“Advancement in Speleothems Petrography and Microstratig- Raphy As Proxies of Climate and Environmental Changes”

“Advancement in speleothems petrography and microstratigraphy as proxies of climate and environmental changes”

VALENTINA VANGHI

(ORCID: 0000-0002-9412-6602)

BSc. Natural Science (Hons.) (University of Florence, Italy)

MSc. Quaternary Geology (University of the Basque Country, Spain)

MSc. Human Evolution (University of Burgos, Spain)

A Thesis submitted in fulfilment of the requirements for a degree of

DOCTOR OF PHILOSOPHY

March 2018

School of Environmental and Life Science

Faculty of Science and Information Technology

University of Newcastle, New South Wales, Australia

Statement of Originality

I declare this Thesis contains no material which has been accepted, or is being examined, for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to the final version of my thesis being made available worldwide when deposited in the University’s Digital Repository, subject to the provisions of the Copyright Act 1968 and any approved embargo.

Acknowledgement of Collaboration

I hereby certify that the work embodied in this Thesis has been done in collaboration with other researchers, or carried out in other institution. Below I have outlined the extent of collaboration, with whom and under what auspices.

John Hellstrom (co-supervisor of this PhD thesis) and Petra Bajo from the University of Melbourne are acknowledged for training me and helping in the U-Th dating procedures presented in chapters 3 and 5. Russell Drysdale, also from the University of Melbourne, is acknowledged for training me in the art of IR-MS stable isotopes analyses presented in chapter 5. Daryl Howard from the Australian Synchrotron (Melbourne) contributed to the data acquisition of SR-µXRF analyses presented in chapter 3. Gerda Gloy from the Bruker Nano Analytics (Brisbane) helped with the µXRF analyses presented in chapter 3. Floriana Salvemini from Australian Nuclear Science and Technology Organisation – ANSTO, assisted at the neutron tomography experiment presented in chapter 4. Vladimir Luzin from ANSTO contributed to the acquisition and processing of neutron diffraction data presented in chapter 4. Jitendra Mata from ANSTO assisted with the small angle neutron scattering analysis and data processing presented in chapter 4. Sandro Montanari from the Osservatorio Geologico di Coldigioco assisted during the fieldwork at Frasassi cave and helped with the interpretation of the data.

Acknowledgement of Authorship

I hereby certify that the work embodied in this Thesis is my own work, conducted under normal supervision. I, Valentina Vanghi, was the primary investigator and lead author of all manuscripts presented in this Thesis.

------(Silvia Frisia) (Valentina Vanghi)

Dedicated to M3

Acknowledgements

This research project has been fully funded by the Australian Government within the Endeavour Award Postgraduate Scholarship program. I am therefore infinitely grateful to the Australian Government for giving me the opportunity to do a PhD and spend 4 wonderful years in this beautiful country that I now call home.

I also acknowledge the financial support, during the last two years of my PhD, through a Post- graduate Research Award from the Australian Institute of Nuclear Science and Engineering (AINSE).

My deepest and sincere gratitude goes to Silvia Frisia and Andrea Borsato my principal super- visors at the University of Newcastle, because during these four years I received a great super- efficient supervision and help every time I needed it. I felt always supported, encouraged and motivated and I have been working in a positive environment, which helped me a lot to get through all the typical ups and downs of a PhD. Thanks for having trusted me for this postgraduate project. A personal thanks for Silvia that, apart of being very competent in her job as professor and students supervisor, for me has been a friend and a point of reference. I should probably say an downunder-mum. Thanks for all the happy moments, the dinners at your place, our conversa- tion about life and Italy (I will never, never forgot your sincere and wise advices) and all the lunch-boxes you prepared many times for me. Andrea, un grazie personale also for all the laughs and funny moments shared together and especially, for all the speleo-adventures! I had so much fun mamma mia!

I am also very thankful to Russell Drysdale for all the (tons) of isotopes analyses I could do at his lab at the University of Melbourne, during these four years. Thanks Russ for being always so welcoming, friendly and helpful; I have always felt like one of your PhD student.

Thanks also to John Hellstrom (my co-supervisor) and Petra Bajo from the University of Mel- bourne for their help during the U-Th dating and for running the age-models.

I am also grateful for the support and supervision received at ANSTO from Floriana Salvemini, Jitendra Mata and Vladimir Luzin.

Thanks to Hugh Dunstan (University of Newcastle) who facilitated access to the fluorescence microscope.

Thanks also to Sandro Montanari from the Osservatorio Geologico di Coldigioco, Sandro Mar- iani, Nereo Preto for their support during the fieldtrips at Frasassi cave and for all the good moments spent together during the speleological campaigns.

Thanks to the Direnzione Regionale, the Soprintendenza Archeologica della Puglia and the municipality of Altamura for giving access and allowing sampling at Lamalunga cave. Thanks to the guys of the Centro Altamurano Ricerche Speleologiche (CARS) for assisting during the field work. Especially thanks to Giovanni Ragone for being very nice and welcoming with us.

I have to sincerely thanks all my uni colleagues. First of all Emma and Andrew, because they have been the first ones I met at Uni and they hold a special place inside my heart. I could not ask for better uni friends. Thanks for being so amazing with me and to have shared all the laughs and cries (many). I love you! ° Thanks to Caio for joking with me all the times, making me laugh and being my friend (and thanks for being so patient everytime I asked you questions about computer stuff) ° thanks to the beautiful, genuine and sweetest persons I know, Azadeh and Tina ° Thanks to Ebony for being a friend, for all the nice moments and talks we had and all the adventures we shared together ° Thanks to Loy for its politeness and kindness and for being a friend ° Thanks to my beautiful sweet friend Laura, it is always so nice to have you around uni ° Thanks to Hannah for all the wise advises and for being so nice to me all the time (you cooked lasagna for me once, I will never forget) ° Thanks to Judy and Danielle but also Hannah and Silvia again, for being such inspiring women in academia ° Thanks to Olivier pour être toujour tres gentil et simpa et pour parler en français avec moi parce que c’est tres important por moi.

Then I want to thanks my Newcastle multicultural family of friends, my best friends here. Thanks for your support and craziness and sorry if I behaved a bit like a weirdo in the past 3 or 4 months. Specifically, grazie al mio Sergione a cui voglio bene come ad un fratello e per il cibo provided in momenti di bisogno e no e per l’aiuto informatico ° gracias a Alicia y Alberto por llevar un poquito de España en Newcastle y gracias por animarme durante mis bajones ° Merci a ma amie française la plus belle du monde Clo, parce que je t’adore et tu sais ca ° Thanks to Elaine and Franci my fantastic austro-italian friends for all the good moments shared together, I am so glad I have met you, I feel a Pisano too ° Thanks to Tamara my running buddy and wonderful wise friend, thanks for being such a beautiful friend for me, you are my sunray ° Thanks to Melissa and Emily for all the quality moments shared and all the laughs ° Thanks to Heather and Svana for all the adventures we went together I miss you too so much ° Thanks to Angy e Andre for

all the good moments and for being always so positive and cheerful and radiant like the sun ° Than thanks to Kim, Larissa, Lyle, Amy, Tim, Pinky and many others that I have met here in Oz.

Thanks to all my beautiful flatmates (Elise, Craig, Vic, Naomi, Andy, Nick) thanks for being patient with me during the writing phase of my PhD. Thanks for sharing your life with me and making our house “home-sweet-home”.

Thanks to all my Uni Melbourne friends for considering me one of them and for hosting me at their places all the times I had to come to Melbourne. First of all, grazie to Michela (v.), because, now, you are like a sister for me and because you make me laugh like a crazy all the time we are together ° Thanks to ‘ompa Andre for all the cazzate che abbiamo combinato insieme and some of the most absurd situations we ended up in! Japan docet, ho detto tutto ° Thanks to Coralie because you are unique. Coralie is Coralie and I love you as you are ° Gracias a Arturo y Brenda para acogerme estupendamente en vuestra casa ° Thanks to Petra for being always so nice and patient with me, for all the things you thought me and your precious help

Now it is time to thank to all my Italian friends that even if so far away they always make me feel as if I am still living close to them and they still share their day-to-day life with me. However, I still miss you a lot. Un grazie speciale a Lola e Laura che sono le mie migliori amiche di una vita e nessuno potra’ prendere il vostro posto nel mio cuore. Grazie a Eu, Cosimo e Marias per essermi venuti a trovare a Newcastle e per avermi fatto rituffare nel passato. Grazie a Simo e l’Ali per le nostre skyppate ma veramente grazie a tutti voi per farmi ridere a crepapelle anche da lontano e farmi dimenticare le brutture (poche, per fortuna) della vita. Io senza tutti voi sarei persa. Grazie Marco, Fra, An e tanti altri. Infine grazie anche alla mia cugina-sorella Sofia perche’ ti voglio un bene dell’anima. A tutti gli altri miei cugini e zii, ai miei fratelli Jacopo, Matte e Baby per avermi fatto sentire sempre il calore della nostra bellissima e unita famiglia.

Grazie ad i miei genitori per il supporto che mi hanno sempre dimostrato e per “lasciarmi andare” ogni volta che ho voluto. Questa tesi la dedico a voi con tutto il mio cuore.

Quiero tambien dedicar esta tesi a M. porque aunque ya no sea parte de mi vida, para mi ha sido un modelo de vida y le debo mucho, porque siento que sin el no haria podido empezar todo esto. Merci pour tout, je ne t'oublierai jamais.

Abstract

Stalagmites are acclaimed accurate proxies of climate variability because they can be precisely dated with radiometric techniques and preserve in their chemical and physical properties records of changes at sub-annual to orbital scales. Over the past two decades, stalagmite records have been used to reconstruct global teleconnections and synchronicity of events. At the same time, traditional proxies applications have not advanced as much as the development of new proxies has. The main goal of this thesis has been to fill this gap, by advancing knowledge on the physical aspects of stalagmites and other cave secondary mineral deposits (speleothems) that can be then be directly linked to climate and environmental processes. This main goal has been accomplished by using both conventional optical petrography and innovative, non-conventional methods such as Synchrotron Radiation based micro-XRF (SR-µXRF) and Neutron scattering.

SR-µXRF has been demonstrated to be the most apt technique to complement petrography in the investigation of speleothems, which have been here considered for their association with a unique paleontological finding. In this thesis, a petrographic, morphologic and microstratigraphic study of some coralloid formations both directly and indirectly associated with the complete skeleton of an early Neanderthal man preserved in in Lamalunga cave (Southern Italy) is presented to prove the reliability of this speleothem as archive of palaeo-hydroclimate. The hypothesis presented in this Thesis is that coralloids formation is linked to hydroaerosol generated by the fragmentation of cave drip combined to enhanced apical evaporation. Thus, their sub-micrometre scale layers preserve records of alternating dry and wet periods. Because of the small scale of the microstratigraphic elements, only SR-µXRF could be used to obtain elemental mapping with the desired high spatial resolution. SR-µXRF maps support the genetic hypothesis here formulated, and demonstrates that the incorporation and distribution of trace element and organic material are related to fabric changes. The overarching conclusion of this investigation is that coralloids, despite their small size are one of the best tools we have to reconstruct the climate and environmental context of early humans in Southern Italy. This will contribute to consolidate and improve the link between speleothem science and paleoanthropology and archaeology.

The absence of stalagmites in Lamalunga cave, which may prevent obtaining a long, continuous and exhaustive palaeoclimate record, has led to the exploration of possible analogue in a highly decorated cave at the same longitude in Central Italy. Stalagmites from Frasassi cave have then

been selected and studied because at both Frasassi and Lamalunga locations, modern precipitations have similar δ18O composition, a phenomenon that is here assumed to have been the same throughout the Quaternary. Modern rainfalls isotopic signal variations, in the Italian peninsula, do not show a longitudinal gradient along the Adriatic coast, where both Lamalunga and Frasassi are located. With the aim of producing a stable isotopic record for Frasassi cave that could be used to complement the palaeoclimate reconstruction from Lamalunga coralloids, the first and necessary foundation has been benchmarking on the petrography the geochemical record of Frasassi cave. This ultimately allowed to interpret the δ13C signal of Frasassi stalagmite as reflecting local/regional scale humid/arid cycles, which coincide well with changes in petrography. By contrast, the δ18O signal does not follow petrographic changes and it is here concluded that it does not reflect drip rate variability (infiltration). It is highly probable that the δ18O reflects a mixed signal that of water stored in the aquifer for a relatively long time, “homogenized” through the years, rather than the immediate response to recharge. The interpretation of Frasassi stalagmite record highlights that, as in Lamalunga coralloids, fabrics best identify an immediate response of the speleothem archive to hydrological changes.

Because of their importance, the analysis of speleothem fabrics has been pushed beyond the conventional microscopy and spectroscopy methods by employing for the first time neutron scattering techniques. This can only been regarded as a pilot study within this Thesis, however the preliminary results are promising. The aim was to help recognizing features that may indicate pathways of crystallization. Neutron diffraction data produced a compilation of different preferred orientations for various types of speleothems characterized by diverse fabrics, which hints at an internal organization characterized by various degrees of rotation of the units composing crystals most likely around a common c-axis. Additional information comes from the application of small- angle neutron scattering (SANS), on the same specimens, which reveals the presence of mass or surface fractals. Tentatively, this is ascribed at the presence of nanocrystals. Both results pave the way to a re-evaluation of speleothem crystallization and, thus, incorporation of geochemical data when non-classical pathways of nucleation and growth influenced speleothem capture of climate change.

TABLE OF CONTENTS LIST OF FIGURES ...... IV LIST OF TABLES ...... XI PUBLICATIONS ARISING FROM THIS THESIS ...... XII 1. INTRODUCTION ...... 1

1.1 BACKGROUND ...... 1 1.2 SPELEOTHEMS FORMATION...... 3 1.3 PETROGRAPHY OF SPELEOTHEMS ...... 5 1.3.1 Architecture of speleothems: Fabrics ...... 9 1.3.2 Speleothem architecture: Laminae ...... 12 1.4 SPELEOTHEM CHEMISTRY ...... 15 1.4.1 Stable isotope ratios ...... 16 1.4.2 Trace elements ...... 18 1.5 STUDY SITES ...... 19 1.5.1 Reasons behind the choice of these two sites ...... 21 1.5.2 Frasassi cave system: speleogenesis and significance for Earth’s history ...... 22 1.5.3 Lamalunga cave current research status ...... 25 1.6 THESIS AIMS AND OBJECTIVES ...... 26 1.7 THESIS STRUCTURE ...... 27 1.8 REFERENCES ...... 32 CHAPTER 2. GENESIS AND MICROSTRATIGRAPHY OF CALCITE CORALLOIDS ANALYSED BY HIGH RESOLUTION IMAGING AND PETROGRAPHY ...... 40

2.1 ABSTRACT ...... 40 2.2 INTRODUCTION ...... 41 2.3 GEOGRAPHIC SETTING AND CORALLOID OCCURRENCES IN LAMALUNGA CAVE...... 43 2.4 MATERIALS AND METHODS ...... 47 2.5 RESULTS AND DISCUSSION ...... 51 2.5.1 Microstratigraphy ...... 51 2.5.2 Mineralogy and petrography ...... 55 2.5.3 Mechanisms of coralloid formation at Lamalunga Cave ...... 57 2.5.4 “Aragonite conundrum” in Lamalunga coralloids ...... 59 2.5.5 Microstratigraphic evidences for Lamalunga coralloids formation ...... 60 2.5.6 Mechanisms of formation of Lamalunga coralloids and hydrological implications ...... 62 2.6 CONCLUSIONS ...... 65 2.7 REFERENCES ...... 67 CHAPTER 3. HIGH-RESOLUTION SYNCHROTRON XRF INVESTIGATION OF CALCITE CORALLOID SPELEOTHEMS: ELEMENTAL INCORPORATION AND THEIR POTENTIAL AS ENVIRONMENTAL ARCHIVES ...... 72

3.1 ABSTRACT ...... 72 3.2 INTRODUCTION ...... 73 3.3 GEOGRAPHIC CONTEXT ...... 74 3.4 MATERIALS AND METHODS ...... 76 3.4.1 Morphological and structural characteristics of the coralloids ...... 76 3.4.2 Water analyses...... 77 3.4.3 U-series dating ...... 78 3.4.4 Micro-XRF and SR micro-XRF ...... 78 3.4.5 Principal component analysis (PCA) ...... 79 3.5 RESULTS ...... 80 3.5.1 Water analyses...... 80 3.5.2 U-series dating ...... 80

3.5.3 Petrography, internal structure and morphology of Lamalunga coralloids ...... 81 3.5.4 Greyscale values and fluorescence stimulated microscopy ...... 85 3.5.5 Distribution maps of trace elements: SR-micro-XRF and micro-XRF maps ...... 88 3.5.6 Elemental quantifications for micro-XRF ...... 90 3.6 DISCUSSION ...... 94 3.6.1 Mg and Sr behavior in Lamalunga coralloids ...... 95 3.6.2 Silicon concentration in Lamalunga coralloids and possible origin ...... 96 3.6.3 Uranium, Iron and Bromine concentration ...... 98 3.6.4 Incorporation and concentration of the elements ...... 99 3.6.5 Models of elements incorporation ...... 101 3.6.6 Humid phase (climatic) ...... 101 3.6.7 Dry phase (climatic) ...... 102 3.6.8 Non climatic episodes ...... 102 3.6.9 Greyscale values significance ...... 103 3.7 CONCLUSIONS ...... 104 3.8 REFERENCES ...... 106 CHAPTER 4. NEUTRON SCATTERING PILOT STUDY ON SPELEOTHEM FABRICS COMPLEMENTARY CHARACTERIZATION OR ADVANCE IN KNOWLEDGE OF SPELEOTHEM PROPERTIES? ...... 112

4.1 BACKGROUND ...... 112 4.2 A BRIEF INTRODUCTION TO NEUTRON SCIENCE ...... 114 4.3 BRIEF INTRODUCTION TO NEUTRON TOMOGRAPHY ...... 116 4.4 MATERIALS AND METHOD ...... 116 4.4.1 Textural analysis ...... 117 4.4.2 Porosity analysis ...... 118 4.5 RESULTS AND DISCUSSION...... 119 4.5.1 Speleothem textural analysis (KOWARI) ...... 119 4.5.2 Significance of textural analysis of speleothems ...... 121 4.5.3 Porosity analysis ...... 123 4.5.4 SANS scattering ...... 126 4.6 CONCLUSIONS ...... 130 4.7 REFERENCES ...... 132 CHAPTER 5. CLIMATE VARIABILITY ON THE ADRIATIC SEABOARD DURING THE LAST GLACIAL INCEPTION: THE FRASASSI CAVE CASE STUDY...... 135

5.1 ABSTRACT ...... 135 5.2 INTRODUCTION ...... 136 5.3 FIELD LOCATION AND SAMPLE ...... 139 5.4 METHODS...... 141 5.4.1 Petrographic and fluorescence microscopy ...... 141 5.4.2 U-Th dating and age model...... 142 5.4.3 Stable isotopes analysis ...... 143 5.5 RESULTS ...... 146 5.5.1 Microstratigraphy and petrology ...... 146 5.5.2 U-Th series results ...... 147 5.5.3 Stable C and O isotopes ratio ...... 149 5.5.4 Grey values ...... 150 5.6 DISCUSSION ...... 152 5.6.1 Environmental interpretation of calcite fabric and FR16 δ13C ...... 152 5.6.2 Moisture provenance and interpretation of Frasassi δ18O signal ...... 155 5.6.3 Regional climatic context of δ18O during the glacial inception ...... 157 5.6.4 Possible factors influencing Frasassi δ18O signal ...... 160 5.7 CONCLUSIONS ...... 162

5.8 REFERENCES ...... 164 CHAPTER 6. CONCLUSIONS ...... 170 CHAPTER 7. FUTURE DIRECTIONS ...... 174 APPENDIX I SPELEOTHEM CHRONOLOGY FROM FRASASSI CAVE SYSTEM (CENTRAL ITALY): TIMING OF SPELEOGENESIS, UPLIFT RATE AND PALEAOCLIMATE EVOLUTION...... 179

I. AIM OF THE RESEARCH ...... 179 II. MATERIALS AND METHODS ...... 179 III. DISCUSSION ...... 179 Hypogenic karst processes ...... 179 Frasassi karstic levels ...... 181 Stalagmite growth rates vs δ18O global records: preliminary hypothesis ...... 184 IV. PRELIMINARY CONCLUSIVE REMARKS ...... 185 V. REFERENCES ...... 186 APPENDIX II COMPOSITE Δ13C AND PETROGRAPHIC 196-355 KA RECORD FROM FRASASSI CAVE (CENTRAL ITALY) STALAGMITES: INVESTIGATING DRIVERS OF CALCITE CARBON ISOTOPE SIGNALS ...... 187

I. INTRODUCTION: C ISOTOPE RATIO IN SPELEOTHEMS ...... 187 II. MATERIALS AND METHODS ...... 187 III. RESULTS ...... 188 IV. DISCUSSION ...... 191 Significance of δ13C changes in Frasassi record ...... 191 V. CONCLUSIONS ...... 191 VI. REFERENCES ...... 192

III

List of Figures

Fig. 1.1 Transfer of the geochemical signal from the outside to speleothems growth layers………………………………………………………………………………………………...5

Fig. 1.2 Step-kink-hole model with adsorbed ions shown in black. Refer to the text for more details. Picture taken from Morse and Arvidson (2002)……..……………………………………………………………………………………...6

Fig. 1.3 Different pathways to crystallization by particle attachment. The classical theory of nucleation (De Yoreo and Vekilov, 2003), only accounts for the monomer-by-monomer crystal growth. Sketch tak-en from De Yoreo et al. (2015)…………………………………………………………………………………………….7

Fig. 1.4 Plots of free energy change as a function of nucleus size as envisaged by the classical, one-step and nonclassical nucleation models with intermediate amorphous phase. Plots taken from Lee et al. (2016)…………………………………………………………………………………………….8

Fig. 1.5 Some of the most common fabrics observed in stalagmites…………………………………………………………………………………………….12

Fig. 1.6 (A) Geographical locations of the cave sites object of this study: Frasassi cave and Lam- alunga cave. (B) Giustini et al. (2016) map of spatial distribution of δ18O (‰) of precipitations in Italy……………………………………………………………………………………………...20

Fig. 2.1 A) Location of Lamalunga Cave near Altamura (Southern Italy). B) Plan and schematic cross section of Lamalunga Cave. The orange dotted line represents the plan of the cross section. The locations of the samples here studied (ABS5 and ABS6) are also indicated. ABS3 coralloid, studied in Lari et al. (2015), was sampled in the same chamber of the “Altamura Man” skeleton. Coralloid speleothems are represented with small dots. The original entrance was a vertical shaft now completely filled by limestone debris. The actual entrance to the cave was artificially enlarged. C) View of the hill where the cave opens to the surface (black cross)…………………………………………………………………………………………….42

Fig. 2.2 Different types of coralloid speleothems from Lamalunga Cave. A) The Neanderthal skeleton almost completely coated by coralloids. B) Main gallery showing coralloids that are mostly concentrated on the floor and on the lower parts of the walls, while they are almost absent on the upper parts of the walls and on the ceiling. This boundary is indicated by a dotted black line; C) rounded nodular coralloids developed upwind (yellow arrows represent the direction of the air-flux) on a large stalagmite, D) cylindrical coralloids; E) rounded nodular coralloids; F) branched coralloids………...... 44

Fig. 2.3 Images of samples ABS5 and ABS6. A) Polished slabs; B) complete thin sections in PPL; C) complete thin sections in XPL; D) Schematic interpretation of the principal features observed in the samples. Some crystals boundaries and major margins defining groups of crystals with same c-axis orientation, are represented with thin blue and black lines respectively. Thick black lines visualise the boundaries between the main microstratigraphic growth phases. Orange lines indicate possible hiatuses identified by dissolution/corrosion features. The black arrow in ABS6 panel D, indicates crystals becoming wider moving from the central axis towards the flanks: from fiber-like Ce (Fb) crystals to Ce crystals. Red layers correspond to micritic layers rich in Fe-

oxides. Green rectangles indicate the regions analysed with fluorescent light. In ABS6, organic- rich bands are coloured with different shades of brown depending on the intensity of fluorescence emitted: e.g. darker brown parts are the most fluorescent. On the left side of ABS5 are also indicated the U/Th ages with 2σ errors published in Lari et al. (2015). Fabric codes: Ce = columnar elongated; M = micrite; OM = organic-rich layer; Ms = microsparite. Abbreviations: Fls = flowstone-like; Crl = coralloid; I. Crl = incipient coralloid……………………………………..45

Fig. 2.4 Coupled PPL (parallel polarizers) thin sections and fluorescence maps (2 wavelenghts combined) for ABS5 and ABS6. The corresponding fluorescence and greyvalues line scans were carried out along the dashed vertical lines. Fabrics and major discontinuities for the thin sections are labelled as follows: Ce = columnar elongated; M = micrite; OM = organic-rich layer; MS = microsparite. To facilitate their correlation of greyscale values and fluorescence intensity, diverse fabrics have been highlighted by diverse coloured bands. Blue has two different shades, dark and light, which are intended to reflect higher (++) or lower (+) concentration of organic material (OM) and higher (ff) or lower (f) fluorescence intensity respectively. The presence of clean, compact Ce calcite parts is illustrated as white bands. Hiatuses are marked by black (clearly recognizable gap) and blue (possible gap) dotted lines. Note that the fluorescence intensity scales are inverted………………………………………………………………………………………….48

Fig. 2.5 A) Ensemble of cylindrical coralloid growing on a bedrock fragment collected from the cave floor. The location of sample ABS6 is marked by a white box; Coralloids speleothems can assume many different shapes and forms and their internal structure is commonly highly laminated: B) Cylindrical coralloid with incipient branching termination; C) complex globular coralloid; D) Cone-shaped coralloid which was growing on the Neanderthal skeleton (Lari et al., 2015)………………………………………………………………………………...…………..49

Fig. 2.6 Discontinuities and hiatuses observed in PPL. A) Dark levels in ABS5 formed by four different hiatuses. B) Detail of A) (red rectangle). The flat terminations of some crystals have been corroded with micrite filling the voids (yellow arrow). C) Detail of B) (red rectangle). D) and E) Hiatuses in ABS5 with micritic veils capping the crystals terminations, showing corroded surfaces (blue arrows). F) Finely laminated axial part in ABS6. The darker laminae are due to the presence of micrite and organic-rich particles. G) Detail of F) (red rectangle) showing a corroded surface where, on top, another level of crystals with spherulitic growth started nucleating. H) Irregular micrite layer at the base of ABS6. The red colour is caused by the presence of iron oxides. Scale bars correspond to 0.5 mm for A) and F), to 100 µm for B), C), D), E), H), and G)………………………………………………………………………..………………………52

