin at around AD 1200 Diaconu et al., 2017). Popa and Kern (2009) also found this and persist until an abrupt termination between AD 1870 and interval to be drier in the Calimani! Mountains ( 1820 m 1900. Previous studies on Magurici! and Gaura cu Musca! a.s.l.), opposed to other areas of Europe that featured cooler guano (Geanta! et al., 2012; Onac et al., 2015) determined and wetter summers (Bradley and Jones, 1993). the onset of the LIA at AD 1240 and 1285, respectively, with Notably, the silty-clay layer interbedded within the guano conclusion at AD 1810 in the first site. However, it is pile (AD 1712 and 1715) occurs within this period of difficult to interpret the climatic conditions through the pollen relatively stable climate. Its occurrence probably suggests the record at this time due to periods of substantial deforestation. presence of a lake originating from increased precipitation A better indicator of the LIA termination in the region is the that temporarily submerged the lower 45 cm of the guano d13C record. Here we interpret the demise of the LIA to have pile within the Circular Room. Previous studies have also occurred at AD 1892 where a clear transition to drier linked the occurrence of detrital layers to wetter periods conditions occurred. The drier conditions are consistent with during which cave passages could have been partly or totally the expansion of Quercus and decrease of Fagus sylvatica inundated (Forbes and Bestland, 2006; Onac et al., 2014). found by Geanta! et al. (2012). During the interval when the silty-clay accumulated in Magurici! Cave, the weather appears to have deteriorated throughout many parts of Europe causing severe flooding 1935 to Present events (Marusek, 2011). The suggested increased precipita- Following the cessation of the LIA, d13C values indicate that tion for the interval of AD 1712–1715 is contemporaneous climate shifted rapidly towards drier conditions until AD with the wettest period in the southern Czech Republic 1938. The extremely low values between AD 1935 and 1939 (Brazdil et al., 2002; Fig. 6E). This interval also coincides suggest this was one of the driest periods experienced at the with one of the higher reconstructed precipitation values site. Severe droughts are recorded in other regions of Eastern (Buntgen€ et al., 2011; Fig. 6F). The event would probably Europe (Wilhite et al., 2000; Buntgen€ et al., 2011; Ionita have resulted in the ceasing of guano deposition due to the et al., 2015) and agree with the drier conditions suggested by development of a temporary lake within the chamber. The the d13C record of Forray et al. (2015) in the south-eastern reason the lower 45 cm of guano was not washed away Apuseni Mountains (Fig. 1; locality 6). Additionally, this event probably relates to the absence of fast moving/turbulent water follows a large increase in charcoal accumulation in the during this event. Therefore, the lower part of the sequence Rodna Mountains at AD 1920 and higher values overall was preserved when guano deposition resumed. This was between AD 1900 and 1950 (Feurdean et al., 2015). The confirmed by the fact that radiocarbon ages in this section of increased burning suggests warmer and drier conditions guano were found to be in stratigraphic order, lacking any during this interval, consistent with the d13C values of this depositional hiatus. study and historical records (Sandu et al., 2008) 13 The d C values indicate that the climate towards the end However, in disagreement with other climate reconstruc- of the LIA (AD 1785–1850) in north-west Romania was tions for Romania are the progressively wetter conditions that gradually becoming wetter. There is a notable wet period that occurred since AD 1938 inferred from the Magurici! occurs from AD 1785 until AD 1811 that apparently affected d13C record (Fig. 6A). The d15N record from this core also multiple areas in Romania as well as Eastern Europe. It suggests a drier winter climate during this interval (Cleary represents the wettest interval at Lake Ighiel since AD 1600 et al., 2017). While the d13C values suggest wetter conditions (Fig. 6B) and an increase in precipitation also occurred in the since AD 1938, this is highly unlikely due to the aforemen- southern Czech Republic (Fig. 6E). This also corresponds with tioned reduced precipitation across other European regions. a shift towards lower temperatures in the Calimani! Mountains This discrepancy can be explained by a shift in the control of (Fig. 6G) and in Hungary (Fig. 6H). Although European plant WUE within the region. Although the d13C values differ precipitation is trending towards drier conditions since AD from both local (Fig. 7) and European (Fig. 6E,F) precipitation 1785, precipitation values remain relatively high between AD records, there are similarities between the Magurici! record 1790 and 1800 (Fig. 6F). Additionally, the drying trend in the and the warming trends of European temperature reconstruc- European precipitation reconstruction between AD 1810 and tions (Fig. 6H,G) and local soil temperature (Fig. 7). Increas- 1823 is contemporaneous with a return towards drier ing temperatures can influence evaporative demand, therein conditions at Magurici! and Lake Ighiel. controlling water available to plants during the growing 13 The d C values over the following 80 years (AD season (Seibt et al., 2008). If temperatures reach a sufficient 1823–1892) indicate that there was a sustained trend towards magnitude this climatic parameter could become a more