Fig. 2.7 Thin sections photos taken under PPL (A, C, E) and XPL (B, D, F). A) and B): columnar elongated fabric (Ce) in ABS5. Dark micritic and organich rich layers interrupt the growth of the crystals. C) and D) ABS6 fiber-like crystals with evidence of spherulitic-type growth. The lamina couplets are formed by micrite (M) and organic rich dark layers alternating with clean compact calcite layers. E) ABS6 acute crystal terminations, which suggest a relatively thick film of fluid; and F) ABS6 flat crystal tips and some possible eroded surfaces capped by a dark material conceivably containing organic matter (see text for details). Scale bars are 0.5 mm for A) and B) and 100 µm for C), D), E) and F)………………………………………………………………………………………………...53

Fig. 2.8 A) Sketch representing the processes involved in the formation of coralloid speleothems in Lamalunga cave. When a drop hits a surface inside the cave, it splits in smaller droplets which then bounce and spread in the surrounding. If an air flux (blue arrows) is present inside the cave, hydroaerosol is produced downwind. B) Sketch representing the mechanisms controlling the formation of lenticular dark bands and white isopachous bands. When the concretion is fed by hydroaerosol the water supply is limited (Case 1). Long periods of evaporation (red arrows), helped

by ventilation (indicated by blue arrows), is the principal mechanism controlling the growth of a coralloid. Also the transport of impurities (colloids) by hydroaerosols which get preferentially deposited on the tips, promotes coralloid type of growth by inducing the nucleation of fiber-like crystals of calcite. When, instead, the water supply is relatively higher because is mostly derived by splashing drops and evaporation is reduced, impurities (colloids) are diluted and clean elongated columnar calcite forms (Case 2)……………………………………………………………………………………………….. 55

Fig. 2.9 Sketch illustrating factors controlling Lamalunga coralloids evolution. Arrowed bars illustrate graphically the concept of increasing importance of each factors on the evolution of coralloids morphology. For example, branched and cylindrical coralloids are the result of the combined action of hydroaerosol feeding and evaporation driving to supersaturation with respect to calcite. In addition, branching coralloids likely form when the amount of impurities reaching the growing surfaces is maximum. Impurities, likely carried by hydroaerosols, are preferentially deposited on the convex surfaces and seem to control the growth of fiber-like crystals and their elongation in the direction of growth. Relatively stronger evaporative processes on the protruded parts of the concretion forester the nucleation of fiber-like crystals which tend to grow faster compared to the crystals on the flanks of the coralloid. The flowstone-like morphologies are directly fed by spray produced by nearby drops splashing thus forming a water film on the top of the speleothem. Because of the smoother morphology, flowstones-like coralloid formation is here inferred to be driven by relatively more intense CO2 degassing and lower evaporation than their branching and cylindrical counterparts. Furthermore, a relatively more constant water supply and reduced impurities input at the top would promote growth of elongated columnar crystals…………………………………………………………………………………………..…...61

Fig. 3.1 A) Map of Italy with the location of Lamalunga Cave near Altamura. B) Plan and schematic cross section of Lamalunga Cave. The orange dotted line represents the plan of the cross section. The locations of the samples here studied (ABS5 and ABS6) are also indicated. ABS3 coralloid, studied in Lari et al. (2015), was sampled in the same chamber of the “Altamura Man” skeleton. Coralloid speleothems are represented with small dots. A vertical shaft completely filled by limestone debris clogs the alleged original entrance. The actual entrance to the cave was artificially enlarged. C) View of the hill where the cave opens to the surface (black cross)………………………………………………………………………………….…………73

Fig. 3.2 Polished slabs of the investigated coralloid samples. A) ABS6-A, B and C are part of the same multiaggregate of coralloids. The corresponding planes of cut are also shown (white rectangles). Notice the different plane of cut for ABS6-C that is orthogonal to the other two. B) ABS5 slab. The sample was growing on a broken stalagmite……………………………………………………………………………….……………..75

Fig. 3.3 Synchrotron elemental (Ca, Sr,) concentrations plotted against grey values intensities from the polarised thin section and the fluorescence map (using 2 wavelenghts, blue and green, combined) for ABS5 and ABS6-A. The corresponding fluorescence and greyvalues line scans were carried out along the dashed vertical lines. Fabrics and major discontinuities for the thin sections are labelled as follows: Ce = columnar elongated; M = micrite; OM = organic-rich layer; MS = microsparite. To facilitate their correlation of greyscale values and fluorescence intensity, different fabrics have been highlighted by diverse coloured bands. Blue has two different shades, dark and light, which are intended to reflect higher (++) or lower (+) concentration of organic material (OM) and higher (ff) or lower (f) fluorescence intensity respectively. The presence of clean, compact Ce calcite parts is illustrated as white bands. Hiatuses are marked by solid blue (clearly recognizable gap) lines. Note that the transmittance intensity and Ca scales are inverted…………………………………………………………………………...……………..80

Fig. 3.4 SR micro-XRF maps (Ca, Sr, Fe and elastic signal) of ABS5 compared to the corresponding image of the polished slab (A) and the petrographic thin section under polarized light (B). Note that the thin section was made 2 mm away from the slab plane and thus it shows a slightly different architecture. Sr and Fe are preferentially concentrated along the dark micritic-rich bands on the thin section corresponding to the opaque milky bands on the slab. This corroded surfaces represent moment of no calcite deposition (hiatuses) indicated by Ht A, B and C. The ages (ka) published in Lari et al. 2015 are indicated in (A). Scale bars unit is in ppm for the elements maps and in percentages for the elastic scatter map……………………………………………………………………………………………...81

Fig. 3.5 A) Synchrotron distribution map (Ca and Sr) of thin section ABS6-A compared to the polished slab (a) and to the thin section under parallel polarizers (PPL) (b). Sr concentrates preferentially in the axial part of lenticular-shaped bands that appears opaque on the slab (a) and dark in the thin section (b). Where the growth band is more isopachous, Sr concentration is lower. Sr- rich layers are also present above hiatuses (Ht A, B and C) identified by clear dissolution features (arrows). The three U-Th ages (expressed in ka) are also indicated. Scale bar unit is in ppm. B) Zoom-in of the region indicated by a white rectangle in (A). Parallel (PPL) and crossed polarized (XPL) micrographs compared to Ca and Sr SR micro-XRF maps of the laminated part in ABS6- B. Dark regions in the thin section corresponds to low values for Ca, thus reflecting a relatively high intercrystalline porosity. Translucent parts are instead very dense as shown by high Ca values. Black circles confine regions with low Ca concentration which in some cases are rich in Sr, when also Sr is low they represent voids possibly filled by other elements (white arrows)………………………………………………………………………….…………………82

Fig. 3.6 Synchrotron micro-XRF elemental maps of ABS6-B compared to the corresponding polished slab (A). The elements concentrate mostly where Ca concentration is low and where the laminae appear white under natural light. Sr and Br tend to be more concentrated in lenticular shaped stacks of layers. Fe and Y mostly are concentrate at the base of the coralloid where micrite layers have been observed. U concentration pattern is similar to Sr distribution. Principal hiatuses (Ht A, B and C), are marked by white arrows.. The three U-Th ages (expressed in ka) are also indicated. Scale bars unit is in ppm for the elements maps and in percentages for the elastic scatter map……………………………………………………………………………..……………….84

Fig. 3.7 Synchrotron elements (Ca, Sr, U, Br, Fe and Mn) distribution maps of ABS6-C compared to image of the polished slab (A) and to the micro-XRF intensity images (squared maps on the left side) (Ca, Si, P, Mg and Sr). The tracers mostly concentrate where Ca concentration is low and where the laminae show a white coloration on the slab. The black rectangle on the slab shows the position of the micro-XRF maps. The small yellow rectangle show the region from where elemental concentrations have been quantified. Principal hiatuses (Ht A, B and C), are marked by white arrows. Scale bars unit is in ppm………………………..…………………………………………………………………….85

Fig. 3.8 A) Element distribution maps obtained with the conventional micro-XRF for Mg, Si and Ca in a small region of ABS6-C (yellow insert in figure 3.7). Si has opposite pattern of distribution with respect to Ca and is the element with the higher concentration after Ca. In the opaque bands Si is very high, up to 16% and Mg can reach up to 3%. In compact translucent bands, Si is very low (~ 0.1 %) whereas Mg is around 0.4%. B) Line scans of Ca, Si, Sr and Mg extracted in the central part of the maps (black rectangle) by geometrically interpolating the pixels. Sr and Mg trends show a similar pattern of distribution. Si seems mostly concentrated in grains with a 50- 100 µm diameter……………………………………………………………………………..….87

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Fig. 3.9 Sr concentration along selected growth layers in ABS6-B. The Sr concentration was measured along the seven 100µm width bands visualised by dashed red lines on the Sr map (black/white scale where white represents the max concentration of 2100 ppm and black the minimum of 0 ppm). The graph on the right plots the Sr concentration along the bands centred on the coralloid growth axis. In band 4 the cycles in the central part have a period of 138 ±24 µm………………………………………………………………………………….……………89

Fig. 3.10 Principal component analysis (PCA) for ABS6-B (SR micro-XRF data) and ABS6-C (conventional micro-XRF data). Only PC1 and PC2 were compared in scatter plots because they show the most significant variances. The red circles enclose the elements with significant loadings. GV = greyscale values…………………………………………………………………...…………………………..90

Fig. 3.11 Linear correlations between Sr, Mg, Si and Ca in ABS6-C (micro-XRF data in Fig. 8) from a selected region of interest. The correlations were then calculated for the whole interval and then separately for the translucent (compact) and opaque (porous) calcite end-members………………………………………………………………………………………..…….91

Fig. 3.12 Schematic figure that shows all the principal characteristics of Lamalunga coralloids……………………………………………………………………………...…………….102

Fig. 4.1 Idealised power law scattering curve showing Guinier, Transition and Porod regimes………………………………………………………………………………………...113

Fig. 4.2 Pole figures of speleothems from the neutron textural analyses carried out at the ANSTO KOWARI beamline. See text for the details.…………………………………………………………..……………………………...118

Fig. 4.3 Tensor view 3D representation of the porosity observed in a laminated portion of FR16…………………………………………………………………………………………...122

Fig. 4.4 Histograms of the different kinds of porosity in FR16 analysed by neutron tomography. For each fabric observed in FR16 (compact columnar, open columnar), two histograms have been created accordingly to the presence/absence of organic material and fluid inclusions. On the x- axes are reported volumes ranges of the pores in microns squared……………………………………………………..…………………………………..123

Fig. 4.5 Cross-section of scattering intensity (= neutrons counts on the area of the detector) (I) vs. scattering vector Q. Q = 4π sin (θ)/λ. The scattering vector represents the distance between incident and scattered directions. Q=0 means no scattering of the neutron beam. Dots represent Frasassi samples scattering curves and the lines corresponds to CL9, CS10, MIK7 and TM19 scattering curves. For FR16 P indicates porous calcite, PP = very porous calcite, Tr = translucid calcite……………………………………………………………………………………..……126

Fig. 4.6 Sketch representing a surface and mass fractal and the typical cross section plot that they yeld (curve slope = 3). I is the scatter intensity and Q is the scatter vector (modified from Hammouda (2008))……………..……………………………………………………………...126

Fig. 4.7 FR16 samples cross-section having scattering intensity (= neutrons counts on the area of the detector) (I) vs. scattering vector Q after Power Law model fitting. Q = 4π sin (θ)/λ. The scattering vector represents the distance between incident and scattered directions. Q=0 means no scattering of the neutron beam. Note that curve fitting has to be improved at high and low Q. Black dots represent the Frasassi samples scattering data and the red line is the Porod curve after

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the fitting. P = porous calcite PP = very porous calcite; Tr = translucent calcite…………………………………………………………….…………………………….127

Fig. 4.8 Plot of scattering intensity (= neutrons counts on the area of the detector) vs. scattering vector Q after Power Law model fitting. Q = 4π sin (θ)/λ. The scattering vector represents the distance between incident and scattered directions. Q=0 means no scattering of the neutron beam. Black dots are the scattering data and the red line is the Porod slope after the fitting. Note that curve fitting has to be improved at high and low Q. CL9 is a flowstone from Collalto cave (Italy); CS10 is a dolostone from Costalta cave (Italy); MIK7 is a microbialite; TM19 is laminated stalagmite from Tam Doum Mai (Laos)…………………...……………………………………………………………………...127

Fig. 5.1 A) Geographical location of Frasassi cave system (red dot in the map of Italy). B) Map of Grotta Grande del Vento of Frasassi karstic system. C) Section of the western part of Grotta Grande del Vento (the plane of cut is indicated with the red dotted line in B) and location of where FR16 was collected (Manhattan chamber, V° karstic level). The altitudes of the karstic levels indicated in the section are calculated from the river bed. Map and section of the cave are simplified versions from the original made by Dottori D., Maccio S., Bocchini A., Coltorti M., Novelli A. and Recchioni R. during the speleological campaigns from 1973 to 1985…………………………………………………………………………………………....135

Fig. 5.2 Geologic cross section of Frasassi anticline modified from Galdenzi et al. (2008b). Infiltration water and sulfuric exhalations are indicated by blue and green shaded arrows respectively. Normal and thurst fault lines are indicated by red arrows...... 136

Fig. 5.3 Principal fabrics observed in FR16 (photo in the centre of the figure): compact columnar (dark grey in the sketch of the stalagmite), open columnar (light grey), micrite and microsparite (yellow). On the right of the sketched stalagmite, the photos at parallel and crossed nicols of some of the thin sections. On the left micrographs of the principal fabrics observed under the optical microscope. The red arrow indicate the hiatus separating the micrite fabric to the columnar fabric. Red scale bars in the micrographs correspond to 1 mm. Thin sections short side measures 1.2 cm…………………………………………………………………………………………...…141

Fig. 5.4 FR16 stable isotope signal (d13C and d18O) compared to petrographic log (light orange line) and greyscale values (blue graph). C = compact columnar fabric; C/Co = laminated part where compact and open columnar fabric alternate; M/Ms alternation between micrite and microsparite fabric. “Hi” indicates the hiatus visually marked in the polished slab as a change from micrite to laminated fabric. The hiatus is also recorded by the abrupt shift in the isotopic profiles. The negative peaks are marked by red arrows and possibly indicate 5 cycles toward arid conditions……………………………………………………………………………………………142

Figure 5.5 Age-depth model for FR16 after A) and before B) the hiatus. Red dots represent the U-Th ages (see Table 1). Dark and light-grey lines represent 1σ and 2σ errors, respectively. The derived growth-rate time series C) and D) where grey lines represent the 2σ errors. Depths are measured from the top of the stalagmite………………………………………………….…………………………………………146

2011) and FR16 ages with respective 2σ-uncertanties are also shown. The blue graph represents the reflectance of the stalagmite surface in greyscale values whereas the bold black line is FR16 growth rate……………………………………………………………………………………..148

Fig. 5.7 Analysis of the isotopic composition (oxygen and deuterium) of modern drip waters collected in Frasassi cave during the period 2014-2015, compared to the water lines of isotopic composition of modern precipitation from all the Mediterranean region (Giustini et al., 2016b). The Italian meteoric water line is also showed as well as the local Sicilian water line………………………………………………………………………………………….….152

Fig. 5.8 Continuous wavelet analysis of FR16 δ18O and δ13C signals using the continuous wavelet transform toolbox for MATLAB (Grinsted et al., 2004). The isotopic time series are represented at the bottom of the power spectra. The thick black contour designates the 5% significance level against red noise and the cone of influence. The period time unit is in ka. The resulting periodicity varies between 1 to 4 ka like the DO events and are especially evident in the pre-hiatus phases of the stalagmite…………………………………………………………………………………..157

Fig. A1.1 Interpretation of the evolution of Frasassi system from an original, modified from an unpublished drawing by A. Mon- tanari………………………………………………………………………..………………….177

Fig. A1.2 H2S vapours chimney (photo on the left side). Gypsum deposit (photo on the right side)…………………………………………………………………………………………....178

Fig. A1.3 Uplift rate for the different karstic levels graph………………………………………………………………………………...…………179

Fig. A1.4 Age models and main fabrics observed for the samples…………………………………………………………………………………….………180

Fig. A1.5 Growth rates of Frasassi stalagmites are compared with NGRIP ice core δ18O record and LR04 marine benthic δ18O record (Lisiecki and Raymo, 2005). Sapropel events and marine isotope stages (MIS) are also indicated……………………………………………………………………………..…………….182

Fig. A2.1 FR11 and FR12 grey scale profiles. High values (>110) correspond to the porous parts of the stalagmites characterized by open and/or porous columnar fabrics. Translucent zones are formed by compact calcite which is indicated by low grey values (<110)…………………………………………………………………………………….……186

Fig. A2.2 Frasassi δ13C record and grey values profile compared to LR04 marine benthic δ18O record (Lisiecki and Raymo, 2005), Antarctic atmospheric CO2 record (Petit, 1999) and summer insolation curve (Laskar et al., 2004). Marine isotope stages (MIS) 7 to 10 and terminations III and IV are also indicated. Blue dots represent the U-Th dates performed on FR11 and FR12 with 2σ error bars. Green boxes highlight interglacials and interstadials…………………………………………………………………………………..…………..187

List of Tables

Table 1.1 Most common chemical proxies commonly used in speleothem science, for more details please refer to the main text………………………………………………………………………………………………15

Table 3.1 Hydrochemical characteristics of the cave waters collected at Lamalunga cave in May 2008..……………………………………………………………………………………………78

Table 3.2 Results of Multicollector ICP Mass Spectrometry U/Th analyses on the coralloid ABS6-A and ABS5 (Lari et al. 2015). The isotope analyses are reported as activity ratios (AR), the errors are reported in brackets as ±2σ…………………………………………………………………………………………...….79

Table 3.3 Mean values in translucent (Trans) and opaque calcite fabric expressed in mg/g ±1σ for selected intervals in coralloid samples ABS6-B, C (cf. Fig. 3.7) and ABS5 (cf. Fig. 3.4) measured in conventional µXRF and with SR- µXRF………………………………………………………………………………………..….88

Table 4.1 Power law slope parameters (m) for the analysed carbonate samples……………………………………………………………………………..………….124

Table 5.1 Results of Multicollector ICP Mass Spectrometry U-Th analyses on calcites. The isotope analyses are reported as activity ratios (AR), and the errors are reported in brackets as ±2σ. The black bold line indicates the position of the hiatus………………………………………………………………………..…………………….145

Table 6.1 List of all the samples collected in Frasassi cave and corresponding time span of growth……………………………………………………………………………………….…172

Table A1 Uplift rate of the different karst levels…………………………………………………………………………………..…………..179

Publications arising from this thesis

Peer-reviewed Publications

Vanghi, V., Frisia, S., Borsato, A. 2017. Genesis and microstratigraphy of calcite coralloids analysed by high resolution imaging and petrography. Sedimentary Geology 359, 16-28.

Vanghi, V., Borsato, A., Frisia, S., Howard, D. L., Gloy, G., Hellstrom, J., Bajo, P. High-resolution synchrotron XRF investigation of calcite coralloid speleothems: elemental incorporation and their potential as environmental archives. Submitted. Sedimentology.

Vanghi, V., Frisia, S., Borsato, A., Hellstrom, J., Bajo, P. Climate variability on the Adriatic seaboard during the last glacial inception: the Frasassi cave case. Accepted. Quaternary Science Re- views.

Conference Proceedings

2016 American Geophysical Union (AGU) Fall meeting San Francisco (USA). Vanghi et al. Composite δ13C and petrographic 195-355 ka record from Frasassi cave (central Italy) stalagmites: investigating drivers of calcite carbon isotope signals.

2015 International Union of Quaternary Research (INQUA) XIX conferences, Nagoya (Japan). Poster presentation: Vanghi et al. Speleothem chronology from Frasassi cave system (Central Italy): timing of speleogenesis, uplift rate and paleaoclimate evolution.

2014 Climate Change: The Karst Record VII (K7) conference, Melbourne (Australia). Poster presentation: Vanghi et al. Coralloid speleothems associated with Neanderthal skeleton in Altamura cave (Southern Italy): environmental constrain and paleoclimate significance.

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1. Introduction

1.1 Background

Speleothems importance as archives of Earth’s climate dynamics and teleconnections linking marine and terrestrial domains has been rapidly increasing over the last two decades (McDermott, 2004; Henderson, 2006; Wong and Breecker, 2015). Although speleothems are comparable to ice and marine sediments as archives of past climate variability, they exhibit numerous distinctive advantages (Woodhead and Pickering, 2012). The most important advantage is that they yield precise chronologies, based on the creation of thorium-230 from the radioactive decay of uranium, which permits to fix paleoclimate events in absolute time (Richards and Dorale, 2003; Hellstrom, 2003). The exceptional time-series provided by U-series decay chain dating applied to speleothems, can go back in time up until 500,000 years ago (Middle to Late Pleistocene) but recently advancement on U-series isotopic measurements has pushed this boundary up until the Early Pleistocene (Richards et al., 1998; Pickering et al., 2011; Bajo et al., 2012; Woodhead and Pickering, 2012) and the Permian (Woodhead et al., 2006; Woodhead et al., 2010) based on U- Pb method. Furthermore, changes in the morphology, the mineralogy and the chemical composition of stalagmites are clearly modulated by climate, such as drip rate variability and temperature- influenced cave ventilation (Frisia and Borsato, 2010). Their ability to preserve, layer upon layer, climate events and environmental processes spanning from orbital to sub-annual time scale, enables giving precise chronological marks to large shifts in hydroclimate. Furthermore, by investigating multiple proxies in speleothems, such as stable isotope ratios, trace elements concentration, petrography, radiogenic isotopes and fluid inclusions, it is possible to distinguish global from local controls of the palaeo-proxy signal (Dreybrodt and Scholz, 2011). For example, stalagmite time-series can refine the temporal boundaries of glacial terminations (Cheng et al., 2009) or record shifts in monsoon activity (Wang et al., 2008; Allu et al., 2015) and/or of the El Niño/Southern Oscillation (ENSO) system (Frappier et al., 2002). Speleothem-based paleoclimate studies, use spelean carbonates δ18O to track the response of global, regional and local hydroclimate to changes in the amount and seasonality of precipitations and/or shifts in moisture sources and storm trajectories (Bar-Matthews et al., 2000; McDermott et al., 2001; Krklec and Domínguez-Villar, 2014). The δ13C signal encoded in the carbonate layers instead is interpreted as a proxy of surface temperature and vegetation cover, or atmospheric carbon dioxide (Dorale et al., 1998; Fairchild et al., 2006; Breecker, 2017). Speleothems can be found over most continental

areas, which permits a direct comparison of climate variability over very distant regions, and even correlate events at hemispherical scale (Henderson, 2006). Exciting new uses of speleothems have been expanding over the palaeoanthropological domain due to the frequent assemblages of both humans and other animals’ remains in cave environments that were used as shelters, or because they simply represented an entrapment (Vaks et al., 2007; Pickering et al., 2011; Lari et al., 2015; Rosenberger et al., 2015). Lamalunga cave speleothems, which are one of the objectives of study in this thesis (see chapters 2 and 3), will contribute to consolidate and improve the link between speleothem science and paleoanthropology and archaeology. Still remaining within the archaeological context, speleothems can be helpful also in the debate surrounding rock, which represents a very important sign of cognition and symbolic thought that would differentiate H. neanderthalensis and H. sapiens and which is mainly dated using relatively chronology (by comparing drawing styles) (Aubert et al., 2014). Given that precise age is absolutely crucial to assign the symbolic use of pigments to one species or the other, Uranium-series dating technique applied on carbonate coatings deposited over the painting(s), allows precise and reliable absolute ages (Aubert et al., 2007; Pike et al., 2012; Aubert et al., 2014; Hoffmann et al., 2018).

Another important application of speleothem science concerns the study of tectonic processes and landscape evolution (Wang et al., 2004; Polyak et al., 2008). This can be especially interesting in areas of relatively (geologically speaking) rapid uplift related to the collision of plates or where the presence of hot spots, earthquakes and volcanism impacts on human’s life (such as Italy) shaping the lands where they live. In appendix 1, I present a preliminary study in which I attempted to date the phases of development of the different karstic levels in Frasassi cave, which are the result of the interaction between tectonic uplift, erosion and aggradation processes during Quaternary glacial cycles.

Speleothem science is a relatively recent but fast advancing branch of research interrogating Earth’s past, with the goal to gain better understanding of future developments in Earth’s system that may affect our quality of Life. Their common association with archaeological and palaeontological findings also makes them one of the best tools to reconstruct the response of early humans and ancient civilizations to geologic, ecologic and climate catastrophes (Drysdale et al., 2006; Holmgren and Öberg, 2006; Webster et al., 2007; Medina-Elizalde et al., 2010; Sletten et al., 2013; Frappier et al., 2014). This is the foundation of the present research. Because much of my focus has been on advancing knowledge in the petrography of speleothems, the following two sections (1.2, 1.3) introduce the basics of speleothem formation and their fabrics. Section 1.4 briefly summarizes the geochemical background and is followed by section 1.5 where my case studies are out into context.

1.2 Speleothems formation

Speleothems are secondary minerals deposits that form in caves (Hill and Forti, 1997). Stalag- mites are a very common type of speleothems, they grow from the cave floor upward and are commonly fed by water dripping from an overhead stalactite (Self and Hill, 2003). Most stalagmites are composed of calcite, the calcium carbonate (CaCO3) phase that is thermodynamically stable at surface temperature (T) and pressure (P). They may be also composed of aragonite, which is the high-pressure polymorph of CaCO3, and a mineral phase that is thermodynamically unstable at surface P and T (Frisia et al., 2002). Speleothems deposit from dripping water with high carbon dioxide content (pCO2) that has dissolved the carbonate host-rock while seeping through its fissures and pores and which finally enters into a cave environment. Cave atmosphere has a relatively low pCO2 therefore carbon dioxide degasses from the solution (high pCO2), and calcium carbonate (CaCO3) precipitates in the form of speleothems, like stalactites and stalagmites (Fig. 1.1) (Ford and Williams, 2007; Fairchild and Baker, 2012). The Saturation Index measure the degree of calcite that can precipitate from the dripping water (SIcc). Supersaturated solutions (SIcc>0) precipitate calcium carbonate whereas undersaturated solutions (SIcc<0) dissolve calcite. When SI=0 the system is at the equilibrium (Langmuir, 1971; Ford and Williams, 2007).

The key reactions leading to the final speleothem formations are (Fairchild and Baker, 2012):

 H2O (aqueous) + CO2 (gas) ↔ H2CO3 (1) occurring in the soil zone where the CO2 is either coming from the atmosphere or as the by- product of biological activities.

 Dissolution of the carbonate bedrock:

2+ - CaCO3 + H2CO3  Ca + 2HCO3 (2)

 Speleothem precipitation:

2+ - Ca + 2HCO3  CaCO3 + H2O + CO2 (3)

The capability of a solution to precipitate calcite is commonly measured by its Saturation Index (SI):

- SIcalcite = log {(Ca2+) (CO3 )/ Kcalcite} (4)

- Where (Ca2+) (CO3 ) is the ion activity product and Kcalcite is the temperature-dependant solubility constant for calcite. Calcite will precipitate when SIcalcite > 0, and dissolve when SIcalcite < 0. When SI=0 the system is at the equilibrium (Langmuir, 1971; Ford and Williams, 2007).

The precipitation of calcium carbonate in caves occurs from water solutions that commonly have low ionic strength and reach supersaturation with respect to calcite (or aragonite) through the mechanism of degassing (Eq. 3), which is the loss of carbon dioxide (CO2) from the solution to the cave atmosphere (Ford and Williams, 2007).

2+ If the Ca ion concentration in the parent solution does not increase, the loss of CO2 from the solution through degassing shifts the equilibrium reaction to the right, and calcium carbonate precipitates (Dreybrodt, 2011) (Eq. 3). Timing for CO2 degassing is dependent upon drip rate: the slower the drip rate the longer the degassing process may proceed thus keeping the solution until it exhausts (Kowalczk and Froelich, 2010). Degassing in caves may be forced by kinetic processes, particularly in ventilated caves, when the pCO2 of the cave air may attain values smilar to those of surface air (which nowaday is circa 400 ppmv), while drips may have a CO2 concentration at least ten time higher (Hansen et al., 2013). Cave ventilation is most commonly modulated by temperature difference between the cave and the surface and, therefore, in mid-high latitude and high altitude settings is one of the most efficient mechanisms to stimulate seasonality in growth rate. When the temperature contrast between the cave air and surface air is at a maximum, with the cave air warmer than the exterior, degassing is forced by the flow of air outward, and calcite deposits faster (Fairchild and Baker, 2012; Borsato et al., 2015).

This short preface about how speleothems grow provides the rationale for the use of speleothems as “weather station” (McDermott, 2004). First, because their growth seems to be modulated by climate, particularly hydroclimate and environmental parameters. Second, because water acts as a transmission belt of external climate and environmental signals, in the form of stable isotope ratios, trace elements, organic colloids into the cave (Fairchild and Baker, 2012) (Fig. 1.1).

Fig. 1.1 Transfer of the geochemical signal from the outside to speleothems growth layers.

1.3 Petrography of speleothems

Petrography is the branch of geology that studies sediments and rocks for their identification and classification, to interpret their environment of deposition and identify possible post-depositional alterations (diagenesis). Petrography is a necessary tool that provides a frame of reference for geochemical studies (Scholle and Ulmer-Scholle, 2005). The mechanisms of crystal growth have been widely investigated at the atomic level, which helped to understand how crystals micro and macro morphologies are determined. The crystal forms and their spatial arrangement, referred as “fabric”, are a result of the crystal nucleation type and morphology, lattice defects and presence of impurity elements or particles.

In the classical approach, nucleation is a one-step process that takes place in a solution of ions that has reached supersaturation, leading to the nucleation of the solid phase by monomer-by- monomer addition of simple chemical species (atom, ion, and molecule) (De Yoreo and Vekilov,

2003; De Boever et al., 2017). The model described for calcite surface accretion, at molecular scale, is the “step-kink-hole model” representing the different surface sites that can adsorb ions (Fig. 1.2) (Morse and Arvidson, 2002; Ford and Williams, 2007b). Classical nucleation explains that under Gibbs low driving force (the free-energy barrier to allow nuclei to become stable is high), when supersaturation ratio of the parent solution is minimal, crystals that nucleate then grow to be relatively large and symmetric. At high Gibbs driving forces (the free-energy barrier is lower than in the first case), or when impurities are present at the interface between the solution and the surface, nucleation occurs relatively faster, crystal size and symmetry is reduced and crystals orientations are multiple (De Yoreo and Vekilov, 2003; Sunagawa, 2005; Fairchild and Baker, 2012). In relation to polycrystalline aggregates, their geometrical relation/orientation with the substrate selects the crystals that will continue growing, which explains the principle of “geometrical selection” (Sunagawa, 2005). Usually, it is promoted the growth of the crystals whose growth vector is orientated perpendicular to the surface, because their tips are in an ambient phase characterized by high free energy. Consequently, a crystal fabric with a preferred optical orientation develops. The different textures of polycrystalline aggregates depends on the shape of the surface and so, for example, spherulites form when the substrate is spherical and banding patterns of layers of polycrystals form on irregular curved surfaces (Sunagawa, 2005).

Fig. 1.2 Step-kink-hole model with adsorbed ions shown in black. Refer to the text for more details. Picture taken from Morse and Arvidson (2002).

In a non-classical approach of crystal nucleation, crystallization is meant to happen following multiple pathways by attachment of particles, or a series of transient, amorphous “precursor” phases ranging from multi-ion complexes to fully formed nanocrystals (Fig. 1.3). The formation of nano-clusters lowers the energy barrier required to start nucleating even at low supersaturation

levels. Specifically, the amorphous phase can mediate the nucleation of the crystalline phase because the system has to overcome a much lower free energy barrier compared to the direct crystallization from a solution (classic nucleation theory) (Fig. 1.4). Thus, the energetics of prenucleation clusters is much more complicated that what estimated in the classical nucleation model (Lee et al., 2016).

Recently, Frisia et al. (2018) TEM and SR-µXRF observations revealed that the mechanisms of formation of speleothem calcite include both classical and non classical crystallization pathways. Depending on which pathway is involved in the process of growth, diagenetic transformations are prevented or fostered.

Fig. 1.3 Different pathways to crystallization by particle attachment. The classical theory of nucleation (De Yoreo and Vekilov, 2003), only accounts for the monomer-by-monomer crystal growth. Sketch taken from De Yoreo et al. (2015).

Following classical nucleation and growth approach, stalagmite fabrics have been classified in a system based on supersaturation state of the parent water and presence of impurities by Frisia (2015). That classification updated two previous works Frisia et al. (2000) and Frisia and Borsato (2010), which, in turn, referred to pioneering work by Folk and Assereto (1976); Kendall and Broughton (1977); Kendall and Broughton (1978); Gonzalez et al. (1992); Kendall (1993). Prior to the seminal paper by Frisia et al. (2000) however, fabric characterization and descriptions were mainly based on optical microscopy techniques and, occasionally, scanning electron microscopy (SEM). Insight on growth mechanisms and processes, which are the basis of the modern classifi- cations, most of which were developed by the speleothem group at the University of Newcastle, are based on transmission electron microscopy (TEM), which allows investigating speleothem crystals microstructures (Frisia et al., 2018). In parallel, researchers based at the University of Bochum (Germany) have pioneered the use and electron backscatter diffraction (EBSD) methods to investigate Radiaxial Fibrous (RFC) and Fascicular Optic Fibrous Mg-calcite in speleothems (Richter D.K. et al., 2015), highlighting the need of textural, in addition to microstructural, investigation.

The characterization and interpretation of fabric and, thus, the internal stratigraphy in speleothems have always received less attention compared to the climatic signals inscribed into stable isotopes or trace elements, despite their significance as indicators of water supply rates, kinetics and flow conditions at the speleothem surface (Dreybrodt, 1988; Dreybrodt, 1999). Furthermore, both geochronology and proxy data interpretations should be framed in speleothem stratigraphy, which varies from simple to highly complex (Muñoz-García et al., 2016), and tests the accuracy of the capture of the chemical signal within the physical context. The first speleothem-based paleoclimate research that benchmarked the interpretation of speleothem stable isotope stratigraphy on fabrics, McDermott et al. (1999) paved the way to recognizing the importance of stratigraphic, in addition to chemical, correlations between stalagmite proxy series along a N-S traverse in Europe. This seminal paper introduced new fabrics in addition to the well-known columnar calcite (Kendall, 1993). In an Irish speleothem, McDermott et al. (1999) found that dendritic (tree-like) calcite fabric was dominant and marked dry periods. Similarly, Onac et al. (2002) found dendritic calcite fabric in a stalagmite from Romania, but contrary to the Irish stalagmite case, they interpreted this fabric as an index of wet conditions. Boch et al. (2011), demonstrated that there is a direct relation between compact/open columnar calcite and a more positive/more negative 13C values, which reflect low/high drip rate. The critical issue in the use of fabrics as paleoclimate

tool has been the difficulty to their quantification. The fabric log provided in McDermott et al. (1999) was a qualitative tool, which allowed distinguishing if fabrics were somehow influencing stable isotope signals. In addition, the importance of fabrics as a stand-alone proxy of climate and environmental changes remained controversial. Until a breakthrough paper, Luetscher et al. (2011), correlated stable isotope (δ13C and δ18O) fluctuations measured in Holocene stalagmites formed below the ice with unusual fabric changes, indicative of bio-mediation. They observed a consistent shift toward depleted isotope values along stromatolite-like layers, implying bacterially mediated calcite precipitation under extreme cold conditions. This discovery confirmed that speleothem fabrics are a powerful tool to reconstruct climate-related growth processes and a necessary benchmark for an accurate interpretation of speleothem geochemistry.

Only in recent works, the internal stratigraphy of speleothems has been described and interpreted from different points of view and the introduction of petrographic and micro-stratigraphic logs finally complemented geochemical analyses (McDermott et al., 1999; Bertaux et al., 2002; Verheyden et al., 2008; Belli et al., 2013; Borsato et al., 2015; Frisia, 2015; Muñoz-García et al., 2016; Martín-Chivelet et al., 2017). Specifically, the petrographic-log proposed by Frisia (2015), by assigning numerical values to observational fabric codes, allows generating fabric time series bearing climatic significance that can be immediately correlated with chemical time-series. This approach has changed the common perception that speleothem petrography is a “qualitative” description. Recent developments in crystallization pathways (De Yoreo et al., 2015) have added value to a petrographic approach supporting accuracy of chemical data from speleothems (Frisia et al., 2018). This Thesis is testimony of the advance in understanding about the importance of fabrics in speleothem-based climate investigation.

1.3.1 Architecture of speleothems: Fabrics

Stalagmites are aggregate of crystals. In the hierarchy of speleothem architectural elements con- ceived by Martín-Chivelet et al. (2017), the smallest element is the individual crystallite (1st order). However, recent development in speleothem growth mechanisms suggest that there are multiple crystallization pathways acting, as documented by precipitation experiments performed in caves Demény et al. (2016) and Frisia et al. (2018). Pre-nucleation clusters, amorphous phases, nanocrystal aggregation and classical nucleation pathways may all operate in caves (Frisia et al., 2018). Thus, crystallites are likely to have been already the product of growth from nano- and meso-crystals (Frisia et al., 2018). The implication is that stalagmite fabrics may be the result of

multiple crystallization processes acting at the same time, and these dictate speleothem architecture and geochemical properties. This concept defies purely thermodynamics in favour of an em- pirical approach (Fairchild and Baker, 2012; Belli et al., 2013; Belli et al., 2017). Nevertheless, the smaller growth units observable at the microscope (micrometre scale) can be reasonably considered the smallest architectural element.

Fabrics are the 3d order architectural element. Changes in stalagmites fabric are good indicator of changes in the environmental parameters relative to cave carbonate formation and are powerful palaeoclimate proxies (Railsback et al., 2011). The mechanisms controlling fabrics development and the spatial arrangement of the crystals are related to the supersaturation state with respect to calcium carbonate (SIcc), fluid flow, Mg/Ca ratio, presence of inorganic and organic compounds in the parent solution. Specifically, in meteoric water when the degree of supersaturation is low, the growth rate is retarded and equant crystals are precipitated. On the other hand, acicular or elongated crystals are found in vadose pores where rapid CO2 degassing and/or evaporation lead to a high degree of supersaturation (Given and Wilkinson, 1985). These are directly or indirectly related to climate parameters, such as rainfall amount, soil efficiency, temperature, vegetation cover (Frisia et al., 2003; Self and Hill, 2003). However, the influence of any of these parameters on crystallization pathways remains largely unknown, although the presence of specific Humic Substances (HS) seems to have a fundamental role (Frisia et al., 2018). Similarly, temperature seems to exert an influence on the nucleation and growth of amorphous phases (Demeny et al., 2017). Crystallization pathways also influence the probability of occurrence of post-depositional processes (diagenesis), such as aragonite inversion to calcite, which has large impact on interpretation of chemical data (Frisia, 2015).

The most common primary calcite fabrics observed in speleothems pertain to columnar types. Dendritic, fabrics and micrite are less common. Microsparite and mosaic calcite fabrics are, to date, considered the product of diagenetic transformation (Frisia and Borsato, 2010; Frisia, 2015).

Columnar fabric types (Fig. 1.5 A): Macroscopically, columnar fabric consists of crystals  1 mm wide and  2 mm long, elongated perpendicular to the growing speleothem surface, with a length to width ratio of about 6:1, uniform extinction under crossed polarizers and serrated or open crystal boundaries. When length to width ratio is > 6:1 it is referred to elongated columnar fabric. In the hand specimens, this fabric has translucent appearance. Columnar types consist of composite crystals where stacking of growth units, which depends on the presence of impurities in the feeding water, determines the presence of pores: the “open columnar type” of columnar fabric. When crystalline margins are closed it is referred to “compact columnar type”. If crystals have defects

that result in serrated and irregular crystal boundaries, it is referred to the “microcrystalline type”. When observed that the SEM, the dominant form typical of emerging crystal tips at speleothem tips is the cleavage rhombohedron. However, the flanks consist of macroscopic (at sub-micrometre scale) steps and kinks (Frisia et al., 2000), which may accommodate attachment of growth units (Frisia et al., 2018). Columnar fabrics, and particularly the compact type, have been observed to form under low drips with constant supersaturation with respect to calcite (SIcc), whereas the open type of the columnar fabric forms under highly variable drips rates.

Dendritic fabric (Fig. 1.5 B): it is formed by branching polycrystals arranged ca. 90° angles one with respect to the other. Due to its high intercrystalline porosity, this fabric has a milky and opaque appearance in the hand specimen. At the microscale, it has high density of crystal defects. Dendritic fabric usually develops under a fast drip rate, and relatively high SIcc attained through degassing and it is usually common at near-entrance settings subject to air currents and evaporation, or in ventilated caves (Frisia and Borsato, 2010). Microbial influence might play a role in its formation (Frisia, 2015).

Micrite (Fig. 1.5 C): it consists of equant 2μm maximum crystals, which appear dark under the optical microscope in both parallel and crossed polars. This fabric has been interpreted, in analogy to its marine counterpart, as requiring high number of pre-existing nuclei, possibly related to the presence of organic compounds. Its formation can then be the result of bio-mediation (Frisia et al., 2012). Alternatively, it could form from highly supersaturated drip waters. Micrite can also be diagenetic when associated to aragonite (Frisia, 2015).

Mosaic (Fig. 1.5 D) and microsparite (Fig. 1.5 E) are considered diagenetic fabrics: mosaic fabric is formed by low-Mg calcite crystals, >30 μm and <1 cm in diameter and is considered a diagenetic product of dissolution and re-precipitation of primary precipitates, including calcite. Porous fabrics such as dendritic, microcrystalline or open columnar are, in fact, prone to allow water percolating through interconnected porosity. Microsparite consists of >2 μm and <30 μm crystals and it could possibly be the product of micrite dissolution and replacement (neomorphism).

A Columnar fabric (scale bar=1mm). B Dendritic fabric (scale bar=1 mm). C Micrite layer (arrow). (From Photo taken from my MSc thesis. Photo taken from my MSc thesis. (Frisia and Borsato, 2010).

D Mosaic fabric. (from (Frisia and E Microsparite fabric (from (Frisia, Borsato, 2010). 2015).

Fig. 1.5 Some of the most common fabrics observed in stalagmites.

1.3.2 Speleothem architecture: Laminae

Some speleothems show a rhythmic alternation of intra- or inter-porosity that produces a macro and microscopically visible lamination. According to Martín-Chivelet et al. (2017), if laminae identify a single growth layer, they are a 2nd-order architectural element and the crystal boundaries of columnar fabrics cross-cut them. In micrite fabric, the crystals are commonly constrained within the boundaries of the laminae, which brings the 2nd and 3d orders of architectural elements to coincide.

Difference in the texture of laminae reflects variations in the environmental conditions of CaCO3 precipitation. Commonly, porous laminae correspond to a relatively rapid growth rate and high crystalline defects whereas compact laminae correspond to slow growth rate, leading to the formation of crystals with only few defects (Genty and Quinif, 1996; Frisia et al., 2000). Crystal

growth rate depends on the SIcc and on the rate of CO2 degassing. During wet periods when water discharge is high, the solution is more diluted and thus less supersaturated and usually compact calcite precipitates. During dry periods, when the infiltration is more concentrated in minerals and more saturated, porous laminae form. This lamination is referred to as “couplets”, because annual lamination is normally the most common. Despite it, laminae found within speleothems may occur at a variety of timescale and other sub-annual or millennial-scale events can be registered in the microstratigraphy of a speleothem (Fairchild et al., 2006) which, accordingly to Martín- Chivelet et al. (2017), represents the 4th order-order architectural element and defines stacked arrangement of laminae sets (at a centimetric scale).

The location of the cave where the speleothems form is also very important. Yet in some cases, even small changes in the hydrochemistry of the drip water or in environmental parameters of the cave atmosphere are enough to provoke rhythmic changes in the internal microstratigraphy of a speleothem. This is especially valid for stalagmites growing close to a threshold condition for a particular fabric or mineralogy. Stalagmites found in sections of a cave experiencing seasonal changes in ventilation (near the entrance) for example, are more likely to show a clear lamination because of the greater excursion in the environmental parameters (air temperature, relative humidity, air CO2 concentration) typically connected to “cave breathing” that controls cave air and drip water composition and subsequently, speleothem growth dynamics (Boch et al., 2011).

Frequently, stalagmite growth can be disturbed, periodically or sporadically, which results in growth discontinuities or inclusion levels whose genetic significance is very important because they potentially record dramatic environmental processes occurred at the surface of the speleothem (Perrin et al., 2014). These laminations correspond to the 5th and the 6th levels of architectural elements described by Martín-Chivelet et al. (2017). In many cases, lamination can be the product of deposition from infiltration waters that periodically carry impurities and can be are visible as detrital layers or inclusion levels (Borsato et al., 2007; Perrin et al., 2014). The impurity-rich part of the lamina couplets are usually incorporating fine detrital material, manly clay particulate, colloidal organic matter (Hartland et al., 2012) deposited and dust that can be transported by the drip water, suspended in the air or as hydroaerosol (Dredge et al., 2013; Vanghi et al., 2017). Laminae can also form when mineralogy changes, normally when calcite and aragonite alternate (Pickering et al., 2010; Wassenburg et al., 2012). Aragonite precipitation is known to be favoured when the Mg2+/Ca2+ ratio in the parent solution is high (Hill and Forti, 1997; Martín-García et al., 2009), which commonly is the case during dry periods in caves cut in dolomite host-rock (Railsback et al., 1994; Frisia et al., 2002; McMillan et al., 2005; Wassenburg et al., 2012; Perrin et al., 2014).

Moreover, a prolonged gap in calcite deposition, due for example to cessation of dripping which is usually followed by dissolution (when << SIcc) of the underlying carbonate crystals, can result in a visible layer, which usually is a band of micrite (deposited post-quem) that identifies a discontinuity in the growth of the speleothem (also referred to as hiatus) (Martín-García et al., 2009; Railsback et al., 2011). Railsback et al. (2011) describe two kinds of discontinuities: Type E and Type L. Type E are discontinuities formed by dissolution over the surface of the speleothem under high discharge, which inhibits sufficient CO2 degassing necessary to bring the feeding water to equilibrium with CaCO3 for calcite precipitation. Alternatively, another explanation for Type E layers is the occurrence of the condensation-corrosion process, where condensation results in undersaturated or acidic waters. Condensation occurs when the temperature of cave walls and the speleothem uppermost surface is below the air dewpoint and, thus, water vapour condenses as a thin water film. Such moisture is undersaturated with respect to CaCO3 because it is in equilibrium with cave air pCO2 and thus is corrosive (Dublyansky and Dublyansky, 1998; Dreybrodt et al., 2005). Type L discontinuities are caused by diminishing flow of water onto the speleothem top leading to a calcite deposition just on the tip of the stalagmite and no precipitation on the flanks.

Dark organic rich laminae can also be mediated by biofilms (mainly Actinomycetes bacteria) developed on the surface of the speleothem and thus may not record periodic influxes of exogenic organic matter following the wet season (Jones, 2011). Biofilms produce a distinctive microstratigraphy via constructive and/or destructive processes, like thin micrite layers, the production of calcified filaments, substrate etching and breakdown. It is possible to recognize such microbial activity in old carbonate cave deposits by observing mineralized microbes or stromatolitic structures the identification of fabrics/ textures that are known to be indicative of microbial activity (like micrite). All of these criteria fundamentally rely on the interpretation of fabrics thus stressing the importance of petrography in speleothem science (Jones 2001).

Set of stacked single growth layers define the 3D internal stratigraphic architecture of the stalagmite and are included in the 4th order of stratigraphical elements by Martín-Chivelet et al. (2017). These relate to the features of the drip rate and to water chemistry. Stacked layers trends have been termed aggradational (growth is almost only vertical), progradational (higher drip rate, thus the stalagmite diameter increases), retrogradational (drip rate decreases and the stalagmite tapers out vertically).

1.4 Speleothem Chemistry

Speleothems can be considered as archives of the environmental chemical signal of the surface (chemical proxy data) and thus can help to indirectly interpret climatic changes occurred in time (Fairchild and Baker, 2012). Specifically, by analysing the isotopes of carbon and oxygen and stalagmite carbonates (CaCO3), it is possible to infer past changes of the atmospheric oxygen and carbon dioxide (Ford and Williams, 2007b). Trace elements partitioned in the calcite layers of a stalagmite can instead give information about the hydrological characteristics of the aquifer (Fairchild and Baker, 2012).

All the environmental signals originate in four domains or realms where they are transferred through and modified during time. These domains are:

1. The atmosphere 2. The soil and the upper epikarst 3. Lower epikarst and the cave environment 4. Carbonate deposit (i.e. stalagmites)

Table 1.1 shows the most common chemical proxies usually examined in speleothem science. Each of these parameters are associated with a measurable variable that reflects atmospheric, hydrologic, soil, cave and ecological changes with effects on the long or on the short term.

Speleothem param- Long term control- Decadal and millen- Annual scale con- eter ling variable nial scale control- trolling variable ling variable

18 δ O Latitude, Altitude, Temperature, rainfall, Drip rate, CO2 evapo- global temperature moisture source δ18O ration, (degassing?) (glacial vs intergla- signal or “storm cial = ice volume) track”

13 δ C Latitude, Altitude, ice Temperature, rainfall CO2 degassing rate, volume, methane soil efficiency, vege- cave air circulation bursts tation type (C4 or (microbial activity in C3), canopy and soil the cave?) cover density

Trace elements and Bedrock and soil Temperature, precipi- Colloidal transport, natural organic composition tation, redox state in crystallization path- matter (NOM) the aquifer, pH ways, speleothem growth rate (microbial associations at stalagmite tips?)

Table 1.1 Most common chemical proxies commonly used in speleothem science, for more details please refer to the main text.

In the Earth system, these parameters are controlled by a multitude of variable within different reservoirs (i.e. the atmosphere, the ocean, caves etc.). These variables can show co-variation or antipathetic covariation. The original environmental signals can be then identified using speleothem proxy signals and, in some cases, quantified using mathematical equations (transfer functions) (Fairchild and Baker, 2012). The speleothem-system is very complex because different proxies are involved at the same time and the difficulty of interpreting these data relies on understanding which one had more impact and their respective weight. Different processes can obscure the original climatic signal in speleothem isotope values.

1.4.1 Stable isotope ratios

The most commonly used stable isotopes in speleothem research for palaeoclimate and palaeoenvironmental reconstructions are those of C and O that are extracted from the CaCO3 molecule of the calcium carbonate. Particularly, the degree of isotopic fractionation between the liquid and the solid phase (which is temperature-dependent) determines the ultimate signal. Isotopic Frac- tionation is based on the assumption that during a reaction from a light to a dense phase, the light phase is enriched in the lighter isotope and this is enhanced with temperature. Thus, in the transformation from water into water vapour, the latter will be enriched in 16O, whilst the water will be enriched in 18O. By contrast, during condensation the heavier isotopes go into the liquid while the gas phase is enriched in light isotopes (Ford and Williams, 2007b).

The proxy used as indicator for atmospheric temperatures and precipitation variations, is δ18O (McDermott, 2004; Lachniet, 2009; Fairchild and Baker, 2012). This because drip water δ18O, ideally reflects a weighted mean δ18O of the annual precipitation at the surface. The oxygen signal of the rain is however itself influenced by atmospheric temperature and Rayleigh fractionation processes during rainout (Dansgaard, 1964; Lachniet, 2009). Rayleigh distillation and advection

paths respectively also control the δ18O values of rainfall, which become more negative from low to high latitudes and low to high altitudes, coinciding as well with a geographic temperature decrease (Johnston et al., 2013). The δ18O also variates accordingly to a longitudinal gradient as a function of the distance from the Ocean (McDermott et al., 2011). Moreover, on the long-term time scale (decadal to millennial), factors controlling the δ18O of meteoric water include the δ18O of the oceans, changes in seasonality of precipitation and in the moisture source and tracks (Bar- Matthews et al., 2003; Krklec and Domínguez-Villar, 2014). The δ18O of precipitation can be modified by evaporation in the soil, which is also a function of temperature (and rainfall amount). In this case, the δ18O of the groundwater would be more positive than the overall signal of the meteoric precipitation. Finally, the water to calcite fractionation is controlled by temperature, with a shift to more positive values indicative of cooling. However, it has been demonstrated that the δ18O of speleothem carbonates is largely controlled by that of the parent waters, whereby a more negative δ18O has been associated with cooling (McDermott, 2004). From this short synthesis, it is clear that the δ18O signal of speleothem calcite is related to many factors, and it becomes difficult to quantify temperatures solely from speleothem carbonate if the δ18O signal of the parent water is not known. One could rely on the signal of original fluid inclusions trapped within the speleothem, but also this can be modified by processes of crystallization (Demény et al., 2016). In the cave, rapid degassing and/or evaporation phenomena may cause disequilibrium (kinetic)

CaCO3 deposition. This condition is favoured during enhanced in-cave ventilation or by humidity decreasing below 100% (Lachniet, 2009). These processes may also favour non-conventional crystallization pathways, most likely the formation of precursors before growth of the final product.

The δ13C of speleothem carbonates is related to that of the dissolved inorganic carbon (DIC) in the drip water. This has three major sources: (i) the soil CO2 deriving from the atmosphere and from the biological activities, (ii) the atmospheric CO2 and (iii) the carbonate bedrock. High bio-

13 logical activity in the soil promotes the production of biogenic CO2 that has negative δ C values, ranging from -26 to -20‰ for grasses type plants (C4 metabolism) and -16 to -10‰ for tree-like plants (C3 photosynthetic pathway ) (Cerling, 1984; McDermott, 2004; Fairchild et al., 2006), which is then up-taken by the percolating water.