68 10 JOURNAL OF QUATERNARY SCIENCE

Figure 7. Comparison of d13C guano record from Magurici! Cave to temperature and precipitation at Baia Mare Meteorological Station, 31 km north of the cave (data from Klein Tank et al., 2002). important factor influencing plant WUE than the amount of Conclusions water received during the growing season. Although infre- These results indicate that the MWP and LIA had a substantial quently considered, there is evidence of mean annual impact on north-west Romanian climate. The d13C record temperature having a significant impact on carbon isotope from Magurici! guano suggests the MWP to have occurred from discrimination in alpine areas (Xu et al., 2015). The atmo- AD 881 until AD 1240, consistent with other studies in the spheric and soil temperatures in the region reaching maxi- region (Feurdean et al., 2015; Onac et al., 2015). The inferred mum values of 19 and 22 ˚C could represent this magnitude drier conditions during this interval agree with other records in (Fig. 7). Because temperature is one of the most critical Romania and Europe. While the timing of the LIA in Romania factors that controls plant growth (Xu et al., 2015), and the appears to vary with location within the Carpathian Moun- primary forest constituents have been trees that prefer cool tains, the presence of the hiatus within our record suggests an and wet conditions (F. sylvatica; Geanta! et al., 2012) it is initiation at AD 1250. From AD 1600 until the conclusion of plausible that a warming climate could become the most this climatic event, d13C values suggest progressively wetter influential on plant WUE. The soil temperature, at Baia Mare conditions. The abrupt shift towards drier conditions at AD meteorological station, shows a positive and significant trend 1893 is interpreted here to signify the termination of the LIA in (99% significance level) over the last 40 years (Fig. 7), while the region. From AD 1938 to 2012, the d13C values suggest the total precipitation amount shows no trend. 13 that temperature had a greater effect on plant WUE than Therefore, we interpret the d C record to indicate a precipitation. The addition of these significant findings to other warming trend beginning at AD 1938 until AD 2012. During guano studies indicates that the proxy will be valuable in this interval the climate in the region during the growing future studies attempting to decipher past climate and season became progressively warmer, consistent with other environments. Romanian and European observations and reconstructions (Ionita et al., 2016). Within the area around Zidita! Cave, plant WUE continued to be controlled by water availability (Forray Acknowledgements. We thank A. Giurgiu who helped during coring et al., 2015), which could be due to its position at a higher and initial sampling activities. This research was supported through Romanian CNCS grant PN-II-ID-PCE 2011-3-0588 to B.P.O. altitude (410 m a.s.l.) within the Apuseni Mountains where annual temperatures are lower. Alternatively, Magurici! Cave Abbreviations. AP, arboreal pollen; CAM, crassulacean acid metabo- lies at a lower elevation onto the SomesS Plateau, where lism; LIA, Little Ice Age; LPAZ, local pollen assemblage zone; MWP, temperatures are comparatively higher during the growing Medieval Warm Period; NAP, non-arboreal pollen; WUE, water-use- season. The inferred warming trend after AD 1938 at Magurici! efficiency. is consistent with the temperature increase at Calimani! Mountains (Fig. 6G) and Budapest (Fig. 6H). Thus, temperature References must be considered as a possible influence of plant WUE in Abdi E, Majnounian B, Rahimi H et al. 2009. Distribution and tensile 13 future guano-derived d C studies, particularly with respect to strength of Hornbeam (Carpinus betulus) roots growing on slopes samples deposited after the industrial revolution. of Caspian Forests, Iran. Journal of Forestry Research 20 : 105–110.

69 A GUANO-DERIVED d13C AND d15N RECORD 11

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