Bedrock δ13C values are commonly moderately positive (0 ‰ to +3 ‰), if the bedrock is a carbonate subjected to diagenesis in the marine realm. Yet, there are cases when the δ13C released during dissolution of elements in the bedrock is as negative as that of the soil water, for example if the limestone was subjected to diagenesis in the meteoric environment (Miller et al., 2012).

Soil vs bedrock isotopic imprint in the drip water is also modulated by open/close conditions during the carbon exchange between the different phases (Fohlmeister et al., 2011). When the conditions of this exchange system are open, soil provides the almost entire CO2 supply (with light C) to the drip water. Under closed system conditions, the up to 50 % of the CO2 contribution comes from the dissolution of the bedrock, which is free of carbon originating from the decom- position of old soil organic matter. The δ13C signature thus, increases. In nature, however, mixed conditions are most commonly encountered (Hendy, 1971; Bajo et al., 2017).

Cave microclimate may influence the δ13C signal recorded in the speleothems: ventilation or prolonged hydrological pathways and processes may increase degassing and/or evaporation leading

12 to kinetic fractionation and a predominant loss of light C to the cave air. Specifically, when CO2 degassing of the percolating water happens before entering in the cave (for example in bedrock pockets filled with low pCO2), a CaCO3 precipitation prior (PCP) to reaching the stalagmite is triggered, and the remaining solution becomes enriched in the heavy carbon isotope. The phenomenon of PCP is common during dry conditions (Fairchild et al., 2000). Degassing triggered by ventilation and/or cave breathing is most typical of caves in climates characterized by strong seasonal contrast (Fairchild et al., 2006).

1.4.2 Trace elements

Other proxies linked to hydrological changes and composition of the host rock are trace elements that include chemical elements whose concentration in the solution or in the solid phase is less than 1% (Morse and Bender, 1990). Impurities incorporation in speleothem growth layers is due to the high capacity of calcite structure to substitute trace elements ions. This is especially true for trace metals with valence two, like Mg, Sr and Ba, which can replace Ca in the carbonate crystal lattice (Fairchild and Treble, 2009). The variety of chemical species found in speleothem is related to interactions of the infiltrating water with the atmosphere, the soil and vegetation and the karst bedrock. Trace elements incorporation has been generally considered as dependent to their respective partition coefficient that may vary to a greater or lesser extent with temperature, precipitation rate and crystal morphology (Fairchild and Baker, 2012). However, a thermodynamics approach implies equilibrium incorporation of tracers and cannot be fully explained nor tested because non-equilibrium processes are common in caves (Belli et al., 2017). Furthermore, it has been demonstrated, that crystal surface irregularities, at atomic scale, lead to a differential ions incorporation and intra-sectoral zoning. Larger ions, like Sr or Ba, are usually enriched in obtuse

(+) kink sites while smaller ions, like Mg or Mn, are enriched in acute (-) kink sites (Reeder, 1996). The intra-sector zoning is typical of crystals grown in the laboratory, under controlled conditions and by classical spiral step growth mechanisms. In the caves however, water chemistry is hardly controlled and carries colloidal particulate of both organic and inorganic origin (Hartland et al. 2012). In addition to the incorporation of trace ions of different size on kinks on advancing steps, impurities are adsorbed on the reactive calcite surface and then incorporated in the lattice. Finally, the new discovery that even speleothems carbonate may grow through particle attachment suggest that particulate may bridge nanocrystals which are attached with a slight lattice mismatch, accommodated by an amorphous phase (Frisia et al., 2018).

Many trace elements are transported in cave dripwaters bound to colloids (solids with a dimension between 1 nm and 1 μm), like natural organic matter (NOM), which can form polymers with inorganic metals (Hartland et al., 2012). For each tracer the dissociation kinetic from NOM complexes varies and this is reflected in a different partitioning between solution and calcite crystal surface. This is also dependent on the affinity for the calcite surface (surface charge), the availability of lattice sites and the amount of crystalline defects (Hartland et al., 2014). In the layers of the speleothems this can be visible as annual, to sub-annual, synchronous variation in organic fluorescence and trace metals. Therefore, the information that we can gain from changes in trace elements and organic ligand concentration and composition can potentially reflect the climate and environmental conditions of the overlying surface and can be linked to the rainfall amount (Richter et al., 2004; Borsato et al., 2007; Zhuo et al., 2008; Jo et al., 2010). However, the link has to be supported by robust petrographic observations, which allow recognizing different mechanisms of incorporation.

1.5 Study sites

In my research, I have considered the speleothems from two caves opening near the Adriatic seaboard in Italy along a N-S transect (Fig. 1.6 A). The selection of the case studies was operated based on their cultural importance, the relative paucity of information relative to the cave formations and the fact that their climatic setting is influenced by the same trajectories and cyclonic circulation (Horvath et al., 2008).

Fig. 1.6 (A) Geographical locations of the cave sites object of this study: Frasassi cave and Lamalunga cave. (B) Giustini et al. (2016) map of spatial distribution of δ18O (‰) of precipitations in Italy.

Frasassi cave (43°24′03″N 12°57′43″E) is the northern case study (Fig. 1.6 A), and its geological setting and climate are described in chapter 5, section “Field location and sample”, which I refer the reader to. Here, I provide a description of Frasassi speleogenesis, which has been widely reported in several studies carried out during the past twenty years (Galdenzi and Menichetti (1995); Galdenzi et al. (1999); Galdenzi and Maruoka (2003); Galdenzi et al. (2008); Galdenzi (2012); Jones et al. (2015)), but identifies one of the most intriguing aspects of this system, with implications for the ages of the stalagmites formed at diverse levels. The evolution of the Frasassi system has been only marginally considered in my PhD thesis, however, it will be the subject of future studies in the light of the results of the present research. Future work will also focus on themes like the Mt. Frasassi anticline uplift rate and the palaeoclimate evolution of the karst system.

Lamalunga cave (40°51'51.9"N 16°34'31.3"E, 508 m a.s.l.) is the southern case study, located close to the town of Altamura (Puglia) (Fig. 1.6 A). Contrary to Frasassi, which owes its fame to the beauty of the cave decorations and spectacular galleries, Lamalunga gained celebrity when in 1993 a complete fossilized hominin skeleton was discovered inside one of its small chambers. This fossilized skeleton, known as the “Altamura man”, is in an excellent state of preservation and is partially covered by coralloid formations that leave most of the bones visible, including the cranium, the mandible, and several postcranial elements (Lari et al., 2015). From the morphological and genetic studies conducted by Lari et al. (2015), the skeleton most likely belongs to a

typical European Neanderthal male of the late Middle/early Late Pleistocene, as it exhibits a mix- ture of archaic and derived features. For the first time after its discovery, the authors could perform a quantitative study on the remains including a preliminary paleogenetic characterization and its first dating. The results of the analyses on mtDNA (mitochondrial DNA) fragments extracted from the right scapula, confirmed its Neanderthal haplotype and, thus, the Altamura man has become the first complete Neanderthal skeleton existing in the palaeontologic record. Spele- othems played a very important role in his story because it was thanks to coralloid concretions, extracted from over one of the long bones, that the hominin could be indirectly dated, for the first time, by using the U-Th method (Hellstrom, 2003). Ten U-Th dates provided a first range of ages for this specimen who possibly lived between 130 ± 2 ka and 172 ± 15 ka and that makes the Altamura man the most ancient Neanderthal from which endogenous DNA has be recovered so far (Lari et al., 2015).

1.5.1 Reasons behind the choice of these two sites

The importance of Lamalunga cave, brought to fame thanks to the discovery of the Neanderthal skeleton, is undoubted. Therefore, it comes along that studying a cave with an added historical and archaeological impact benefitted to the research project per se. The need to provide a climatic context to the remains follows the palaeoanthropological investigation whose aim is to characterize, genetically and morphologically, this primitive form of Neanderthal from the Southern Med- iterranean region. However, due to preservation issues and the scarcity of stalagmites that could represent good candidates for studying the paleoclimate of the cave, there was the necessity to find other possible solutions and put a remedy to this lack of archives.

Firstly, we considered to use the coralloid speleothems that are very abundant inside Lamalunga cave. This kind of speleothems is normally not utilised in palaeoclimate science because of their minute dimensions, which make it difficult sampling for dating and geochemical analyses. In this thesis I have demonstrated instead, that coralloids can be a valuable archive and can be used when stalagmites are absent or not good candidates (please refer to chapters 2 and 3). Nevertheless, before performing conventional geochemical studies, it is very important to describe their microstructures and microstratigraphy that permits to connect coeval phases of growth and thus the use of geochemical data across different samples.

Secondly, we selected another cave site in support to Lamalunga cave. We aimed to choose a cave with a vast assortment of stalagmites that would have allowed a good selection of adequate samples. Frasassi cave, hosts many kilometres of galleries well decorated with speleothems of different dimensions and forms, and is easily accessible as it has been explored and mapped since the early 70’s. In addition to this, I could count on many years of collaboration and reciprocal trust between my research group and the local geological research institute (Osservatorio Geologico di Coldigioco) and the speleological society (Gruppo Speleologico di Fabriano).

More importantly, it has been demonstrated by Longinelli and Selmo (2003) and Giustini et al. (2016) that there is an affinity between Frasassi cave and Lamalunga cave areas, in relation to the mean yearly modern δ18O precipitation signal (Fig. 1.6 B). From the map published by Longinelli and Selmo (2003) and Giustini et al. (2016), it is clear that, despite the latitudinal extension of the Adriatic coast of Italy, no latitudinal δ18O gradient has been found. Thus, at both Frasassi and Lamalunga locations, modern precipitations have similar δ18O composition. Assuming that this relationship was maintained over the past, it is possible to make the hypothesis that Frasassi and Lamalunga past δ18O precipitation signals were similar.

Another appealing justification to choose Frasassi cave as case study, is its location. Frasassi cave is indeed located in a strategic position relative to Corchia cave which is a very well-known cave in speleothem community, that has been largely studied Drysdale et al. (2004); (Drysdale et al., 2005; Drysdale et al., 2007; Zanchetta et al., 2007; Bajo et al., 2012; Bajo et al., 2017) and that is located on the opposite side (the Thyrrhenian side) of the Apennine range. This allows the direct comparison of the two caves records and potentially help to understand different responses to local circulation changes and the related orographic effects over time, in a complex geographical region like the Mediterranean.

In the following sections, information about each of the two caves, not provided in the specific studies of their speleothems, are reported to provide a framework of my speleothem research.

1.5.2 Frasassi cave system: speleogenesis and significance for Earth’s history

Frasassi cave is particularly interesting first because of its geographic position, which is longitu- dinally related to the other cave object of this research, then because of its genesis and exceptional

biota content. Thus, although my thesis is not focused on palaeontological and speleogenetic aspects, the interest in working in this cave system stemmed from its being famous and well apt for safe research in a tourist cave.

Frasassi provides a typical example of galleries developed by sulphuric acid dissolution, where sulphuric acid is derived from the oxidation of H2S present in the geologic setting (Eq. 5). Sulfuric Acid Speleogenesis (SAS) has produced some of the largest caves in the world, like Lechuguilla Cave and Carlsbard Caverns in New Mexico (Palmer et al., 2009). Frasassi ground waters in the aquifer are enriched in H2S and salts due to the rise of mineralized waters from the underlying evaporitic layers of the Triassic Burano Formation. Oxidation happens when sulfidic-rich water meets fresh water forming sulfuric acid that reacts with the carbonate rock causing its dissolution (Eq. 6).

Main SAS chemical reactions (Galdenzi and Maruoka, 2003; Jones et al., 2015) are as follows:

+ + 2- H2S + 2O2 ⇔ H + HSO4– ⇔ 2H + SO4 (5)

+ 2- 2H + SO4 + CaCO3 ⇔ CaSO4 · H2O + CO2 (6)

The final product of the reaction is gypsum (Eq. 6) that replaces the limestone on the walls of the cave in the vadose zone. Gypsum replacement crusts, when desiccated, detach from the walls for gravity and form thick deposits on the floor. These deposits are eventually weathered by gypsum- undersaturated ground water or remain as gypsum floor deposits (Galdenzi and Maruoka, 2003; Ford and Williams, 2007b; Jones et al., 2015).

The other type of groundwater is carbonate-rich and derives from the meteoric water that infil- trates through the limestone. Meteoric infiltration is believed to have played a secondary role in speleogenesis at Frasassi (Galdenzi and Maruoka, 2003). Meteoric water is found in the vadose zone and has low salinity, low sulfate and high dissolved oxygen.

In some cases, sulfide-oxidizing bacteria can have a role in caves enlargement processes. Yet, microorganisms can accelerate the oxidation of hydrogen sulfide much faster than abiotic processes alone, increasing the acid production and the aggressiveness of the water (Galdenzi et al., 1999; Galdenzi and Maruoka, 2003; Engel et al., 2004). Sulfur-oxidizing microbes colonize Frasassi streams and pools, however Jones et al. (2015), found that most of the sulfide lost from cave streams is released in to the cave atmosphere and sulfuric acid is not an important end-

product of microbial sulphide oxidation. This implies that microbiota is not responsible for subaqueous SAS. Predominating degassing in Frasassi flowing waters is only due to high dissolved hydrogen sulfide (H2S) concentration and rapid stream flow.

Due to its sulfuric groundwater, Frasassi cave system hosts a chemolithoautotrophic sulfide-oxidizing microbial ecosystem (like bacteria belonging to the genera Thiobacillus, Acidithiobacillus and Sulfobacillus) whose population structures have been studied during the past 10 years in Sarbu et al. (2000), Vlasceanu et al. (2000), Macalady et al. (2006), Macalady et al. (2007), Macalady et al. (2008). These ecosystems are consistent with the extremely acidic, oxic, aphotic, iron-poor and sulphur-rich geochemistry of this environment comparable to sulfureta at hot springs and deep-sea vents. The interest for these organisms arises because they can be considered analogs for microbially dominated, early earth biotic communities such as those during the early Proterozoic era that might have developed after the initial rise of oxygen (Macalady et al., 2006). Most of these bacterial colonies that have been found in this cave, are present in biofilms covering various types of surfaces like cave walls or air-water and solid-water interfaces. These microbial biofilms appear to be covered by a thick layer of mucous glycocalyx (snottite), often with protruding filaments, which protects the very acidic microhabitat where these organisms live (Vlasceanu et al., 2000). The low-pH liquid created by the sulfur-oxidizing bacteria reacts with the carbonate cave walls releasing carbonic acid together to sulfate minerals (like gypsum and barite).

Peterson et al. (2013) studied the ostracod assemblages found in Frasassi cave, in the adjacent sulfidic spring and in the Sentino river. What they found is an extremely diverse, endemic population (21 species) likely related to the heterogeneity of the groundwater and associated habitats. The interest of this works also relies on the fact that ostracod fauna is unknown for most of the documented sulfidic cave and karst aquifers because these assemblages have been adapting very well to both the toxicity of H2S and the hypoxia in these sulfidic springs and hypogean lakes. Sulfur bacteria, which thrive in these sulfidic waters, provide a limitless and constant food source whereas in non-sulfidic epigean waters the food source normally consists of algae and animal matter.

Sub-fossil eels (Anguilla anguilla) remains have been found “glued” against the rock of Frasassi phreatic lake's bank scattered on the shores up to 5 metres above the water table (Mariani et al., 2007). Those eels were actually permanently living in the cave thanks to the rich endemic ecosystem of invertebrates hosted in Frasassi karst system, which includes amphipods, gastropods and ostracods (Sarbu et al., 2000). Beside the biological interest of this finding, these fossil eels

have been used as a record of the vertical variation of the water table resulting from the tectonic uplift of the Frasassi massif combined to the incision of the Sentino River. The 14C dates of the eels thus, helped to precisely date the uplift rate of the the Apennine area around Frasassi that in the past 8000 years has been rising at a mean rate of 0.6 mm/yr (Mariani et al., 2007).

1.5.3 Lamalunga cave current research status

In this section, I briefly introduce the story of the research project about Lamalunga cave and the current state of the works. Despite the Altamura man discovery happened in 1993, research about the cave has not progressed as much as it would have been expected in over 20 years. What we know about this cave is still mostly based on sporadic on-site observations and photographs and, before the published results from this Thesis, Vanghi et al. (2017), the only peer-reviewed paper was Lari et al. (2015). Most publications relative to the cave are written in Italian and difficult to access by the international community. Currently, the works in Lamalunga cave are led by Prof. Giorgio Manzi from the University “La Sapienza” in Roma in collaboration with Prof. David Caramelli from the University of Florence, Dr. Andrea Borsato, Ass. Prof. Silvia Frisia and my- self from the University of Newcastle. Due to its invaluable importance, it is very complicated to obtain permissions for scientific investigations in Lamalunga cave. On one side, there is the local caving club that, even though very helpful and friendly, is very protective and not open to invasive interventions. On the other side, there are the local academic institutions that claim for their active participation to the project. Because many institutions are involved in the project, it is very complicated to manage the works and obtain all the necessary official permissions from the local Authority for Cultural Heritage (Sovrintendenza dei Beni Culturali della Puglia). The collection of speleothems for paleoclimate studies, that is the core of this PhD thesis, have been carried out during two campaigns, in 2011 and in 2015, when we sampled the coralloid speleothems that I then used for petrological and geochemical analyses. These data are reported and discussed in one published paper and one submitted paper, which are presented in chapters 2 and 3. An exhaustive description of the geological and climatic context for Lamalunga cave is presented in chapters 2 and 3 of this Thesis.

My research is the first of its kind, which uses petrographic investigation to understand past environments and, overall, provides information by interrogating speleothems that are not commonly used as climate archives.

1.6 Thesis aims and objectives

This PhD thesis has as main objective that is of demonstrating the paramountcy of petrographic and stratigraphic investigation in any paleoclimate study based on cave secondary carbonate mineral deposits also known as speleothems. This complies with the current multi-proxy approach to speleothem-based paleoclimate science, and is an indispensable requirement to improve the understanding of the significance of climate proxy data extracted from stalagmites, stalactites and other types of cave formations (Fairchild and Baker, 2012). Thus, although this thesis has four main themes (presented in chapters 2 to 5), each theme deals with a common subject: the advancement of the knowledge of the physical aspect of speleothem.

Specific aims of this thesis are:

- To inaugurate the palaeoenvironmental and climate study of the Altamura man in the absence of suitable, conventionally used, stalagmites from the site and test the validity of coralloid speleothems as archives of climate changes by implementing petrography and microstratigraphy;

- To test the potential of Synchrotron Radiation based micro X-Ray Fluorescence (SR- µXRF) mapping as non-conventional petrographic tool in the investigation of the inter- relation between trace elements, organic compounds and speleothem fabrics;

- To employ materials science techniques, such as neutron diffraction and small angle neutron scattering to characterize the internal structure of speleothems, specifically the crystal orientations (texture), crystal size and crystallite boundaries, porosity as growth-related properties that may influence post-depositional changes and re-setting of significant climate signals.

- To lay the foundations of the palaeoenvironmental and climate study of Frasassi cave combining stable isotope geochemistry data and petrography.

1.7 Thesis structure

The present thesis is structured in 5 chapters that include manuscripts that have been published, submitted or in preparation and two appendixes referring to works presented as posters at the International Union of Quaternary Research (INQUA) 2015 and the American Geophysical Un- ion (AGU) 2016 conferences.

Chapters 1 and Chapter 2 provide the introduction and the background of a multi-disciplinary project, outline the research questions and the strategies taken to answer them. Specifically, the focus of the research, the petrographic investigation, is set in the context of speleothem science and of the specific expertise I developed at the University of Newcastle.

In Chapter 3, which presents the part of my research that has been published in the peer reviewed Journal Sedimentary Geology (Vanghi et al., 2017). I investigated the processes involved in the formation of non-conventional speleothems, known as coralloids, found in Lamalunga cave. This research is at the basis of subsequent palaeoclimatic and palaeoenvironmental reconstruction from the petrographic characteristic of these geological archives. This approach has been necessary because many theories were proposed regarding the formation of these speleothems that contributed to create confusion regarding their ontogenesis. Furthermore, due to their small dimensions, coralloids have been commonly discarded for palaeoclimate investigations. Thus, there was the need to comprehend the mechanisms of formation of Lamalunga coralloids formation prior to start interpreting them as climate archives that may potentially provide an environmental context to the Neanderthal found inside the cave. My petrographic investigation was coupled with microstratigraphy using descriptive tools devised by the speleothem research group at UoN, whose unique expertise is in carbonate petrography. I was able to support the hypothesis that coralloid formation is linked to the effects of hydroaerosols generated from the fragmentation of nearby drip water and evaporation. Fabric changes allowed me to reconstruct the environmental context during growth, which post-dates the Neanderthal death. Mineralogic and petrographic information also demonstrated that coralloids from Lamalunga are pristine, that is, they were not subjected to diagenetic processes. This is crucial in any palaeo-investigation from speleothems, because diagenesis may alter their most important characteristic: the preservation of ages. This is particularly true when, as in the case of Lamalunga coralloids, their ages is used to date unique skeletal remains. Consequently, I could confirm that their U-Th analyses produced reliable ages. Chronostratigraphy, which consists on the identification of similar phases of growth and types of

laminae over diverse specimens, was then performed to correlate time equivalent layers and ex- tend the chronological constraint to coralloids that could have not been dated, due to their reduced dimensions. Thus, the paper contributes to the paleo-sciences by providing researchers with the tools by which non-conventional speleothems can be used with confidence to date archeological and palaeoanthropological findings.

Chapter 4 is a continuation of the study presented in Chapter 3, which can be considered its foundation. In fact, in Chapter 3 I demonstrated that elemental incorporation and crystal growth have a mutual dependency. The originality of the work presented in Chapter 4 relies on the application of Synchrotron-Radiation based micro x-ray fluorescence (SR-µXRF), which allows investigating the spatial distribution of the non-Ca elements in speleothems at an unprecedented scale (Frisia et al., 2018). For low Z elements (like Mg, Si, S and P) present in concentrations < 1000 ppm, we applied conventional micro XRF. I used SR-µXRF and micro-XRF maps as useful and original tools to complement petrographic observations through their 2D (two dimensional) representations of trace elements distribution. Commonly, in speleothem research trace element investigation is carried out by running a single transect using laser ablation inductively-coupled plasma mass spectrometry (LA-ICPMS). This is not sufficient to highlight the lateral variability that I present in Chapter 4. The study revealed that elemental incorporation in Lamalunga coralloids is influenced by climate, discrete environmental events and hydrophysical characteristics of the cave that control evaporation and aerosol transport. To achieve such understanding, I analysed two different morphologies of coralloids that grew under different hydrological conditions, which then resulted in a diverse trace element concentration distribution. The research highlighted that in the typical “evaporative” coralloid trace elements are three times more concentrated than in coeval flowstone-like morphology, which suggests more abundant availability of water. Thus, the two petrographic tools, one optical and the other chemical, contributed to provide the hydroclimate significance of speleothem morphology. Moreover, I observed that Siliconis present in ele- vated quantity relative to more conventional speleothems (stalagmites), and maps show that it is intimately associated to particulate. I could deduce that Si could have caused the ineffective coalescence of the crystalline margins that resulted to the opening of the fabric. It is also speculated that Si could derive from volcanic ashes, produced by explosive Southern Italian volcanism that has been particularly active during the past 200 ka. Its overarching achievement is that coralloids identify a potential archive of volcanic eruptions in a region adjacent to Campi Flegrei, one of the few “super-volcanoes” in the World. This opens the pathway to use Lamalunga coralloids as archive of catastrophic eruptions, with potential for the study of their recurrence. The content of this chapter is part of a manuscript in revision, submitted to Sedimentology Journal.

Chapter 5 identifies the most innovative part of my research, where I applied material sciences techniques to speleothem science. It is a pilot study on speleothem fabrics characterization that uses neutron tomography, small-angle neutron scattering (SANS) and strain scanner, all performed at the Open Pool Australian Lightwater (OPAL) reactor of the Australian Nuclear Science and Techonology Organisation (ANSTO). The main scopes were to investigate the internal structural features of different speleothems below the micrometre scale. This research stemmed from recent findings by our group relative to speleothem nucleation and growth, which discovered that some speleothems consist of nanocrystal aggregates as seen with the Transmission Electron Mi- croscope (TEM) (Frisia et al., 2018). It also explored another recent finding, that of “criptic diagenesis” (Bajo et al., 2016) in speleothems where dissolution and re-precipitation may occur in small interconnected pores. Diagenesis investigation in speleothems fabric is important because it can compromise the original climate imprint and thus the palaeoclimate interpretation of the geochemical signal archived in the calcite. The results of this pilot study are preliminary, none- theless, some have overarching implications for the understanding of how speleothem growth. Firstly, in terms of technology, I have found that neutron tomography does not produce any further improvement to what is already offered by x-ray tomography (XCT) (for further details refer to the application of XCT in speleothems explained in Vanghi et al. (2015), Walczak et al. (2015) and Chawchai et al. (2018)). The acquisition time for high-resolution investigation is preferred in XCT, which is also more accessible considering that it does not require a nuclear source like neutron tomography. By contrast, the results obtained from the diffractometer and the small-angle neutron scattering (SANS) revealed that preferred crystallographic orientation varies, most likely according to different crystallization pathways. The SANS further permitted to estimate and eventually quantify the properties of particles (e.g. nanocrystals) and their aggregates, like the morphology, the porosity and the surface area (Besselink et al., 2016). The internal structures of the different carbonate samples analysed revealed complex morphologies and different degrees of fractal nature. This find elegantly fits within the framework of the non-classical crystallization model, which suggests that crystals may form because of the aggregation of primary particles (nanocrystals) (De Yoreo et al., 2015; Frisia et al., 2018). The overarching conclusion is that speleothems (or some speleothems) may indeed consist of nanocrystal aggregates, which opens up a whole new interpretation of their trace element and organic particulate incorporation, thus explaining phenomena that were previously ascribed to “kinetic effects. Chapter 5 is the core of a manuscript in preparation.

In Chapter 6, I employed petrography in support to the interpretation of geochemical data in a stalagmite formed of pristine columnar calcite sampled at Frasassi cave (Central Italy). The stalagmite under study, FR16, offers a continuous 16 ka stable isotope record spanning from 112.8

±1.5 ka until 96.6 ±1 ka, corresponding to the marine isotope stages MIS 5c and MIS 5d (Lisiecki and Raymo, 2005). The period covered by FR16 is encompassed in the last glacial inception (MIS5e to MIS4), which is an interesting climatic phase but that received less attention in comparison to other transitions, specifically terminations (end of a glacial cycle). Nevertheless, shifts towards glacial conditions are important to understand the causes of global changes. Specifically, considering that the warm period preceding the plunge into cold conditions recorded by Frasassi stalagmite (MIS5e or Eemian) is an analogue to the present warm period (Milankovitch, 1941; Kelly et al., 2006; Ganopolski et al., 2016).

FR16 δ13C trend, which is a measure of vegetation response to rainfall, highlighted two major shifts toward aridity linked to DO (Dansgard-Oeschger) events recorded by the Greenland Ice Cores and known as GS24 and GS25. Petrographic observations coincide only partially with these conclusions indicating that in-cave hydrophysical processes would have prevailed during crystals growth.

The δ18O signal, which usually is mainly influenced by the isotopic composition of the rainwater, does not record the DO cycles as well as the carbon isotope ratio profile. The trend of the oxygen appears thus attenuated by a factor/s contrasting the effect of the cold and arid stadials, which I interpreted as due to changes in insolation.

One of the most interesting conclusions that can be drawn from the Frasassi record, is that its δ18O signal is more negative compared to other available, coeval speleothem records from the Mediterranean, and particularly those from the Tyrrhenian coast (Drysdale et al., 2005; Drysdale et al., 2007; Columbu et al., 2017) and the Eastern Mediterranean (Bar-Matthews et al., 2003). This difference has been explained as due to different storm track history and moisture source origin, which highlights complex climate dynamics for the region. The main conclusion of this part of my research is that trajectories and moisture provenance influence the rainfall O isotope ratio (and thus the speleothem signal) more than the amount effect (more rain = more negative δ18O signal). Chapter 6 is part of a manuscript accepted in Quaternary Science Review and currently under review.

Chapter 6 summaries the findings of this research. Chapter 7 presents the future developments expected from this thesis.

In Appendix I, I present some preliminary results in the framework of a broader study whose objectives are to (1) timing the development of the different karstic levels of Frasassi cave, (2) calculate the uplift rate of the area and subsequently (3) reconstruct the climate evolution of the

Italian Adriatic coast during the Quaternary. The first two tasks have been accomplished by dating the three stalagmites, spanning a ~260,000 years period from MIS9 to MIS5, to determine the minimum age of the karstic level in where they were growing. For the last objective, we compared the stalagmites growth rate with the petrographic observations and the δ18O global record (Lisiecki and Raymo, 2005) to have an insight onto the hydroclimatic and thermic conditions of the Adriatic region.

In Appendix II, I present an introductory study relative to the δ13C interpretation variation of the oldest Frasassi cave records, which spans from MIS7 to MIS10 (195 ka to 354 ka). Isotopic data have been complemented by greyscale profiles from high resolution images of stalagmites coupled with petrographic observations. A clear correspondence occurs when all these three variables are compared together suggesting that the carbon variations are in part influenced by the same mechanisms that control the crystal growth. My plan is to publish this material in the following 12 months (2018-2019).

1.8 References

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Webster, J.W., Brook, G.A., Railsback, L.B., Cheng, H., Edwards, R.L., Alexander, C., Reeder, P.P., 2007. Stalagmite evidence from Belize indicating significant droughts at the time of Preclassic Abandonment, the Maya Hiatus, and the Classic Maya collapse. Palaeogeography, Palaeoclimatology, Palaeoecology 250, 1-17.

Wong, C.I., Breecker, D.O., 2015. Advancements in the use of speleothems as climate archives. Quaternary Science Reviews.

Chapter 2. Genesis and microstratigraphy of calcite coralloids analysed by high resolution imaging and petrography

-Vanghi, V., Frisia, S., Borsato, A., 2017. Sedimentary Geology 359, 16-28.-

2.1 Abstract

The genesis of calcite coralloid speleothems from Lamalunga cave (Southern Italy) is here investigated from a purely petrographic perspective, which constitutes the basis for any subsequent chemical investigation. Lamalunga cave coralloids formed on bones and debris on the floor of the cave. They consist of elongated columnar crystals whose elongation progressively increases from the flanks to the tips of the coralloid, forming a succession of lens-shaped layers, which may be separated by micrite or impurity-rich layers. Organic molecules are preferentially concentrated toward the centre of convex lenses as highlighted by epifluorescence. Their occurrence on cave floor, lens-shaped morphology and concentration of impurities toward the apex of the convex lenses supports the hypothesis that their water supply was hydroaerosol, generated by the fragmentation of cave drips. Evaporation and degassing preferentially occurred on tips, enhancing the digitated morphology and trapping the organic molecules and impurities, carried by the hydroaerosol, between the growing crystals, which became more elongated. Micrite layers, that cap some coralloid lenses, likely identify periods when decreasing in hydroaerosol resulted in stronger evaporation and higher supersaturation with respect to calcite of the parent film of fluid. This interpretation of coralloid formation implies that these speleothems can be used to extract hydroclimate information.

2.2 Introduction

In Lamalunga cave (Southern Italy), which is best known for the discovery of a complete fossilized Neanderthal skeleton, coralloids are the most common speleothem type, and due to their association with bones, they had been used to date the fossil (Lari et al., 2015). Coralloid speleothems, also known as cave popcorns (Hill and Forti, 1997), consist of knobs with digitate form, usually growing in clusters. Their internal structure is characterized by layers of calcium carbonate, which gradually thicken from the depressions toward the protrusions. Coralloids speleothems are one of the most common speleothem type found in caves after stalactites, stalagmites and flowstones (Hill and Forti, 1997). Usually, coralloids are calcite (Gradzinski and Unrug, 1960; Webb, 1994; Hill and Forti, 1997; Niggemann et al., 1997; Dublyansky and Dublyansky, 1998; Cuevas-González et al., 2010; Leél-Őssy et al., 2011; Merino et al., 2014; Richter et al., 2015; Ammari et al., 2016) but they can also be aragonite (Thrailkill, 1968), in most of the cases alternating with calcite (Bar-Matthews et al., 1991; Cañaveras et al., 2001; Ortega et al., 2005; Caddeo et al., 2015; Martín Pérez et al., 2015). Speleothem-bearing caves can also develop in non-carbonate bedrocks yielding silicate speleothems and among them, coralloids have also been reported. Siliceous coralloids have been found in sandstone (Wray, 1997; Lundberg et al., 2010), granite (Willems et al., 2002) and also lava tube (Miller, 2015; Miller et al., 2016).

Nucleation and growth of coralloids are controlled by “substrate selection” Self and Hill (2003), a process described as when “the mineral individual (or mineral aggregate) growing from a convex substrate protrusion during competitive growth will continue its growth at the expense of its neighbours growing from flat or concave surfaces”. One of the main differences with other conventional speleothems is that coralloids ontogenesis is not linked to dripping water. Different mechanisms have been invoked to explain coralloid formation, which include: 1) spray from splashing drops producing hydroaerosols, which in turn supplies the fluid that feeds the nascent coralloid (Gadoros and Cser, 1986; Dublyansky and Dublyansky, 1998), 2) high evaporative conditions and capillary film of water, which moves from the base toward the prominence of the speleothem (Gradzinski and Unrug, 1960; Self and

Hill, 2003; Caddeo et al., 2015) and 3) strong CO2 degassing (Thrailkill, 1976). Evaporation and CO2 degassing of the parent water is greater at the speleothem protuberance, resulting in greater carbonate precipitation at the apex of convex surfaces (Thrailkill, 1976; Caddeo et al., 2015), due to the high surface to volume ratio. The apex can then be larger than the coralloid base resulting in a grape-like form (Hill and Forti, 1997). Ventilation is believed to favour the growth of coralloids showing a preferred orientation facing upwind direction, by enhancing degassing and, consequently, the saturation state of the film of fluid relative to a phase of calcium carbonate (Onac and Forti, 2011). Studies on cave microbial communities have also revealed that some cave bacteria may contribute to coralloid formation. In particular, Banks (2010) isolated 51 culturable bacteria from a coralloid speleothem with

the aim to test their ability to precipitate calcium carbonate in a nutrient-poor, Ca-rich environment. They discovered that numerous bacteria colonizing coralloid speleothems counteracted calcium toxicity by calcification, thus providing nuclei for subsequent calcite growth (Banks et al., 2010). Critically, microbial metabolism has been also hypothesized as potential mechanism in the formation of coralloid speleothems consisting of calcium carbonate and silica (Legatzki, 2011).

Coralloids can also grow underwater, in cave pools. In this case, their formation requires mass transfer by diffusion from the bulk water, which is favoured by still water (Caddeo et al., 2015). Similarly, to their subaerial counterparts, subaqueous coralloids show higher convexity towards the centre of each

“finger” because carbon dioxide is lost at the surface of the water table and the gradient of CaCO3 supersaturation decreases with depth (Hill and Forti, 1997).

The importance of coralloids is that they can be the only speleothem present in a cave, or that they occur, as in the case of Lamalunga cave, in association with bones or artefacts of Cultural Heritage interest. Being small, their removal has little visible impact compared to sampling of whole stalagmites, and they can be dated by U-Th method (Aubert et al., 2014; Lari et al., 2015). Thus, these speleothems can be a valid alternative to the removal of stalagmites to put an age on events. Little is known, to date, about their possibility to yield additional information in their petrographic and chemical characteristics. Caddeo et al. (2015), for example, analysed the δ13C and δ18O signatures of coeval layers sampled along coralloid digitate accretions stemming from stalactites and mostly composed of aragonite. They show that, due to more intense evaporation upon the convexities, the δ13C and δ18O fractionation is enhanced compared to depressed zones. Their results, therefore, would implicate that the chemical signature of these speleothems is strongly modified by kinetics, which would hinder their use as palaeohydrological archives. Furthermore, Ortega et al. (2005) and Devès et al. (2012) used a coralloid characterised by alternating aragonite and calcite to study the remobilisation of uranium and strontium, which can lead to errors in age dating with the U-series dating method. These geochemical approaches suggest that the crucial step to the characterization of the significance of coralloids as archives of the “radiometric” clock and paleohydrology is the characterization of their petrographic characteristics as related to their mechanisms of formation and/or alteration.

In the present study, we focus on the physical aspect of the coralloids collected at Lamalunga cave to elucidate their accuracy as chronological tools and investigate the potential of their fabrics as archive of hydrological changes in the cave. Specifically, we here hypothesize that fabrics of Lamalunga coralloids are linked to aerosol-induced deposition, which then implies that they are related to infiltration and drip activity in the cave. Our petrographic and mineralogical data also demonstrate that the coralloids from Lamalunga formed as calcite and were not subject to significant early and/or late diagenetic processes resulting in U-leaching (Bajo et al., 2016) and consequently, their U/Th analyses can produce

reliable ages. Finally, it is shown that a microstratigraphic approach for coralloids could be very useful to correlate time equivalent layers, thus allowing extending the chronostratigraphy over diverse specimens.

Fig. 2.1 A) Location of Lamalunga Cave near Altamura (Southern Italy). B) Plan and schematic cross section of Lamalunga Cave. The orange dotted line represents the plan of the cross section. The locations of the samples here studied (ABS5 and ABS6) are also indicated. ABS3 coralloid, studied in Lari et al. (2015), was sampled in the same chamber of the “Altamura Man” skeleton. Coralloid speleothems are represented with small dots. The original entrance was a vertical shaft now completely filled by limestone debris. The actual entrance to the cave was artificially enlarged. C) View of the hill where the cave opens to the surface (black cross).

2.3 Geographic setting and coralloid occurrences in Lamalunga Cave.

Lamalunga cave (40°51'51.9"N 16°34'31.3"E, 508 m a.s.l.) (Fig. 2.1A, B), is located near the town of Altamura, in Puglia, a region of Southern Italy, ca. 50 km from the Adriatic Sea and ca. 70 km from the Ionian Sea. Climate is Mediterranean, characterized by wet winters and autumns (rainfall mainly concentrated during October-December), and dry summers (July is the driest month). Mean annual precipitation ranges from about 500 to 800 mm (Brandimarte et al., 2011) with mean precipitations < 30 mm from June to August (data from S.I.M.N., Italian National Hydrographic and Mareographic Service,

Bari compartment, Cassano Murge weather station over the period 1921–1990) (Andriani and Walsh, 2009). Today, the vegetation above the cave consists of grasslands and the soil is not well developed, and restricted to pockets between exposed rocks (Fig. 2.1C). Rather unfortunately, the whole karst in Puglia has been strongly impacted by human activities through history, with stone clearing, quarrying and deforestation, which changed both surface and underground drainage (Andriani and Walsh, 2009). This suggests that the present day setting is not representative of the conditions under which most of the studied samples formed.

Lamalunga cave is a small (less than 100 m long) (Fig. 2.1B), shallow gallery cut into the well-bedded Calcare di Altamura limestone of Upper Cretaceous (Coniacian) age (Zezza, 2000) where, in 1993, a complete Neanderthal skeleton was discovered (Fig. 2.2A). The development of the cave gallery is sub-horizontal and gently dips to a NE direction. Today, rainfall percolates through soil pockets and highly fractured bedrock. It must be also mentioned that, currently, access to the cave is via an artificially enlarged pit closed by a hatch. By contrast, the entrance at the time of the Neanderthal dwelling was likely through a 20 m deep shaft, which is now obstructed by a limestone debris forming a talus cone along the main gallery (Fig. 2.1B). Sub-horizontal bedding planes, fractures and joints of the bedrock favoured detachment of blocks from the ceiling and walls, and formation of numerous collapse deposits in the cave. The cave floor and part of the walls are also coated by red clay, which most likely infiltrated into the cave from the soil zone.

Spot microclimate measurements were conducted within the cave in mid-February 2011 and early Sep- tember 2015. The temperature of the cave air fluctuated around 15.5 ± 2.5°C. Relative humidity was circa 100% in winter but decreased to 97.0 ±2.5% at the end of summer. The CO2 concentration in the air had almost atmospheric values (350 to 410 ppmv), without significant differences throughout the cave. Perceivable air currents were not detected during both surveys.

Today, water infiltration into the cave has a seasonal trend, and during summer, the cave is usually almost dry. Infiltration recovers a few days following major rainfall events and activates stalactite drips feeding small pools and flowstones for several weeks. Coralloids are mostly concentrated in the northern section of the cave (small dots in Fig. 1B), where an impassable and very narrow fissure connects with another passage which remains still unexplored (Zezza, 2000; Agostini, 2011). Coralloids form on bedrock or stalagmites and flowstones protrusions, and in the “abside dell’Uomo”, the small chamber hosting the Neanderthal skeleton, coralloids almost entirely cover the floor and the bones. It is noteworthy to note that coralloids grow preferentially on the protruding supraorbital torus, the projecting nasal bone and midface bones. Similarly, coralloids abound on ridges of the skeleton long bones and on fossil animal bones scattered on the floor of the northern section of the cave (Giacobini, 2011). Several different morphologies of coralloids have been found at Lamalunga cave: rounded to elongate

cylindrical (Fig. 2.2D-E) and arborescent-branched morphologies (Fig. 2.2C-F). All coralloids in Lam- alunga cave do not grow following a preferential orientation, which commonly is upwind.

Fig. 2.3 Images of samples ABS5 and ABS6. A) Polished slabs; B) complete thin sections in PPL; C) complete thin sections in XPL; D) Schematic interpretation of the principal features observed in the samples. Some crystals boundaries and major margins defining groups of crystals with same c-axis orientation are represented with thin blue and black lines respectively. Thick black lines visualise the boundaries between the main microstratigraphic growth phases. Orange lines indicate possible hiatuses identified by dissolution/corrosion features. The black arrow in ABS6 panel D, indicates crystals becoming wider moving from the central axis towards the flanks: from fiber-like Ce (Fb) crystals to Ce crystals. Red layers correspond to micritic layers rich in Fe-oxides. Green rectangles indicate the regions analysed with fluorescent light. In ABS6, organic-rich bands are coloured with different

shades of brown depending on the intensity of fluorescence emitted: e.g. darker brown parts are the most fluorescent. On the left side of ABS5 are also indicated the U/Th ages with 2σ errors published in Lari et al. (2015). Fabric codes: Ce = columnar elongated; M = micrite; OM = organic-rich layer; Ms = microsparite. Abbreviations: Fls = flowstone-like; Crl = coralloid; I. Crl = incipient coralloid.

2.4 Materials and Methods

Two coralloids, ABS3 and ABS6 (Fig. 2.3), were collected from the human skeleton and from a fragment of bedrock, respectively. The age of ABS3 is known and reported in (Lari et al., 2015), whilst for ABS6 radiometric dating was not carried out. An incipient coralloid, characterized by a smoother morphology and grown on a broken stalagmite, ABS5, was collected following the rationale that it could be coeval with ABS3, but have thicker and more continuous layers, which could be radiometrically dated and offer robustness to the hypothesis that coralloid yield accurate ages. Critically, ABS5, once dated by U-Th method, revealed some growth phases that are time equivalent with ABS3. ABS5 growth spans from 121.9 ka to 7.6 ka, whereas ABS3 grew from 130.1 ka until 7.04 ka (Lari et al. 2015).

For the present study, the three coralloid samples were embedded in epoxy resin to minimize damages to fabrics during cutting, then sectioned along their vertical growth axis, and polished by using diamond- polishing paper of progressively finer grits. The polished samples were subsequently imaged by using an Epson Expression 11000XL digital scanner scanning at 2400 dpi, which enables to highlight microstratigraphic changes at the scale of the whole specimen. On digital images, we identified samples of interest for petrographic observations. These were carried out on thin sections 30 µm thick by using a Leica MZ 16A stereomicroscope and a Zeiss Axioplan microscope in polarized light (PPL) and cross polarized light (XPL) at the University of Newcastle, Australia. The thin sections were made only for ABS5 and ABS6 because ABS3 was too small and would have broken during the cutting procedures compromising the feasibility of other analytical techniques. Fluorescent light images stimulated at blue (488 nm) and green (543 nm) wavelength lasers were obtained by using a Zeiss Axio Imager A1 fluorescence microscope with an LED Colibri controller and Olympus software at the University of New- castle, Australia. Given that a suitable method for fluorescence standardization is still unavailable, focal plane depth, laser power and exposure time were held constant during the acquisitions in an attempt to normalize image intensities (Orland et al., 2012). A short exposure time of 825.8 ms was used in order to avoid images, which were too bright and might not reflect the real fluorescence intensity (Waters, 2009). The images were obtained by simultaneous excitation using both blue and green wavelength lasers. A series of 6 and 8 overlapping images (3400 µm x 2550 µm) for ABS5 and ABS6a respectively, were collected with a 2.5x objective along the working traverse of each sample (Fig. 2.4).

As a classification of fabrics for coralloids does not exist, the Lamalunga sample fabrics were classified following the criteria proposed for stalagmites and flowstones in Frisia (2015), which may, however, be inadequate, as the feeding mechanism for coralloids is clearly not related directly to drips. Disconti- nuities within the samples were classified according to Martín-Chivelet et al. (2017). Microstratigraphy, which describes a succession of growth phases bound by distinctive surfaces that can be traced across several samples, was reconstructed by identifying sequences of fabrics changes, including fluorescent observations (Fig. 2.4) in the three samples and by correlating fabrics with similar characteristics.

In addition to standard and fluorescence petrography, greyscale values were used to refine microstratigraphy. The greyscale allows visualizing colour changes in thin sections, which are diagnostic of fabrics (open vs. compact, impurity ridden or impurity free) and type of discontinuities along the vertical growth axis (Fig. 2.4). Each pixel value represents the level of grey intensity of the 16-bit images, ranging from black (value 0) to white (value 255). Grey pixel values were measured on high resolution RGB (red, green, blue) images of polished slabs observed under transmitted and fluorescent light. Grey values were obtained as average of the intensities of the red, green and blue light (grey level= 0.299R + 0.587G + 0.114B) in each point along the line-scan (Muangsong, 2011; Duan et al., 2014). The values were calculated using the ImageJ software, along a line-scan in which each 4 μm step corresponds to 1.6 pixels. A 10-average points smoothing was applied to the greyscale values to capture the most important patterns of the data and to reduce the noise.

The mineralogical composition of the coralloids was determined via Raman spectroscopy at the Mark Wainwright Analytical Centre, University of New South Wales (Australia), using a Renishaw inVia 2 Raman Microscope equipped with a 532 nm (green) diode laser. Twenty spot measurements (spot diameter 1.5 µm) were carried out on polished sections, guided by the microscope system, which allows to recognize fabrics. The spot measurements were conducted on all major coralloid fabrics identified at the optical microscope, and specifically on fiber-like crystals, which could have been interpreted, by their morphology as seen in the hand specimen, as aragonite.

Fig. 2.4 Coupled PPL (parallel polarizers) thin sections and fluorescence maps (2 wavelenghts combined) for ABS5 and ABS6. The corresponding fluorescence and greyvalues line scans were carried out along the dashed vertical lines. Fabrics and major discontinuities for the thin sections are labelled as follows: Ce = columnar elongated; M = micrite; OM = organic-rich layer; MS = microsparite. To facilitate their correlation of greyscale values and fluorescence intensity, diverse fabrics have been highlighted by diverse coloured bands. Blue has two different shades, dark and light, which are intended to reflect higher (++) or lower (+) concentration of organic material (OM) and higher (ff) or lower (f) fluorescence intensity respectively. The presence of clean, compact Ce calcite parts is illustrated as white bands. Hiatuses are marked by black (clearly recognizable gap) and blue (possible gap) dotted lines. Note that the fluorescence intensity scales are inverted.

Fig. 2.5 A) Ensemble of cylindrical coralloid growing on a bedrock fragment collected from the cave floor. The location of sample ABS6 is marked by a white box; Coralloids speleothems can assume many different shapes and forms and their internal structure is commonly highly laminated: B) Cylindrical coralloid with incipient branching termination; C) complex globular coralloid; D) Cone-shaped coralloid which was growing on the Ne- anderthal skeleton (Lari et al., 2015).

2.5 Results and discussion

2.5.1 Microstratigraphy

Coralloids in Lamalunga cave can have many different shapes, from rounded to arborescent and their internal structure is commonly laminated (Fig. 2.5). Specimen ABS6 was part of an aggregate of connected cylindrical coralloids grown on a bedrock fragment. However, at the extremities of the aggregate, the calcite crust shows relatively smoother surfaces, resembling a flowstone at a smallest (mm) scale. ABS6 consists of two digitated coralloids (Fig. 2.3; Fig. 2.5A) towards the convex morphology of the substrate however on its left hand side it shows stacking of flat and quasi-parallel layers typical of a flowstone. Thus, ABS6 has been here labelled “flowstone-like” to distinguish its morphology from the cylindrical (cuspate) coralloid morphology (right hand side) (Fig. 2.3).

Sample ABS5 also shows a flowstone-like morphology. However, its internal structure illustrates that its growth may have commenced as an incipient coralloid, as clearly marked by the cuspate trend of dark laminae laminae at ca. 4 mm from the contact with the substrate (Fig. 2.3). Subsequently, ABS5 evolved into a morphology characterized by isopachous layers. Both ABS5 and 6 show visible banding and lamination. In the sagittated, cylindrical morphology of ABS6 and lower part of ABS5, laminae are clearly visible and regularly spaced for almost their entire length, whereas in the isopachous “flowstone- like portion, laminae are faint and concentrated in bands. Both specimens grew on red, iron-oxides rich clay cemented by micrite and microsparite (Fig. 2.6H).

In both ABS5 and ABS6, the boundaries between laminae are marked by dark surfaces bearing evidence of corrosion and/or dissolution, which yield to micro-topographic features, like “mesa-like” surfaces (Fig. 2.6A-B, E) and micro-cavities (Fig. 2.6D blue arrows). These dark laminae appear to consist of micro-sparite or micrite associated with impurities of both organic and inorganic origin similar to those documented in stalagmites from temperate settings (Frisia and Borsato, 2010; Frisia, 2015). In some cases, dissolution/corrosion is limited to small regions (like for example on the protruded areas), in other cases it can be observed all along the coralloid surface. Dissolution/corrosion in ABS5 is especially evident in the last set of dark layers at ca. 4 mm from the top (Fig. 2.6A-C). In ABS6, erosion features are mainly observed where dark laminae are predominant (Fig. 2.6G-F). Some of the dissolution/corrosion layers can be confidently classified as hiatuses, particularly when the evidence of dissolution (corrosion) can be followed on the entire fossil surface of the speleothem (Figs. 2.3, 2.4).

The cylindrical coralloid portion of ABS6 shows bands of <10 µm thick laminae consisting of clean, transparent calcite capped by a black veil of micrite and organic matter (Fig. 2.2, 2.6F). Flat crystal tips

mark boundaries between the white and the dark layers, and the subsequent generation of clean crystals grows in optical continuity with the dark impurities-rich substrate (Fig. 2.7F) (Grigor’ev, 1961; Kendall, 1978; Self and Hill, 2003). In the cylindrical morphologies, white/dark laminae sequence fades away moving toward the outer flanks, where lamina couplets become almost indistinguishable. In practice, the dark layers taper out towards the flank of the cuspidate coralloid, resulting in a lenticular shape for the dark band (Fig. 2.3, 2.6F). By contrast, the compact, clean calcite bands appear to be isopachous from the apex to depression of each cylindrical coralloid. Typically, the cylindrical coralloids either merge or are separated by a pore space, which gets larger towards the bottom (Fig. 2.3; Fig. 2.2A).

The greyscale values in the thin sections vary between 25 (darker) and 250 (brighter) and reflect visible changes in the way the light is transmitted through the sample according to porosity and impurity content. The specimens have a diverse colour in the hand specimen and in thin section, with honey-brown (dark), non-laminated parts coinciding with the brighter greyscale levels in thin section, which pertain to compact, dense translucent calcite (Fig. 2.3). By contrast white or yellowish lenticular layers showing visible lamination in the hand specimen, coincide with low grey scale levels corresponding to the “dark”, impurity-rich, laminated fabric (Fig. 2.4).

Fluorescence intensity ranges from 5 (no fluorescence) and 120 (high) (Fig. 2.4). By using the same imaging settings, ABS6 fluorescence intensity seems to be twice that of ABS5. It is reasonable to infer that ABS6 has a higher concentration of organic compounds (Baker A., 1996), which is consistent with the positive correlation between grey scale and fluorescence.

Irregular micrite layer at the base of ABS6. The red colour is caused by the presence of possible iron oxides. Scale bars correspond to 0.5 mm for A) and F), to 100 µm for B), C), D), E), H), and G).

Fig. 2.7 Thin sections photos taken under PPL (A, C, E) and XPL (B, D, F). A) and B): columnar elongated fabric (Ce) in ABS5. Dark micritic and organich rich layers interrupt the growth of the crystals. C) and D) ABS6 fiber- like crystals with evidence of spherulitic-type growth. The lamina couplets are formed by micrite (M) and organic rich dark layers alternating with clean compact calcite layers. E) ABS6 acute crystal terminations, which suggest a relatively thick film of fluid; and F) ABS6 flat crystal tips and some possible eroded surfaces capped by a dark material conceivably containing organic matter (see text for details). Scale bars are 0.5 mm for A) and B) and 100 µm for C), D), E) and F).

2.5.2 Mineralogy and petrography

Raman spectroscopy mesurements revealed that the studied coralloids consist entirely of low magnesium calcite. No aragonite spectra were identified, even in areas consisting of “acicular” or “fiber-like” crystals. This confirms petrographic observations, which did not show aragonite relicts embedded in the calcite (Frisia et al., 2002). The pristine succession of lenticular and isopachous laminae of columnar crystals, without evidence for mosaic fabric, is further evidence of the primary origin of the calcite. The only indication of diagenesis is related to the dissolution features, which, however, are restricted to single laminae and do not cut across several layers (Fig. 2.6).

Samples ABS 5 and 6 are formed by elongated columnar (Ce) calcite fabric (Frisia, 2015) (Fig. 2.7A- B) consisting of columnar crystals with length to width ratio >> 6:1. The boundaries between crystals are commonly straight and the calcite is compact, with no clear evidence of intercrystalline porosity. The portions of the samples characterized by proper Ce crystals are translucent when observed in polarized light (PPL) and do not emit fluorescence when irradiated with fluorescent light (Fig. 2.3). Crys- tals surfaces are usually coated by a dark veil, whose nature is likely to be micrite associated with impurities (Fig. 2.4A-G), as observed in stalagmites (Frisia and Borsato, 2010).

Compared to ABS5, which is entirely formed by compact Ce, ABS6 shows a sub-type of elongated columnar fabrics with very thin, elongated crystals (fiber-like) seeming to radiate from a centre, similarly to spherulitc growth (Fig. 2.5C-D). The extinction of these crystals, arranged in fan-like structures, is sweeping and similar to that of fascicular-optic calcite (Cfo) described by Kendall (1977), which is defined as an aggregate of crystals characterized by sweeping extinction from the centre outwards (sensu Richter et al. (2011) and Frisia (2015)). The crystals are arranged in bundles, with slightly di- verging lattice orientation, typically observed in crystal-split growth, which has been ascribed to the presence of foreign ions such as Mg (Folk, 1974; Kendall, 1977). Fiber-like crystals tips are both acute or flat (Fig. 2.5E-F). This fabric in ABS6 is only present in the convexities and the crystals become thinner by moving toward the growth axis where they acquire a highly elongated, fibre-like aspect (Fig. 2.2C-D). Toward the flanks, the crystals are wider, progressively resembling the elongated columnar type. Fluorescence is high in correspondence to fiber-like crystals (Fig. 2.3), which suggests that the development of the fabric may be related to the presence of organic compounds (Kendall, 1993; Morse et al., 2003; Banks, 2010).

Micrite (M) and micro-sparite (Ms), whose crystal sizes range from less than 2 µm to between 2 and 30 µm respectively, are associated to dark horizons (Fig. 2.6A-G), likely ridden with presence of organic compounds, as indicated by their bright fluorescence (Fig. 2.4), and, in some cases, also containing iron

oxides (red colour) (Fig. 2.6H). Likely, particulate was abundant at the time of their formation, which confirms the hypothesis that micrite formed when nucleation sites are more bountiful than during the time of formation of the other fabrics (cf. Freytet and Verrecchia, 1999). Furthermore, evidence of corrosion or dissolution associated with micrite layers, and bright fluorescence also suggest that development of this fabric may have been mediated by the presence of microbes, as hypothesized in the case of stalagmites (Frisia et al., 2012; Frisia, 2015). Microsparite could then be the product of dissolution and replacement of micrite in a very early stage or diagenesis (Frisia, 2015) interrupted then by the deposition of the successive columnar calcite layer.

Fig. 2.8 A) Sketch representing the processes involved in the formation of coralloid speleothems in Lamalunga cave. When a drop hits a surface inside the cave, it splits in smaller droplets which then bounce and spread in the surrounding. If an air flux (blue arrows) is present inside the cave, hydroaerosol is produced downwind. B) Sketch representing the mechanisms controlling the formation of lenticular dark bands and white isopachous bands. When

the concretion is fed by hydroaerosol the water supply is limited (Case 1). Long periods of evaporation (red arrows), helped by ventilation (indicated by blue arrows), is the principal mechanism controlling the growth of a coralloid. Also the transport of impurities (colloids) by hydroaerosols which get preferentially deposited on the tips, promotes coralloid type of growth by inducing the nucleation of fiber-like crystals of calcite. When, instead, the water supply is relatively higher because is mostly derived by splashing drops and evaporation is reduced, impurities (colloids) are diluted and clean elongated columnar calcite forms (Case 2).

2.5.3 Mechanisms of coralloid formation at Lamalunga Cave

Petrographic evidences

The most recurrent fabric in Lamalunga coralloids is elongated columnar (Ce) (Fig. 2.7A-B). However, a subtype of Ce crystals, with fiber-like aspect, characterizes ABS6 convex surfaces. The arrangement of these fiber-like crystals is typical of spherulitic growth, which is when crystal splits apart during growth forming different subindividuals (Sunagawa, 1987; Self and Hill, 2003) (Fig. 2.7C-D). Spheru- litic growth and elongated crystals suggest relatively high driving force relative to a rhombohedral form (Sunagawa, 1987), which in the case of Lamalunga speleothems should be the supersaturation of the parent solution with respect to calcite (SIcc) (Frisia, 2015 suggests SIcc up to ~0.5). Alternatively, a large number of highly elongated crystals arranged in fans with sweeping extinction have been observed to form under presence of impurities, most notably the magnesium ion (Fairchild and Baker, 2012; Richter et al., 2015). Increasing SIcc in the film of fluid wetting a speleothem surface with respect to the parent “drip” can be achieved by CO2 degassing and/or evaporation at the water/air interface (Dreybrodt, 1999), which can be favoured by in-cave ventilation (Van der Weijden et al., 1997; Baker et al., 1998; Dreybrodt, 1999).

Due to the higher length-to-width ratio of fiber-like crystals compared to Ce crystals, it is reasonable to assume that the fiber-like crystals grow under the influence of a larger quantity of impurities (organic or inorganic) trapped within intercrystalline spaces and that favoured their splitting, than the latter. Likely, given that fiber-like crystals are only present at the tips (convex surfaces) of cylindrical shape coralloids (Fig. 2.6F), it is reasonable to infer that the feeding mechanism delivered fluid and impurities preferentially at the apex of the convex surface, exposed to the cave atmosphere.

Elongated columnar fabric (Ce) in stalagmites has been associated to relatively stable drip rate conditions (Frisia, 2015). However, coralloids are not fed by drips, which implies that this fabric may have a different hydrological significance. If an analogy in the mechanism of formation with the same fabric in stalagmites held true, then it can be hypothesized that Ce formed when hydroaerosol were constantly

carried to the growing speleothem surface. The hydrological conditions under which fiber-like (highly elongated) crystals arranged in fans with sweeping extinction form, are similar to those of proper columnar fabric in terms of supersaturation (SIcc of circa 0.5). The difference is in a lower drip rate and the presence of impurities, most notably foreign ions such as Mg (Frisia, 2015). It is reasonable to infer that in Lamalunga cave coralloids formation, the factor which favours fiber-like rather than elongated crystals growth is an enhanced evaporation at the apex of the cylindrical morphologies, which results in less moisture at their tips (Fig. 2.8A-B).

In stalagmites, Ce and open fabric are characterized by impurities at intercrystalline boundaries (Kendall, 1993; Frisia, 2015). By contrast, in Lamalunga coralloids, impurities are mostly concentrated between fiber-like crystals boundaries, whereas elongated fabric, with low intercrystalline porosity, is mostly “clean”. This difference is likely to be related to the different mechanism of transport of impurities to the coralloid. Firstly, we here consider the nature of impurities on the basis of the sole petrographic and fluorescence observations. In cave systems, impurities are most likely to be in colloidal form and as ions in solution. Colloids were defined by Hartland (2013) as “molecules or particles dispersed in a medium with at least in one direction a dimension roughly between 1 nm and 1 µm” and include organic compounds and minerals generated during weathering of rocks. Colloids mobilization from soil depends on the combination of multiple factors like the amount and the duration of rainfalls, the structure of the soil and the regolith and the ionic strength of the incoming solution (Kaplan et al., 1993; Grant et al., 1996; Jacobsen et al., 1997; Laegdsmand et al., 1999; Rousseau et al., 2004; Hartland, 2013). The colloids are then transported into the cave by the infiltration waters and their incorporation in stalagmites is principally regulated by the saturation index (SIcc) of the solution, which affects crystals growth rate, adsorption and absorption into calcite (De Yoreo et al., 2009; Hartland, 2013). Thin fiber-like crystals, which should have formed under relatively higher SIcc, driven by greater evaporation than in the case of Ce, are associated with intense fluorescence, which indicates that organic colloids are best incorporated in the “dark lenses”, where hydroaersols carrying colloids preferentially reach the surface and the particulate can be accommodated between crystal boundaries (Fig. 2.8B) (Kendall, 1993). Although particulate may induce spherulitic-type growth, it is well known that ions in solution, such as Mg, favour development of highly elongated calcite crystals (Sunagawa, 1987; Fairchild and Baker, 2012). It is thus probable, that greater evaporation is also accompanied by some other mechanism that increases the Mg/Ca ratio in the fluid, which will be discussed in the following sections.

Crystal tips can also give information related to the hydrological condition at the time of calcite deposition. The most common type of crystalline terminations observed in both ABS5 and ABS6, is the flat type that has been associated to organic matter oxidation (which releases CO2) because it causes localized dissolution resulting in “smoothing” of the crystal tips (Fig. 2.7F). Flat crystal tips commonly

indicate a thin film of water at the speleothem surface (Turgeon and Lundberg, 2001). This is in agreement with the hypothesis of limited water supply (hydroaerosols) required for coralloids formation. Acute tips are mostly found along the condensed dark layers at the bottom of ABS6 (Fig. 2.7E), indicating that the speleothem commenced to grow under a different hydrological regime, possibly characterized by higher water supply. Protruding terminations, in fact, commonly develop when the thickness of the film of fluid at the surface increases (Turgeon and Lundberg, 2001). Acute crystal surfaces in ABS5 are observed, instead, along the dark horizons (Fig. 2.6A-E), and as they are associated to micrite they could be the product of a corrosion during low water supply (Frisia, 2015; Wróblewski et al., 2017).

2.5.4 “Aragonite conundrum” in Lamalunga coralloids

The absence of aragonite in the fabrics of Lamalunga coralloids is likely to provide further evidence of the dominant evaporative mechanisms of formation, where the feeding fluid is transported as hydroaerosols. Although Bajo et al., (2016) demonstrated that aragonite transformation in stalagmites may be criptic, the specimen that they studied had aragonite layers. Raman spectroscopy of Lamalunga coralloids revealed that they consist of calcite, and there is no petrographic evidence of aragonite relicts or presence of aragonite layers. The chronological results performed on ABS5 and published in Lari et al. 2015 are stratigraphically consistent and do not show any age inversion which also suggests the absence of post-depositional processes that could have led to a change of aragonite to calcite. . Thus, Lamalunga coralloids should have precipitated as calcite.

Nevertheless, in relatively dry settings, Caddeo et al. (2015) reported coralloids of aragonite and Mg- calcite, which likely formed by evaporation and capillary flow. The caves from which they obtained their samples are cut in dolomitized carbonate rocks and dolomitic marble, which are both source of Mg2+ (Martín-García et al., 2009). Thus, the “capillary” film of fluid, moving from the depression of the pore separating two digitate individuals, when reaching the apex is likely to be a residual Mg-rich solution, which would favour the formation of aragonite (Cabrol, 1978; Frisia et al., 2002). Aragonite coralloids are also reported by Cabrol (1978) in several French caves cut in dolomite. Ortega et al. (2005) also report aragonitic coralloids from a very deep cave (Pierre Saint-Martin cave, 1342 m deep) where, typically, water-bedrock interaction is long and prior calcite precipitation can occur, increasing the dripwater Mg/Ca ratios and thus favouring aragonite precipitation over calcite (Musgrove and Banner, 2004; Fairchild, 2009; Rossi and Lozano, 2016).

Lamalunga is a very shallow cave (< 30 m deep) cut in fractured limestone, which allows a limited water residence time in the aquifer. Some magnesium must be have been present in solution at the time of formation of the coralloids, because this is, commonly, the most important inorganic element influencing development of fiber-like fabrics. Mg incorporation in speleothem calcite is expected when Mg/Ca molar ratio in the parent water is > 0.3 and up to 1.5 if the cave is cut in dolomite (Sunagawa, 1987; Frisia et al., 2000; Davis et al., 2004; Richter et al., 2011; Martín-Chivelet et al., 2017). As we do not know the chemistry of the parent water responsible for coralloid formation, we can only infer that it must have had the right combination of SIcc and Mg/Ca to keep precipitating calcite (De Choudens-Sánchez and González, 2009). Calcite coralloids without traces of aragonite have been sig- nalled in Hungarian caves and interpreted as formed by solutions condensed from vapour above warm pools (Leél-Őssy et al., 2011). The absence of aragonite in Lamalunga could, then, be interpreted as another factor favouring the hydroaerosol feeding of the speleothems, and evaporation driving their morphology and fabrics (Fig. 2.8B). By contrast, capillary flow from the bottom to the top would have played a negligible role, as it would have deposited calcite along the pathway, possibly rising the Mg/Ca ratio of the fluid reaching the tip, where aragonite may have then been the “stable” phase to form. This inference is in agreement with the fact that organic impurities are concentrated at the tip, which would not be expected by capillary flow from bottom to top.

2.5.5 Microstratigraphic evidences for Lamalunga coralloids formation

Greyscale profiles of the polished slabs are critical to highlight petrographic changes along the coralloid that likely have an environmental (hydrological) significance (Fig. 2.4) as well as the presence of hiatuses. These are marked by surfaces that have undergone dissolution and corrosion and cut into previous growth layers (Fig. 2.6D, G). Dissolution surfaces have been observed in coralloids fed by condensation of water vapor, possibly related to periods of non-deposition (Leél-Őssy et al., 2011). Dissolution horizons in stalagmites and flowstones are commonly ascribed to aggressive drip waters, or by the release of CO2 during organic matter oxidation, or by the action of microbes (cf. Frisia (2015) and references therein). In this last case, dissolution may be followed by micritization of the former calcite (Fig. 2.6B- C) (Frisia et al., 2012). A progressive reduction of the drip rate can eventually result in carbonate non- deposition (“hiatus”), and the surface could become colonized by microbial communities. This seems to be the case of the dark micritic levels interrupting ABS5 growth between 2.5 and 3.5 mm from the top (indicated by red rectangles in Fig. 2.4), which are characterized by corrosion/dissolution features and are also associated with micrite (Fig. 2.6A-E). That this dark horinzon is a hiatus is also confirmed by the U-Th ages dates obtained just below (36.9 ±0.17 ka) and above (7.6 ±0.04 ka) (Fig. 2.3D) which interrupted ABS5 slow growth (Lari et al., 2015). The relatively brighter fluorescence further indicates

that organic matter is present, corroborating the idea that the dry surface was colonized by microbes (Banks, 2010; Frisia et al., 2012). Similarly, other horizons showing evidences of corrosion, coated by a dark veil of micrite and thus classified as hiatuses in ABS5 and ABS6 (Fig. 2.4), suggest periodic interruption of growth and microbial colonization of the coralloid surfaces cannot be excluded. As for the petrographic evidence, also the microstructural data point to alimentation of the coralloids from the cave “air” to the speleothem surface (Fig. 2.8), rather than from the bottom of the speleothem to the tip. Generally, microbes colonize surfaces rich in nutrients (Banks, 2010). In Lamalunga coralloids, impurities are concentrated in the apexes that could have been used by bacteria as nourishment.

Fluorescence in speleothems can be attributed to humic and fulvic acids derived from the overlying soil and/or to microbial mats (Baker A., 1996; Brennan, 2013; Orland et al., 2014). When there is no clear evidence of corrosion, dissolution or hiatuses, it is reasonable to infer that fluorescence of the fibrous- calcite lenses reflects changes in the flux of organic particulate from the soil, which is commonly related to the water supply and, subsequently, to the hydrochemistry of the feeding fluid (Martín-Chivelet et al., 2017). This also supports the hypothesis that hydroaerosol carry and then deposits the particulate preferentially on coralloid tips (the most protruded areas) (Fig. 2.8). Subsequently, progressively stronger evaporation acting (dehydration) from the flanks toward the center promotes more calcite deposition and solute concentration at the top of the coralloid at the expenses of its sides. This is exactly opposite to what commonly happens in speleothems formed by gravity-induced deposition. In cone shaped stalagmites, which are analogous in having a convex surface to our coralloids, impurities-rich layers are commonly located along the flanks, because particles, suspended or dissolved in solution, are transported along the flanks under gravity flow. The evaporative-driven mechanism of coralloid formation, instead, results in concentration of impurities at the tip, with fiber-like fabric close to the central axes of the coralloid, and depletion on the flanks, where Ce forms.

On the other hand, the isopachous, clean calcite bands observed in the two coralloid samples from Lamalunga, likely reflect a period of relatively less impurities transported by hydroaerosol combined with moderate to low evaporation (Fig. 2.8B). The uppermost part of ABS6 even though is still laminated, is relatively less fluorescent (Fig, 2.3B and light blue rectangles in Fig. 2.4) suggesting that the hydrological regime was more constant and the input of both organic and inorganic foreign particle was reduced.

Fig. 2.9 Sketch illustrating factors controlling Lamalunga coralloids evolution. Arrowed bars illustrate graphically the concept of increasing importance of each factors on the evolution of coralloids morphology. For example, branched and cylindrical coralloids are the result of the combined action of hydroaerosol feeding and evaporation driving to supersaturation with respect to calcite. In addition, branching coralloids likely form when the amount of impurities reaching the growing surfaces is maximum. Impurities, likely carried by hydroaerosols, are preferentially deposited on the convex surfaces and seem to control the growth of fiber-like crystals and their elongation in the direction of growth. Relatively stronger evaporative processes on the protruded parts of the concretion forester the nucleation of fiber-like crystals, which tend to grow faster compared to the crystals on the flanks of the coralloid. The flowstone-like morphologies are directly fed by spray produced by nearby drops splashing thus forming a water film on the top of the speleothem. Because of the smoother morphology, flowstones-like coralloid formation is here inferred to be driven by relatively more intense CO2 degassing and lower evaporation than their branching and cylindrical counterparts. Furthermore, a relatively more constant water supply and reduced impurities input at the top would promote growth of elongated columnar crystals.

2.5.6 Mechanisms of formation of Lamalunga coralloids and hydrological implications

Petrographic and microstratigraphic data of Lamalunga coralloids strongly suggest that evaporation of hydroaerosols reaching protuberances is the main mechanism of formation of the coralloids (Fig. 2.8). Yet, it remains to be explained the provenance of hydroaerosols and of the organic and inorganic compounds, and the role of cave ventilation, evaporation and degassing to the different existing morphologies observed in Lamanlunga cave coralloids.

Commonly, coralloid mostly grow windward on cave surfaces, because it is where hydroaerosol get intercepted to a greatest extent (Hill and Forti, 1997). In Lamalunga cave, concentration of coralloids is highest in the northern sectors of the cave, suggesting air current flowing in a North direction. How- ever, coralloids do not show a preferential orientation of growth, which should have been against the current airflow. In addition, there is no evidence for an exit (via fractures or fissures) in the northern part of the cave, neither there are perceivable air fluxes. The presence of coralloids ubiquitously distributed and without a preferential direction of growth thus, suggests that the northern section of the karstic system was the site of active dripping which, through splashing, formed aerosol (Fig. 2.8A). Critically, coralloids predominantly develop on the floor, on the bones scattered on the floor and in the lowest parts of the walls, and they do not grow on the ceiling or the upper walls (Fig. 2.2B). This supports the hypothesis of hydroaerosol formation originated from fragmentation of drops into myriad of droplets that formed hydroaerosol, which then reached the lowest levels of Lamalunga cave (Fig. 2.8A). An imperceptible cave ventilation, required for hydroaerosol transport, onto protuberances, was possibly induced by a system of small fractures in the bedrock. This hypothesis is supported by the internal air CO2 level, similar to atmospheric values, which suggests that the cave should “breathe”. A small, albeit constant, “cave breathing” is expected because Lamalunga cave has a small “entrance” and a large chamber (Fairchild and Baker, 2012) (Fig. 2.1). Because the cave is relatively dry and warm, cave breathing would support the evaporation hypothesis in relation to coralloid formation.

Capillarity flow, as a mechanism to explain the formation of coralloids, or incipient coralloids, could have potentially acted in Lamalunga, but through intercrystalline porosity (Maltsev, 1996; Cuevas- González et al., 2010; Merino et al., 2014), rather than an inter-coralloids flow as hypothesized by Caddeo et al. (2015). Capillarity flow, which is the ability of a liquid to flow in any porous solid contrary to gravity (Leverett), would necessitate the existence of a film of fluid at the base of the coralloid, which then flows upward and, through progressive degassing and evaporation, becomes suffi- ciently supersaturated to deposit more calcium carbonate at the top of the speleothem. In the case of coralloids described by Caddeo et al. (2015), capillarity flow moves a thin film from bottom to top. This mechanism implies that there is a film of water feeding the bottom of the coralloids, which, in their case, is fluid flowing through the center of stalactites. The studied Lamalunga coralloids were not fed by fluid extruded from the substrate porosity or microcracks or by a film flowing through stalactites. In fact, they occur on fragments of rocks and speleothems fallen on the floor thus hydrologically inactive, and on bones. Furthermore, the lens-shape morphology of fibrous-like calcite layers, and the concentration of impurities at the apex of the coralloid individuals suggests that there was little, or no contribution to calcite deposition from fluid sourced from the bottom of the inter-coralloid cavity (pore). All evidence points to a source that preferentially feeds the jutting surfaces from the top (Fig. 2.8B). In fact, the substratum at the base of both ABS5 and ABS6 consists of micrite and microsparite mixed with

iron oxydes (Fig. 2.3; 2.6H), which likely originated from the dissolution of the stalagmite and of the bedrock during a period when flowing waters transported clay into the cave. The basal layer could have absorbed water and then released it upward by capillary forces during “evaporative” phases, and eventually precipitating the first fibrous calcites. However subsequently, both ABS6 and ABS5 bands of isopachous compact calcite likely prevented capillary flow within the speleothem through crystal boundaries. Crucially, the capillary mechanism invoked by Caddeo et al (2015), based on stable isotope ratios analyses, it is not tested by thin sections observations, whereby it is uncertain whether they had nanometer-scale aragonite inclusions which may have resulted in C isotope ratio enrichment towards the tips (cf. Frisia et al., 2002). Particularly, when specimens are characterized by extremely thin layers, the “bulk” powder used for isotope analyses may include diverse phases of growth. Therefore, our petrographic approach remains unbiased by geochemistry, and provides a hydrological explanation rooted on the physical characteristics of the samples. On this basis, it is here excluded that fluid was mostly present at the base of the cavity between individual coralloids and migrated upward driven by evaporation.

The role of degassing in Lamalunga coralloid formation can be inferred by observing the morphological characteristics (Fig. 2.9). When degassing prevail over evaporation, flowstone-like accretions, similar to ABS5, characterized by a smooth topography and uniform thick layers, form (Caddeo et al., 2015). Similarly, to conventional vadose flowstones fed by seepage water, flowstone-like coralloids need the constant presence, on their active surface, of a film of water and CO2 degassing is the main process driving calcite precipitation (Gradziński et al., 2012; Wróblewski et al., 2017). The film of fluid is likely to be directly supplied by nearby nebulised splashing drips. On the other hand, when the supply of water is relatively lower and the cave is ventilated, the fragmentation of drips forms hydroaerosol, then CO2 degassing occurs all over the area reached by the hydroaerosol and is followed by evaporation, given that the speleothem is not continuously fed by a drip (Gadoros and Cser, 1986; Lohmann, 1988; Hill and Forti, 1997; Dublyansky and Dublyansky, 1998) (Fig. 2.8). Hydroaerosol is preferentially intercepted by the most exposed areas (Dredge et al., 2013). Similarly, evaporative processes are enhanced on the protuberances, where the CaCO3 depositional rate is increased (Onac and Forti, 2011; Caddeo et al., 2015). These processes influence the morphology of the nascent accretion whose development progressively pass from incipient to fully cylindrical and eventually to the branched type, which represents the final stage in coralloid evolution (Fig. 2.9). It is then reasonable to assume that surface protrusions, where both hydroaersol deposition and evaporation dominated, grew relatively faster at the expenses of depressed, less exposed and relatively drier areas. ABS6 morphology falls between the incipient and the cylindrical types. Precipitation of calcite could also have been induced by bacterial metabolic activity (bio-mediation) at the surface of the coralloids. This was likely favored by hydroaerosol reaching the surface, because micro-organisms preferentially grow when the surface is only slightly wet and the

film of water is stagnant (Banks, 2010; Fairchild and Baker, 2012; Martín-Chivelet et al., 2017). Finally, the impurities content appears to be higher at the tips of convex coralloids, whereas flowstone-like parts are relatively “cleaner” (Fig. 2.4). This also suggests that Lamalunga coralloids were preferentially fed by nebulised droplets resulting from the fragmentation of particulate-rich drops of water infiltrated through the soil (Fig. 2.8). In this perspective, Lamalunga coralloid fabrics and microstratigraphy have the potential to provide information about hydrological changes through time.

Lastly, the presence of impurities-rich layers in Lamalunga coralloids (ABS5 and ABS3), did not negatively influence the precision of the U-Th dating. Lari et al. 2015 showed, in table 1, that the average concentration in Uranium for both ABS5 and ABS3 is relatively high: 617 ± 196 (n=7) and so is the average 230Th/232Th activity ratio: 833 ± 1127 (n=7). This indicates that the Uranium content is suitable for U-Th dating and that detrital contamination is negligible. In addition, considering that the samples respect a correct stratigraphical order, it is possible to conclude that the age determinations are reliable. The lower average 230Th/232Th activity ratio in the cylindrical coralloid ABS3 (61 ± 58, n=3) compared to the flowstone-like coralloid ABS5 (1412 ± 1223, n=4), demonstrates that colloidal particles are preferentially incorporated in cylindrical coralloids, supporting the hypothesis of coralloid formation controlled by hydroaerosols fluid supply. The dates published in Lari et al. (2015) also indicate that Lam- alunga coralloid speleothems grew at slow rates: despite ABS3 total length is only 5 mm, its intermittent growth lasted for ca. 123 ka whereas ABS5, that is 10 mm long, grew intermittently in ca. 114 ka.

2.6 Conclusions

Fabrics and microstratigraphic relationships of Lamalunga coralloids mostly support the hypothesis that their formation is linked to hydroaerosol produced by dripping water and ventilation inside the cave combined to enhanced evaporation at the tips which lead to the peculiar cylindrical shape of these concretions (Fig. 2.8; 2.9). These two mechanisms also explain why impurities has been found preferentially on the convex surfaces of the speleothems. In this perspective, coralloids become a valuable archive, which gives reliable information about the periods when hydroaerosols production in the cave was enhanced and when they carried soil-derived particulate.

Microstratigraphy identified correlated bounding layers characterized by hiatuses supported in the radiometric dating (Lari et al. 2015). Microstratigraphy also shows a sequence of columnar and fibrous- like crystals arranged in isopachous and lenticular morphologies respectively (Fig. 2.3). These preserve a history of drip rate in the cave. Specifically, clean Ce calcite in isopachous bands reflects periods when the cave hydroaerosol were devoid of particulate and reached the speleothem surface at a constant

rate when evaporation was, relatively speaking, at a “minimum”. In such perspective, Ce suggests relatively low seasonal contrast with less temperature fluctuations and thus, less evaporation in the cave. By contrast, laminated, lenticular bands associated to fiber-like crystals and impurities (organic compounds, bacteria, detrital particles etc.), are likely to have formed when enhanced evaporative processes on the tips of the coralloids counteracted hydroaerosol supply, but also when hydroaerosol droplets were loaded with particles. In this perspective, the fiber-like crystals would identify periods when seasonal contrast (driving ventilation) was at a maximum, resulting in fast infiltration during a warm-wet phase followed by intense evaporation (very dry phase).

Finally, Lamalunga coralloids fabrics investigation reveals that they were formed originally as calcite, and, therefore, they are apt to be dated by the U-series methods. In conclusion, Lamalunga coralloids mechanisms of formation appear to be directly related to dripwater in the cave, via splash and fragmentation, and hydroaerosol transport onto protuberances (Fig. 8). Therefore, they identify a powerful tool for subsequent investigation of hydroclimatology by coupling petrography and chemistry.

The present research supports the notion that petrographic research in speleothems is a powerful analytical tool that should be used prior to and in conjunction with conventional geochemical analyses. Without a petrographic investigation, any interpretation based solely on chemical data could lead to equivocal reconstructions. We hope we demonstrated that a detailed petrographic and microstratigraphic investigation of these small speleothems has the full potential to make them valuable archives of climate change. Given that coralloid are commonly associated to valuable Cultural Heritage artifacts, their accuracy as precisely datable palaeoclimate archives is invaluable.

2.7 References

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Chapter 3. High-resolution synchrotron XRF investigation of calcite coralloid speleothems: elemental incorporation and their potential as environmental archives

-Manuscript Vanghi, V., Borsato, A., Frisia, S., Howard, D. L., Gloy, G., Hellstrom, J., Bajo, P. High-reso-lution synchrotron XRF investigation of calcite coralloid speleothems: elemental incorporation and their potential as environmental archives. Submitted to Sedimentology Journal.

3.1 Abstract

Synchrotron high resolution elemental mapping of coralloids from Lamalunga Cave revealed an exceptionally high concentration of Mg, Sr and Si, especially in correspondence of convex surfaces. Evapo- ration, which is especially intense at the tips of the coralloids, is likely responsible for elements concentrations like Sr, which is, in average, 70 times greater than commonly expected for spelean calcite. Similarly, Mg is 8 to 15 times more concentrated than average. Unusually high Si content (in average 84 ppm) is here interpreted as related to dissolution of volcanic ash derived from the explosive activity of the Neapolitan Volcanic Province. Non-Ca elemental concentrations are here deemed responsible for fabric changes in the coralloids. Elemental concentrations are also related to fabric changes in the coralloids. Elongated columnar calcite forming clean isopachous bands, and fiber-like crystals associated to laminated, lenticular bands and impurities (organic compounds, bacteria, detrital particles etc.) are the most common fabrics forming the coralloids. Elongation of the crystals is here ascribed to the presence of Mg, whereas the opening of the fabric, from compact elongated to porous fiber-like, is here interpreted as due to input of detrital particles (like Si) and organic particulate. These two fabrics and their elemental content have here been interpreted as indicative of changes in rainfall, with the clean compact calcite marking higher infiltration and trace elements transport in solution and as water-borne particulate, and the open fabric typical of dry periods, when aerosol transport is dominant. The aim of this work is to expand the conventional concept of carbonate petrography beyond microscopy and spectroscopy by encompassing also geochemical mapping. Thus, the two tools, one optical and the other chemical, allow studying the structural features of the coralloids, correlate specimens sampled in different zones of the cave and contributed to provide the hydroclimate significance of speleothem morphology.

3.2 Introduction

Speleothems are geologic climate archives that can be precisely dated by U-Th method, providing that they have not undergone diagenesis (Hellstrom, 2003; Richards and Dorale, 2003; Fairchild and Baker, 2012; Frisia, 2015; Bajo et al., 2016). This resulted into breakthroughs in our understanding of causes of global, hemispheric and regional climate changes through their physical and chemical properties (Bar-Matthews et al., 1999; Frisia et al., 2000; Wang et al., 2001). Stalagmites are the most common speleothem type used for past climate reconstructions, specifically when they show an internal structure consisting of time-equivalent stacked layers, which, similarly to tree rings, may encode annual variability. The thickness of these layers ranges from few micrometres (< 10 µm) up to millimetres (< 2 mm), permitting high-resolution time series (Fairchild and Baker, 2012).

By contrast, coralloid speleothems, due to their small dimensions (usually < 2 cm), have been rarely considered apt to paleoclimate reconstruction. However, in some cases, these are the most common speleothem type available, because they can grow in semi-arid settings, when the water supply inside a cave is relatively low whereas other speleothems, like stalagmites, require an active drip. By coralloids it is here intended a type of speleothems characterized by botryoidal morphology and curved internal structure (Thrailkill, 1965; Hill and Forti, 1997). Their formation is linked to: 1) hydroaerosols (Gadoros and Cser, 1986; Dublyansky and Pashenko, 1997; Vanghi et al., 2017), originated from cave drip-water sprays via splash and drops fragmentation, which transport and distribute particulate and dissolved chemicals onto their tip; 2) seeping water combined to capillary forces that move the film of water upward through intercrystalline porosity (Maltsev, 1996; Cuevas-González et al., 2010; Merino et al., 2014) or, externally, from the base towards the prominence of the coralloids (Caddeo et al., 2015); 3) enhanced evaporation on the curved surfaces increasing the supersaturation with respect to calcite

(SIcc) of the solution and leading to a major CaCO3 precipitation on the protruding surfaces (Caddeo et al., 2015; Vanghi et al., 2017). If coralloid growth is driven by strong evaporation (Caddeo et al., 2015; Vanghi et al., 2017) this might imply that their stable isotopic composition is kinetically modified compromising the use of δ18O and δ13C as climate proxies (Caddeo et al., 2015). Evaporation can also affect trace elements incorporation by preferentially concentrating them on the tips.

Lamalunga Cave is one of those cases where coralloids are the most abundant speleothem. By taking into account the importance of the site, which is related to paleoanthropological finding of a complete Neanderthal skeleton lying in the depths of the cavern (Lari et al., 2015), the more suitable material for dating the skeleton and reconstructing the environmental conditions of the cave are coralloid speleothems developed on the cave walls, floor and on the Neanderthal bones. Here we investigate the potential of coralloids as reliable paleoclimate and paleoenvironmental archives, comparable to stalagmites,

by using a multidisciplinary approach and performing petrographic, microstratigraphic and geochemical analyses on 4 coralloids, both directly and indirectly associated to the Neanderthal remains. Micros- copy has been complemented with synchrotron and conventional micro-XRF elements concentrations maps to characterize the nature of layering in two dimensions (2D), thus developing a new concept of speleothem petrography, which accounts for the distribution of elements both intra- and extra-lattice. When the chemical information is only available for one or two single transects, it can be more challenging to accurately reconstruct the transfer function from the climate proxy to the chemical datum, especially in heterogeneous media and at a small scale such as that of coralloid convex layers. By using micro-XRF geochemical maps, we aim at identifying the relationships between trace elements incorporated in spelean calcite, how they affect the development of the crystalline fabric of calcitic layers and, finally, provide an understanding of their provenance as potentially related to environmental factors.

3.3 Geographic context

Lamalunga cave opens at 508 m a.s.l. (Fig. 3.1) in Southern Italy (40°51'51.9"N 16°34'31.3"E) close to the town of Altamura at ca. 50 km from the Adriatic Sea and ca. 70 km from the Ionian Sea (Fig. 3.31). The present day climate in the region can be classified as Mediterranean with wet winters and autumns (rainfall mainly concentrated during October–December) and dry summers (July is the driest month). The mean annual precipitation in the area ranges between about 500 to 800 mm (Brandimarte et al., 2011) with mean precipitations < 30 mm from June to August (data from S.I.M.N., Italian National Hydrographic and Mareographic Service, Bari compartment, Cassano Murge weather station over the period 1921–1990) (Andriani and Walsh, 2009). This results in infiltration deficit during June, July and August. Mean temperatures range between 30°C in summer and 5°C in winter (Gioia del Colle weather station).

Lamalunga Cave is cut in the well bedded shallow marine Calcare di Altamura limestone of Upper Cretaceous (Coniacian) age (Zezza, 2000). The cavity is 5 to 30 m deep and developed during the Pliocene and Pleistocene in the uppermost vadose level of the karstic system, which consists in a single sub-horizontal gallery less than 100 m long (Agostini, 2011). In 1993, the members of the local speleological society (C.A.R.S. - Centro Altamurano Ricerche Speleologiche) artificially enlarged the natural opening to the cave and entered the main gallery where they discovered a complete human skeleton embedded in calcite crusts, named afterwards the “Altamura man”. Uranium-series dating on coralloid coatings served to provide a post-quem age to the “Altamura man”, which resulted to be older than 55.9

±1.8 ka and possibly older than 130.1 ±1.9 ka (Lari et al., 2015). DNA extracted from the bones revealed that this human belonged to Homo neanderthalensis (Lari et al., 2015) and the old age attracted the attention of both the scientific community and the general public. The actual access to the cave is a 12 m deep narrow vertical shaft whereas the original entrance was possibly a larger vertical shaft now completely obstructed by a debris and collapse deposits.

The temperature measured inside the main chamber fluctuates around 15.5 ± 1.5° C and coincides with the mean annual temperature at the surface. Cave air CO2 ranges from 4000 ppmv to almost atmospheric values (400 ppmv), which indicates effective cave ventilation. The relative humidity is near the saturation in winter (100%) slightly decreasing down to 97.0 ± 1.5% during summer.

Coralloid formations are mostly abundant in the northern branches of the cave where the thickness of the overlying bedrock is only ca. 5 m and internal temperature fluctuations are larger compared to the other sectors (Fig. 3.1). Coralloids represent the last phase of cave calcite deposition as they systemat- ically coat flowstones, stalagmites and fossil animal bones that are scattered on the floor (Zezza, 2000; Agostini, 2011).

Fig. 3.1 A) Map of Italy with the location of Lamalunga Cave near Altamura. B) Plan and schematic cross section of Lamalunga Cave. The orange dotted line represents the plan of the cross section. The locations of the samples here studied (ABS5 and ABS6) are also indicated. ABS3 coralloid, studied in Lari et al. (2015), was sampled in the same chamber of the “Altamura Man” skeleton. Coralloid speleothems are represented with small dots. A vertical shaft completely filled by limestone debris clogs the alleged original entrance. The actual entrance to the cave was artificially enlarged. C) View of the hill where the cave opens to the surface (black cross).

3.4 Materials and Methods

3.4.1 Morphological and structural characteristics of the coralloids

Samples ABS5 and ABS6 were collected in the NE branches of the cave from a broken stalagmite and a bedrock fragment respectively (Fig. 3.2). Another coralloid sample, ABS3, was removed from a bone of the human skeleton to perform chronological analyses, whose results were published in Lari et al. (2015). ABS5 was collected following the rationale that it could be coeval with ABS3, but have thicker and more continuous layers, which could be radiometrically dated and offer robustness to the hypothesis that coralloid yield accurate ages. Critically, ABS5, once dated by U-Th method, revealed some growth phases that are time equivalent with ABS3 (Lari et al., 2015). ABS6 is a multiaggregate of digitate coralloids, from which three sub-samples were cut: ABS6-A and ABS6-B and ABS6-C (Fig. 3.2). Whilst ABS6-A and B were cut along parallel planes, ABS6-C cut was along a perpendicular plane to ABS6-A and B. All samples consist of calcite (Vanghi et al., 2017). From ABS5 and ABS6-A two 30 µm thick thin sections were obtained and the petrographic observations were carried out by using a Leica MZ16A stereomicroscope and Zeiss Axioplan microscope. Fluorescent light images of the thin sections, stimulated at blue (488 nm) and green (543 nm) wavelength lasers, were obtained using a Zeiss Axio Imager A1 fluorescence microscope with an LED Colibri controller at the University of Newcastle, Australia.

Petrography has been complemented by greyscale profiles for both fluorescence thin sections and polished slabs 16-bits micrographs generated using ImageJ (Schneider et al., 2012) by averaging the pixel brightness intensity (on a grayscale from 0 to 255). This system highlighted porosity and generated profiles that can be matched with chemical profiles. The traverses used to generate greyscale values profiles for each sample were identical for both fluorescence and thin sections and polished slabs images.

Fig. 3.2 Polished slabs of the investigated coralloid samples. A) ABS6-A, B and C are part of the same multiaggregate of coralloids. The corresponding planes of cut are also shown (white rectangles). Notice the different plane of cut for ABS6-C that is orthogonal to the other two. B) ABS5 slab. The sample was growing on a broken stalagmite.

3.4.2 Water analyses

Two cave water samples were collected in May 2008 in polypropylene vials, pre-cleaned with 5%

HNO3 and rinsed with MilliQ® water. Water temperature and electrical conductivity (corrected to 20°C) were carried out using a WTW TetraCon 325 dual probe (resolution ± 3 μS cm−1 and 0.01°C, accuracy ± 2 μS cm−1 and ± 0.2°C), pH measurements were carried out using a WTW MultiLine P3 instrument (accuracy ± 0.2 pH units) and bicarbonate concentration was determined by titration. The samples were analysed at the Hydrochemistry Laboratory of Fondazione Edmund Mach, S. Michele all’Adige, Italy, following the analytical procedure described in Borsato et al. (2016). The major cations (sodium, potassium, calcium and magnesium) and anions (sulphate and chloride) were measured with a Dionex 320 Ion Chromatography instrument. Silica was measured on undiluted samples on a Varian– Cary 50 bio Spectrophotometer and Sr was measured on a Perkin Elmer 3300DV quadrupole ICP-OES using yttrium as an internal standard. Analytical reproducibility, calculated as the differences between

10 repeated analyses of the same sample, was 2% for Ca, Mg and SO4, and 5% for Na, K, Cl, SiO2 and Sr. The saturation indexes, defined as the logarithm of the ion activity product divided by the solubility product (SI = log IAP/KS) were calculated with the program PHREEQC (Parkhurst and Appelo, 1999), by accounting for the pH and temperature measured at the time of sampling.

3.4.3 U-series dating

Three new samples for U-series dating were collected from the coralloid ABS6-A. ABS5 was already dated and the results can be found in Lari et al. (2015). The powders for dating where collected using a manually navigated dental drill along the growth axis of the coralloid. The samples were chemically processed for U-Th dating and analysed following the methods described in Hellstrom (2003) and Drysdale et al. (2012). 15-30 mg of powder were dissolved in nitric acid and then spiked using a mix of 229Th-233U-236Ut tracer solution. The U and Th were eluted in Eichrom TRU-spec selective ion exchange resin. Dried samples were then dissolved in diluted nitric acid. The measurements were performed using a Nu Instruments Plasma MC-ICPMS at the University of Melbourne. An equilibrium reference material (HU-1) was used to correct for instrumental drift and an additional in-house speleothem standard of known age (YB-1) was used to check for the reproducibility of results. An initial 230Th/232Th ratio of 1.5 ± 1.5 was used to calculate corrected ages following Hellstrom et al. (2006).

3.4.4 Micro-XRF and SR micro-XRF

Micro-X-ray fluorescence microscopy was performed at the XFM beamline at the Australian Synchro- tron (Paterson et al., 2011). The XFM beamline is equipped with a Maia 384 detector array which is mounted upstream 10 mm away from the sample target. The beam passes through the detector with an annular configuration which enables a large solid-angle without imposing severe restrictions on sample size or the scale of scanning (Ryan et al., 2014). A 2 μm beam spot size and monochromatic incident energy at 18.5 keV were used. Single element foils Mn, Fe and Pt (Micromatter, Canada) were utilised as references to calibrate the final elemental spectra. The dwell time of the beam on each pixel (2 μm x 2 μm) varied between 0.8 and 4 milliseconds. In this fast-acquisition configuration the detected elements were Ca, Mn, Fe, Br, Sr, Y and U, with typical detection limits of 220 ppm for Ca, 70 ppm for Mn and Fe, 7 ppm for Br, 5 ppm for Sr, 2 ppm for Y and 1 ppm for U. The attenuation depths for the 18.5 keV energy are as follow: Ca = 6 µm, Mn = 21 µm, Fe = 27 µm, Br = 150 µm, Sr = 240 µm, Y = 280 µm, U = 370 µm. The Maia XFM spectral data were analysed using the GeoPIXE software suite, which uses a fundamental parameters approach, with spectral deconvolution and imaging performed using dynamic analysis (Ryan et al., 2014). Line scans with quantified concentrations of Sr and Ca were extracted for ABS5 and ABS6-A thin sections through rectangular selections (50 µm wide) along straight lines in the axial part of the coralloids and by avoiding porous parts and evident crystal defects

on the sample. An 11-average points smoothing was applied to ABS5 and ABS6-A data, to capture the most important patterns of the data and to reduce the noise.

The high excitation energy of the hard X-ray XRF beamline is not suitable to detect low Z elements such as Mg, Si, S and P when present in concentrations < 1000 ppm. For this reason, we utilised a Bruker M4 Tornado micro-XRF to test a selected area on the polished slab of sample ABS6-C. The experimental conditions were as follow: vacuum = 2 mbar; tube voltage =30 keV; current =200 μA; acquisition time = 45 ms; beam spot size = 20μm; pixel size = 20μm. The resulting intensity maps are represented with different arbitrary colours for each element; the quantification of the intensity data was made by using internal standards and normalised to 100% by taking into account the stoichiometry for Oxygen and Carbon in the carbonate and oxide species. In order to minimise the signal to background ratio a successive clustering of 5 pixels along near-horizontal laminae was utilised in order to quantify more accurately the elemental distribution (Devès et al., 2012).

3.4.5 Principal component analysis (PCA)

PCA were performed on ABS6-B SR micro-XRF results and on ABS-C micro-XRF results. The variables used for the PCAs were Ca, Sr, Mg, Si, Fe, Br, Y, and U and the greyscale values measured on the polished slab. The traverses of trace element concentrations however, had a different resolution compared to the traverses where the greyscale values were obtained. Therefore, a linear interpolation was necessary in order to assign an identical scale for the different series to make them suitable and comparable for principal component analysis (PCA). PCA and interpolation analyses were performed with R Studio (the integrated development environment for R) using the built-in princomp and the approx functions respectively (R Development Core Team, 2013). The interpolated spatial resolution for samples ABS5 and ABS6 SR-micro-XRF trace elements and greyscale transects are at 4 μm. The PCA was performed on six different transects (30 μm resolution) of ABS6-B characterised by different elemental concentration and fabric.

3.5 Results

3.5.1 Water analyses

Given the impossibility of directly sampling the film of water at the top of the coralloids, one stalactite drip and one pool water were sampled in the Northern branch of the cave in May 2008. Stalactite drip LL-w1 and pool water LL-w2 are characterised by a similar composition (Table 1) reflecting the direct dissolution of an almost pure limestone host rock as testified by the low Mg/Ca ratio (0.13 and 0.11 mol/mol). The ion content, as well as the calcite saturation, are remarkably similar to the water composition collected at Bus del Diaol, a low-elevation cave developed in Jurassic pure limestone in Northern Italy (Borsato et al., 2016).

2+ 2+ + + 2- - EC Ca Mg Na K HCO3 SO Cl SiO2 Sr Mg/Ca SI SI SI CO2 SI Point Type T(°C) pH 4 (µS/cm) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (µg/l) (mol/mol) calcite dolomite (gas) Quartz LL-w1 drip 15.50 7.62 420.00 88.40 6.80 4.90 0.70 295 4.80 11.20 7.50 64.50 0.13 0.51 0.12 -2.15 -0.14 LL-w2 pool 15.30 7.57 409.00 86.50 5.80 5.50 0.65 289 6.20 10.30 10.30 68.90 0.11 0.44 0.13 -2.20 0.02

Table 3.1 Hydrochemical characteristics of the cave waters collected at Lamalunga cave in May 2008.

3.5.2 U-series dating

A previous series of 4 and 3 U-Th dating were carried out on ABS5 and ABS3 respectively, by Lari et al. (2015). Results for ABS5 are shown in table 3.2. These analyses revealed an ancient phase of Lam- alunga coralloids formation dated between 130 ka and 121 ka which is also related to the age of the Neanderthal remains. To confirm this old age for the skeleton, U-Th dating was performed also on ABS6-A (Table 3.2). The oldest phase of this coralloid is 50.4 ka but it is not excluded that an older phase is present and that, due to the small size of the sample, it was not possible to acquire. The date recorded at 36.8 ka was sampled in the translucent region of ABS6-A, between 5 and 6 mm from the base (Fig. 3.5). This age corresponds to the phase at 36.9 ka sampled in the translucent part of ABS5 just underneath the micritic layer recognized as a hiatus (Fig. 3.4). The last phase of ABS6-A is dated at 8.9 ka and it correlates to the Holocene phase at the top of ABS5 dated 7.5 ka. The high 230Th /232Th activity ratios in both ABS5 and ABS6-A indicate the absence of detrital contamination, thus confirming the reliability of the U-Th results.

230 238 234 238 230 232 Age Initial 238 Th/ U Th/ U Th/ Th ABS6-A Mass (g) U (ng/g) corrected 234Th/238U AR AR AR (ka) AR 50.425 1 0.025 1130.3 (85) 0.381 (0.002) 1.019 (0.002) 88.7 1.022 (0.003) (0.802) 36.873 2 0.027 1372.4 (103) 0.297 (0.002) 1.030 (0.002) 290.1 1.033 (0.002) (0.352) 3 0.024 4570.6 (343) 0.079 (0.000) 1.012 (0.002) 803.7 8.910 (0.054) 1.012 (0.002)

230 238 234 238 230 232 Initial ABS5 (Lari 238 Th/ U Th/ U Th/ Th Mass (g) U (ng/g) Age cr (ka) 234Th/238U et al. 2015) AR AR AR AR 64.654 1 0.063 616 (46) 0.485 (0.001) 1.079 (0.002) 2015.2 1.096 (0.002) (0.331) 36.901 2 0.035 475 (36) 0.299 (0.001) 1.042 (0.002) 2793.5 1.047 (0.002) (0.172) 3 0.067 708 (53) 0.073 (0.000) 1.097 (0.002) 770.1 7.571 (0.045) 1.100 (0.002)

121.904 4 0.008 738 (56) 0.700 (0.003) 1.029 (0.003) 68 1.040 (0.005) (2.220)

3.5.3 Petrography, internal structure and morphology of Lamalunga coralloids

Lamalunga samples have different external morphologies. ABS6 is an aggregate of coralloids characterized by a cuspate cylindrical morphology. In some cases, the coralloids merge together (like ABS6- A), in others, they are separated by a gap (ABS6-B and C). ABS5 morphology is not cylindrical and only slightly cuspate. The external surface is smoother and flatter compared to ABS6 and, accordingly to its morphological aspect, ABS5 is similar to a flowstone at mm-scale and to an incipient coralloid (cfr.Vanghi et al. (2017)).

Lamalunga coralloids consist of calcite as shown by Raman spectroscopy measurements and confirmed by petrographic observations (Vanghi et al., 2017), which did not revealed aragonite relicts embedded in the calcite cfr. (Frisia et al., 2002). Samples ABS 5 and 6 are formed by elongated columnar (Ce) calcite fabric (Frisia, 2015) consisting of columnar crystals whose length to width ratio exceeds 6:1 (Fig. 3.3). Ce fabric forms isopachous, compact and translucent bands. Compared to ABS5, which is entirely consisting of compact Ce (Fig. 3.3 and 3.4), ABS6 also shows a sub-type of elongated columnar fabrics with very thin, highly elongated crystals (fiber-like) arranged in bundles with slightly di- verging lattice orientation and undulatory extinction pattern. Fiber-like fabric is mostly present close to

the central axis along the curved layers and it is associated to dark impurities-rich layers and high intercrystalline porosity. Towards the flanks of ABS6 coralloids, the fabric gradually passes to Ce compact type. Micrite (M) and micro-sparite (Ms), whose crystal sizes range from less than 2 µm to between 2 and 30 µm respectively (Frisia, 2015), are also associated to dark horizons and to corrosion and dissolution features in both ABS5 and ABS6.

Fig. 3.3 Synchrotron elemental (Ca, Sr,) concentrations plotted against grey values intensities from the polarised thin section and the fluorescence map (using 2 wavelenghts, blue and green, combined) for ABS5 and ABS6-A. The corresponding fluorescence and greyvalues line scans were carried out along the dashed vertical lines. Fabrics and major discontinuities for the thin sections are labelled as follows: Ce = columnar elongated; M = micrite; OM = organic-rich layer; MS = microsparite. To facilitate their correlation of greyscale values and fluorescence intensity, different fabrics have been highlighted by diverse coloured bands. Blue has two different shades, dark and light, which are intended to reflect higher (++) or lower (+) concentration of organic material (OM) and higher (ff) or lower (f) fluorescence intensity respectively. The presence of clean, compact Ce calcite parts is illustrated as white bands. Hiatuses (Ht) are marked by solid blue (clearly recognizable gap) lines. Note that the transmittance intensity and Ca scales are inverted.

Fig. 3.4 SR micro-XRF maps (Ca, Sr, Fe and elastic signal) of ABS5 compared to the corresponding image of the polished slab (A) and the petrographic thin section under polarized light (B). Note that the thin section was made 2 mm away from the slab plane and thus it shows a slightly different architecture. Sr and Fe are preferentially concentrated along the dark micritic-rich bands on the thin section corresponding to the opaque milky bands on the slab. This corroded surfaces represent moment of no calcite deposition (hiatuses) indicated by Ht A, B and C. The ages (ka) published in Lari et al. 2015 are indicated in (A). Scale bars unit is in ppm for the elements maps and in percentages for the elastic scatter map.

ABS6-A, B and C internal structure is finely laminated (Fig. 3.5A-B) with alternating clear and dark micrometre-thick, curved layers grouped in millimetre-thick dark bands. Dark layers taper out towards the flanks of the coralloid and this imparts an overall lenticular shape to the dark bands. By contrast, clean compact calcite bands maintain the same width all along the coralloids. ABS6-C was cut along a plane almost orthogonal to ABS6-A and B at the extremity of ABS6 aggregate of coralloids. For this reason the internal structure of the sample, as showed by SR and Bruker micro-XRF maps, results

slightly different from the other two samples: ABS6-C layers curvature is very accentuated and they almost close up like rings near the base. ABS5 internal anatomy is mostly formed by clear isopachous bands of dense Ce calcite, the lamination is less profuse than in ABS6 coralloids and dark layers are not curved.

Dissolution and corrosion features mark the boundaries between clear and dark laminae and in most of the cases are associated to micrite, microsparite and organic material as indicated by fluorescence (Fig. 3.3). Some layers can be confidently considered as hiatuses if the evidence of dissolution and corrosion can be followed on the entire fossil surface of the speleothem even if U-Th ages are not available (Vanghi et al., 2017). After performing the petrographic observation of Lamalunga coralloids thin sections, several discontinuities have been recognized (Fig. 3.3 to 3.7). In ABS6, coralloids erosion is mainly observed along dark laminae whereas in ABS5 is especially evident on the three stacked dark layers at ca. 4 mm from its top. These three stacked dark layers likely represent a complex hiatus as confirmed by the U-Th ages obtained just below (36.9 ± 0.17 ka) and above (7.6 ± 0.04 ka) (Ht C in figure 3.3) which interrupted ABS5 slow growth (Lari et al., 2015). Between 36.9 ka and 64.4 ka and between 121.9 ka and 64.4 ka (Lari et al. 2015) two possible hiatus (A and B in figure 3.3) have also been recognized for the presence of dark micritic layers. Similarly, for ABS6-A, two layers, just above 36.8 ka, have been identified as hiatuses (Ht B and C in figure 3.3) because their surfaces are considerably serrated. Hiatus A above 50.4 ka is characterised by abrupt change in fabric and colour, while erosion features are less accentuated. All these discontinuities have been observed in ABS6 coralloids supporting the hypothesis that they represent gaps in deposition.

Fig. 3.5 A) Synchrotron distribution map (Ca and Sr) of thin section ABS6-A compared to the polished slab (a) and to the thin section under parallel polarizers (PPL) (b). Sr concentrates preferentially in the axial part of lenticular-shaped bands that appears opaque on the slab (a) and dark in the thin section (b). Where the growth band is more isopachous, Sr concentration is lower. Sr-rich layers are also present above hiatuses (Ht A, B and C) identified by clear dissolution features (arrows). The three U-Th ages (expressed in ka) are also indicated. Scale bar unit is in ppm. B) Zoom-in of the region indicated by a white rectangle in (A). Parallel (PPL) and crossed polarized (XPL) micrographs compared to Ca and Sr SR micro-XRF maps of the laminated part in ABS6-B. Dark regions in the thin section corresponds to low values for Ca, thus reflecting a relatively high intercrystalline porosity. Translucent parts are instead very dense as shown by high Ca values. Black circles confine regions with low Ca concentration which in some cases are rich in Sr, when also Sr is low they represent voids possibly filled by other elements (white arrows).

3.5.4 Greyscale values and fluorescence stimulated microscopy

The greyscale values in the thin sections reflect changes in the way the light is transmitted through the sample according to porosity and impurity content. Grayscale values in ABS5 and ABS6-A vary between 25 (dark impurity-rich) and 250 (translucent) (Fig. 3.3). Stimulated fluorescence was measured on ABS5 and ABS6-A thin sections using greyscale. Grayscale values for the fluorescence range from 5 (no fluorescence) to 120 (high fluorescence) (Fig. 3.3). By using the same imaging settings, ABS6 fluorescence intensity seems to be twice that of ABS5. When greyscale values were extracted from the thin sections under transmitted light, compact elongated columnar (white) has higher greyscale levels compared to the micrite or fiber-like columnar (dark) values.

In both samples the mixed micrite and fiber-like layers show the highest fluorescence signal (Fig. 3.3), while the compact columnar elongated fabric is characterized by less intense fluorescence. Bright fluorescent zones are commonly characterized by low greyscale values (Fig. 3.3).

Fig. 3.6 Synchrotron micro-XRF elemental maps of ABS6-B compared to the corresponding polished slab (A). The elements concentrate mostly where Ca concentration is low and where the laminae appear white under natural light. Sr and Br tend to be more concentrated in lenticular shaped stacks of layers. Fe and Y mostly are concentrate at the base of the coralloid where micrite layers have been observed. U concentration pattern is similar to Sr distribution. Principal hiatuses (Ht A, B and C), are marked by white arrows.. The three U-Th ages (expressed in ka) are also indicated. Scale bars unit is in ppm for the elements maps and in percentages for the elastic scatter map.

3.5.5 Distribution maps of trace elements: SR-micro-XRF and micro-XRF maps

The SR-micro-XRF maps (Ca, Sr, Fe, Br, Y, U) for samples ABS5 and ABS6 (A, B and C) and Bruker micro-XRF maps (Ca, Si, Mg and Sr) for sample ABS6-C are shown in Fig. 3.4, 3.5, 3.6 and 3.7. The Ca concentration in the SR-micro XRF maps highlights porous regions and crystal boundaries as well as areas where Ca is partially replaced by other divalent cations (Mg, Sr) or other species (Si, Fe, etc.). Elastic scattering maps provide information on porosity, crystal orientation, crystal defects and boundaries and illustrate the elongation of the columnar fabric (Fig. 3.6).

In ABS5, Sr is generally between 120 and 180 ppm (Fig. 3.5) increasing up to 300 ppm in the dark micritic layers and up to 600 ppm in correspondence of three stacked discontinuities at ca. 4 mm from the top (Ht C). Iron maximum values (5000 ppm) are reached at the base of the sample associated with the red micrite layer, which most likely contains iron oxi-hydroxides. Similarly to Sr, Fe is also present along hiatus Ht C, marking crystals terminations at the top of the corroded layers.

In ABS6-A Sr is especially concentrated on the cusped parts of the two coralloids, where it reaches up to 2400 ppm and where Ca concentration records minimum values (Fig. 3.5A-B). Higher Sr concentrations are thus linked to fiber-like crystals, which are mostly observed on the convex parts of the coralloid (Fig. 3.5B). Toward the flanks, characterized by compact calcite, Sr concentration progressively decreases and so the lenticular dark bands (Fig. 3.5B). However, in the bottom half of the samples, the bands do not seem to restrict laterally and the concentration of Sr seems to be more continuously distributed along the layers in recessed and curved parts (Fig. 3.5A).

Also in ABS6-B the concentration of Ca increases moving away from the central axis while the other concentration show a Gaussian-shape distribution centred in the axial part and diminishing by 60-75% moving from the apex of the cusp to the flanks. Br concentration is maximum at the apex of the bands (50 ppm), but its distribution pattern does not coincide exactly with that of Sr distribution. Mean Ura- nium concentration in ABS6-B is ca. 4 ppm and can reach up to 10 ppm on the lenticular bands. Y as well is mostly concentrated on the cusps (< 50 ppm) and it considerably increases at the micritic layer where it reaches 200 ppm. Fe is only present at the base of the coralloid in correspondence of the basal micritic layer where it reaches the maximum value of 25,000 ppm. Similarly to ABS6-A, the bottom half of ABS-B, where the two adjacent coralloids are fused together, the pattern of distribution of the elements is continuous along the layers.

Similarly to ABS6-A and B, in ABS6-C the highest elemental distribution is along the curved bands: maximal at the apex and decreasing toward the flanks of the coralloid (Fig. 3.7). Sr highest values is ca.

2100 ppm whereas Br reaches values of ca. 20 ppm. Uranium concentration in some lenticular bands exceeds 12 ppm. Iron as for the other samples analysed, was only detected at the base of the coralloid associated to the red micritic horizon.

For ABS6-C were produced also micro-XRF maps for Ca, Si, Sr and Mg (Fig. 3.7). Si is the element with the higher concentration after Ca and its distribution is opposite to the Ca. Three other maps (for Si, Mg and Ca) were acquired in at a smaller area but with higher dwell time to better constrain the spatial distribution of the elements at single laminae level (Fig. 3.8). Si and Mg distribution tends to anti-correlate with Ca and mostly concentrate in the porous opaque bands of the sample.

of Ca, Si, Sr and Mg extracted in the central part of the maps (black rectangle) by geometrically interpolating the pixels. Sr and Mg trends show a similar pattern of distribution. Si seems mostly concentrated in grains with a 50- 100 µm diameter.

3.5.6 Elemental quantifications for micro-XRF

SR micro-XRF Ca and Sr line scans and greyscale values

In order to compare the elemental distributions in samples ABS5 and ABS6-A with their petrographic and microstratigraphic characteristics, chemical line scans analyses were conducted in the axial region by averaging 25 pixels for each point (50 µm wide box selection). Concentrations of Sr and Ca obtained by SR-micro XFR are illustrated in figure 3.3 and were calculated along line scans and then compared to the greyscale levels of the thin sections. Ca and Sr profiles are both very similar to the optical scans but they show opposite trends. In ABS5 the highest Sr concentrations from 400 ppm to 600 ppm are observed in the micrite layers. In ABS6-A, the highest Sr concentration ranges from 2000 ppm to 2250 ppm and coincides with dark, impurities-rich layers in thin section. Sr average values for fiber-like crystals in ABS6-A are the highest (1100 ppm) compared to ABS6-B (650 ppm) and ABS6-C (770 ppm). Sr average values calculated for Ce fabric are lower compared to fiber-like fabric. Strontium average concentration in compact elongated fabric is 178 ppm for ABS5 and 520 ppm for ABS6-A. Fiber-like fabric is less dense than compact elongated columnar fabric as showed by ABS6-A Ca average concentrations of 280,000 ppm and 380,000 ppm respectively. Similarly to ABS6-A, in ABS5 Ca average concentration for translucent calcite is ca. 390,000 ppm.

ABS6-C ABS6-B (SR) ABS5 (SR) Trans Opaque Trans Opaque Trans Opaque Ca 391.6 ± 5.3 353.5 ± 9.3 386.9 ± 3.5 304.4 ± 7.9 398.7 ± 3.2 327.8 ± 46.6 Si 4.3 ± 2.6 84.6 ± 23.5 Mg 4.8 ± 3.9 9.1 ± 4.3 Sr 0.1 ± 0.1 0.3 ± 0.3 0.38 ± 0.12 1.27 ± 0.16 0.1 ± 0.02 0.49 ± 0.1 Y 0.003 ± 0.002 0.021 ± 0.003 0.001 ± 0.001 0.006 ± 0.002 U 0.001 ± 0.001 0.005 ± 0.002 n.d. 0.001 ± 0.001

Table 3.3 Mean values in translucent (Trans) and opaque calcite fabric expressed in mg/g ±1σ for selected intervals in coralloid samples ABS6-B, C (cf. Fig. 3.7) and ABS5 (cf. Fig. 3.4) measured in conventional µXRF and with SR-µXRF.

Fig. 3.9 Sr concentration along selected growth layers in ABS6-B. The Sr concentration was measured along the seven 100µm width bands visualised by dashed red lines on the Sr map (black/white scale where white represents the max concentration of 2100 ppm and black the minimum of 0 ppm). The graph on the right plots the Sr concentration along the bands centred on the coralloid growth axis. In band 4 the cycles in the central part have a period of 138 ±24 µm.

Brucker micro-XRF

ABS-6B SR micro-XRF and ABS6-C Brucker micro-XRF results have been compared with ABS5 SR micro-XRF results (Table 3.3). The data have been grouped accordingly to the type of calcite: translucent or opaque. Translucent calcite is characterized by compact elongated crystals and opaque calcite by fiber-like crystals. Sr values obtained with Synchrotron radiation are higher than the values extracted from the conventional XRF method. In ABS6-B opaque calcite yielded higher mean values for Sr: (1270 ppm), whereas in translucent calcite is 380 ppm. ABS5 shows lower Sr values with respect to ABS6-B with mean values of 100 ppm and 490 ppm for translucent and opaque calcite respectively. Mg and Si appear mostly concentrated in ABS6-C fiber-like fabric regions. Si has very high mean concentration (84,600 ppm) in the opaque calcite whereas in translucent regions is 4300 ppm. Mg vary

from 1,000 up to 30,000 ppm with mean values of 4,800 and 9,100 ppm respectively in the translucent and opaque layers. Y and U are present at trace levels and their mean concentrations appear to be higher in opaque parts compared to translucent parts. Fig.8A shows the element distribution maps obtained in a small region of ABS6-C (yellow insert in figure 3.7) and how Si tends to be concentrated in 100 µm- thick bands consisting of “grains” up to 100 µm and spaced by about 150 - 200 µm along the band. This distance is similar to the Sr cycles along single layers in the opaque Cf lenticular bands (138 ±24 µm) (Fig. 3.9).

Fig. 3.8B shows Ca, Si, Sr and Mg concentration profiles extracted from the map. The elemental profile show how Si pattern is opposite to Ca but this behaviour is less evident between Mg-Sr and Ca.

Fig. 3.11 Linear correlations between Sr, Mg, Si and Ca in ABS6-C (micro-XRF data in Fig. 8) from a selected region of interest. The correlations were then calculated for the whole interval and then separately for the translucent (compact), in green, and opaque (porous), in orange,calcite end-members.

Element correlations and principal component analyses (PCA)

Significant correlations amongst trace elements, greyvalues and fluorescence were calculated by using the Pearson r-value for ABS5, ABS6-A, B and C. We fixed a threshold of significant correlation between two elements at a value of r = ± 0.4. The choice of this threshold is somewhat arbitrary and it allows the correct distinction of different microstratigraphic and petrological phases. All the correlations among the elements and between the elements and the greyscale values range between ± 0.1 and ± 0.8. Due to the large number of samples (> 1000) considered for the Pearson correlations, each p value was <0.001 and thus the relationships can be considered significant. The highest r-value observed is in ABS6-C between Sr and U (r = 0.9) and Fe and Mn (r = 0.8). Ca and Sr are always negatively correlated (r = - 0.4) in all the samples, apart from ABS5, where the correlation is weaker (r= -0.15). Pearson correlation with fluorescence is significant only for Ca in ABS6-A (r = -0.4) and for Sr in ABS5 (r = 0.4). Correlation between Sr and fluorescence in ABS6-A is insignificant (r = 0.1). In ABS6-B and C, greyvalue levels (GV) are well correlated to Sr (positive) and Ca (negative).

The principal component analysis (PCA) was performed on ABS6-B and ABS6-C to compare the results from Sr micro-XRF and conventional micro-XRF. Figure 10 shows how different elemental concentrations can display common pattern of variability where elements that group together should reflect the same forcing mechanisms. For ABS6-B, PC1 total variance is 51% whereas PC2 explains 17%. In PC1 greyscale value (GV) groups with Sr and Y and they are anti-correlated to Ca. In PC2 Br groups with U. For ABS6-C, PC1 total variance is 60% whereas PC2 explains 34%. In PC1 Si correlates with

the greyscale value and have opposite loadings with Ca. In PC1 and 2 Mg and Sr group together and they anti-correlate with Ca and Si respectively.

Linear correlation analyses were also performed and represented in figure 11. Correlations have been calculated between elements distributed in opaque and translucent calcite. Si and Ca show an overall good anti-correlation (R2 = 0.8) which primarily reflects the correlation existing in opaque calcite (R2 = 0.9) whereas in the translucent the correlation is weak (R2 = 0.2). Mg and Ca show a general weak anti-correlation, yet, when compared separately, in the translucent calcite these are linked by a strong anti-correlation (R2 = 0.9) which is lower in the opaque areas (R2 = 0.4). Mg and Sr are positively correlated, especially in the translucent parts of the sample, whereas in the opaque portions the correlation is weaker (R2 = 0.4). Mg and Si no clear correlation has been observed in both translucent and opaque calcite (R2 < 0.2).

3.6 Discussion

Calcite coralloid in Lamalunga, akin to speleothems from temperate regions, preferentially grow during mild climatic phases, namely interglacials or interstadials. The phases of growth occurred during the Marine Isotope Stages (MIS) 1.0, 3.1, 5.1, and 5.5 (cf. (Martinson et al., 1987)) matching coeval speleothem growth phases from other Mediterranean caves (Bar-Matthews et al., 2003; Badertscher et al., 2011). By being coeval, Lamalunga coralloids patterns of elemental incorporation could have been similar, despite their different morphological aspects (digitate vs. flowstone like conformations) suggesting different mechanisms of growth (more vs. less evaporation). Elemental incorporation in calcite coralloid speleothems is mainly influenced by the factors controlling their formation. Evaporation, which is one of the main factors controlling Lamalunga coralloid growth, is stronger in digitate shapes and at their tips (Vanghi et al., 2017) explaining why all the non-Ca elements are mainly distributed along the lenticular layers. In some cases, however, non-Ca elements appear distributed along the entire growth surface of the coralloid and they are also commonly associated with dissolution/corrosion features and along the major hiatuses.

3.6.1 Mg and Sr behavior in Lamalunga coralloids

Lamalunga cave coralloids are characterized by unusually high Mg concentrations (~ 7000 ppm see Table 3.3), compared to stalagmites from temperate climate settings that usually have less than 1000 ppm (Neuser and Richter, 2007; Richter et al., 2011; Frisia, 2015; Belli et al., 2017). Commonly, magnesium incorporated in spelean calcite is believed to derive from the dissolution of the bedrock (Fairchild et al., 2000; Sinclair, 2011; Sinclair et al., 2012) and is then transported into the cave in solution as simple inorganic complex. Mg concentration in cave dripwater is a function of the residence time of the water in the aquifer and prior calcite precipitation (PCP) (Fairchild et al., 2000; Sinclair et al., 2012; Belli et al., 2017). Bands characterized by high Mg content in the coralloid could then be due to PCP and/or a prolonged water-bedrock interaction during particularly arid periods, which increases the Mg/Ca ratio of the parent solution. In this case, Mg could be considered as proxy for paleo-aridity. Lamalunga cave however, is very shallow and the thickness of the overlying bedrock is not enough to allow a prolonged water residence time in the aquifer. Modern drip water analyses indeed, shows low Mg content and Mg/Ca ratio (Mg2+ = 6 mg/L, Mg/Ca 0.12 mol/mol) (Table 3.1).

By taking into account the present-day dripwater composition (Ca ~87 mg/L, Mg ~6.3 mg/L; Table 3.1) and a conservative distribution coefficient DMg of 0.02 ±0.002 (Fairchild and Baker, 2012), the expected Mg concentration in speleothem calcite should be circa 600 ±100 ppm. Mg average concentration in Lamalunga coralloid ABS6-C is much higher than the expected value, ranging from 4800 ppm to 9100 ppm. Thus, bedrock dissolution alone is unlikely responsible for the coralloid high Mg content, and other mechanisms must have had an important role.

The effect of Mg has been demonstrated to be crucial in influencing speleothem fabric by poisoning calcite growth sites and favors the development of elongated crystals with ondulatory extinction (Kendall, 1973; Folk, 1974; Turgeon and Lundberg, 2001; Han and Aizenberg, 2003; Neuser and Richter, 2007; Frisia, 2015; Richter et al., 2015) akin to the Ce and the fiber-like fabric of the coralloids from Lamalunga. However, in ABS6 high Mg concentration characterizes not only fiber-like fabric, but also columnar elongated (Ce) fabric as shown by figure 3.8 whereas fiber-like fabric tend to be more intimately associated with Si as inferred by PCA (Fig. 3.10). This suggests that Mg alone would not induce to the formation of fiber-like crystals. The shift from elongated (translucent) to fiber-like (opaque-porous) fabric could then be linked to a series of variables including, in addition to high Mg content, the presence of other elements and/or organic molecules (Fairchild and Baker, 2012).

Due to the positive high linear correlation between Sr and Mg (R2 = 0.8) and in the PCA (Fig. 3.10),

we assume that their provenance and incorporation pattern should have been similar. Thus the variability of Sr can be used to complement hydrological information for Mg concentrations. However, the correlation between Sr and Mg is weaker in the opaque (R2 = 0.4) relative to the translucent zones (R2 = 0.9) suggesting that in the opaque bands their concentrations are unbalanced.

Common source of Sr found in speleothems are host rock dissolution, as well as allogenic input (cf. Belli et al., 2017). Sr incorporation in the calcite layers has been related to growth kinetics and drip rate (Fairchild and Baker, 2012, and references therein), and it is commonly also positively correlated with

CaCO3 supersaturation (Wasylenki et al., 2005) and negatively correlated to temperature (Tang et al., 2008).

Altamura coralloids show a high Sr content, especially in the opaque porous bands (Table 3.3). Com- monly, high Sr content can be index of diagenesis as Sr is preferentially incorporated in aragonite rather than in calcite because its orthorhombic crystal structure accommodates larger ions than the rhombohedral calcite (Speer, 1990; Railsback, 2000; Ortega et al., 2005). However, Lamalunga coralloids consist of calcite and there is no petrographic evidence of an aragonite precursor (see Vanghi et al. (2017)). In addition, the drip water analysis results (Table 3.1) do not differ from what expected for a pure limestone cave developed in a temperate or Mediterranean climate setting.

In view of the present-day dripwater composition (Ca ~87 mg/L, Sr ~67 µg/L; Table 1) and a conservative distribution coefficient DSr of 0.1 ±0.02, which takes in to account the very slow growth rate (cf. Huang and Fairchild (2001) and Belli et al. (2017)), the expected Sr concentration in speleothem calcite should be around 30 ±7 ppm. This is one order of magnitude less than the average Sr concentration in ABS5 and about 40 times less than the maximum concentration detected in ABS6. Therefore, as in the case of Mg, the dissolution of the bedrock cannot be regarded as the sole contributor to the Sr incorporated in the speleothems. It has been suggested that Sr incorporation above few tens of ppm occurs not only in the calcite lattice but also in crystal defect sites and along intercrystalline boundaries (Frisia et al., 2012; Belli et al., 2017). In this specific case, a speleothem distribution coefficient was found to be more appropriate than the distribution coefficient (DSr) to describe the elemental incorporation in the speleothem regardless to the sites of incorporation of any element within the speleothem (not the calcite crystals) (Frisia et al., 2012; Belli et al., 2017).

3.6.2 Silicon concentration in Lamalunga coralloids and possible origin

Si has been investigated in ABS6-C which can be regarded as the representative of the whole aggregate

of coralloids ABS6 where is preferentially present in the opaque bands with high inter-crystalline porosity characterized by high fluorescence. There is no apparent significant correlation between Si and Mg and thus, as a consequence, Si does not correlate with Sr (Fig. 3.11). This could be explained with the fact that Si came from a source other than the dissolution of the Lamalunga bedrock, which is pure limestone. Given the present-day moderate to low content of dissolved SiO2 in the dripwater (Table 3.1) it is unlikely that Si derived exclusively from dissolution of silicates in the soil and in the aquifer, and, therefore, an additional source and/or concentration mechanism has to be invoked.

Increases in dripwater Si/Ca has been attributed to arid climatic conditions which results in less dilution of dissolved Si in the dripwater, more prior calcite precipitation and enhanced weathering of silicate rocks (Hu et al., 2005). The relationship between silica content in speleothem and past rainfall has been investigated by Hu et al. (2005) in a stalagmite from Southern China. The authors demonstrated that high Si/Ca content in the stalagmite coincide with more positive δ18O values, and, thus reduced rainfall amount. Furthermore, microbial mediation in silica precipitation cannot be entirely ruled out as amorphous silica was observed associated with micrite laminae in stalagmites from the Nullarbor (Australia) showing strong fluorescence, and associated with S and P (Frisia et al. 2012). It is very well known, in fact, that microbes are ubiquitous in caves (Northup and Lavoie, 2001) and that they may exert an influence in mineralization (Banks et al., 2010). Enhanced dissolved Si in dripwater can also be related to the weathering of wind-blown particles in the overlying soil (Belli et al., 2017). Wind-blown minerals deposits (loess) can be particularly significant during glacial periods (Pye, 1995; Újvári et al., 2016) as well as be related to discrete inputs following major volcanic eruptions (Badertscher et al., 2014). Thus, it seems plausible that Si concentration could be interpreted as an aridity indicator. However, given the location of Lamalunga in the Alte Murge plateau, where middle to late Pleistocene ash deposits from the Campanian volcanic Province have been documented at the surface (Wulf et al., 2004) as well as in karstic depression and caves (Sauro, 1991), it is also plausible that Si excess in coralloids could be linked to volcanic eruptions, which have been very intense in Southern Italy over the last 200 ka (Siani et al., 2004; Santacroce et al., 2008; Wagner et al., 2008).

The products of explosive Southern Italian volcanism is extensively recognized in Adriatic and Ionian sea cores (Siani et al., 2004; Sicre et al., 2013), terrestrial (Bertagnini et al., 1998; de Vita et al., 1999; Zanchetta et al., 2000; Scarciglia et al., 2008) and lacustrine deposits (St Seymour et al., 2004; Wulf et al., 2004; Magny et al., 2006; Wagner et al., 2008). During explosive volcanic events, pyroclastic deposits may rapidly cover vast areas around the volcano and disperse over long distances. This is especially valid for Plinian volcanic eruptions, which are typical of the Neapolitan volcanic zone (Vingiani et al., 2014). Tephra (air-borne pyroclastic material) fallouts from Mount Etna or from the Neapolitan volcanoes have been recognized at more than 500 km from the source as for the case of Lake Ohrid in the Balkans (Wagner et al., 2008) or in the Philippi peat basin, in Northern Greece (St Seymour et al.,

2004). Pyle et al. (2006) identified ash layers from across south-western Russia as a distal equivalent of the ca. 39.3 ka Campanian Ignimbrite eruption (Phlegrean Fields, Italy) deposits, demonstrating that ash particles can travel even longer distances (< 2500 km) during large magnitude explosive eruptions (Marti et al., 2016). Volcanic soils (andosols) are known to be very fertile (Nanzyo et al., 1993; Iamarino and Terribile, 2008; McDaniel et al., 2013), which is why anthropic settlements are very frequent on the down the slopes of volcanoes. In Vingiani et al. (2014) the occurrence of andosols have been recognized in Southern Italy in non-volcanic landscapes (Calabria) like Altamura area and they have been mainly attributed to the Eolian volcanic arc and, in minor part, to the Campanian volcanism. It seems thus reasonable to infer that the high Si concentration in Lamalunga coralloids is related to volcanic ash, which consists of small, reactive particulate. In this scenario, Si-rich dust deposited over the soil would be rapidly dissolved and transported into the cave by infiltrating waters. This can also explains the observed pattern of Si concentration, which increases abruptly, whilst its decrease is gradual. It would also explain why Si appearance and demise does not match with Sr and Mg. Si incorporation in coralloid is eventually enhanced in biconvex lens-shape bands by evapo-concentration, akin to Sr and Mg.

3.6.3 Uranium, Iron and Bromine concentration

Uranium commonly correlates with the organic content of sediments because organic-matter rich sediments deposited under anoxic and reducing conditions are an effective sink for uranium from water (ten Haven et al., 1988). Uranium commonly occurs as trace element in calcite and it is used in speleothem U-series chronology. Dissolved U in natural solutions can be present with hexavalent (U6+) which is

2+ very soluble and can be incorporated in the calcite and divalent (U2 ) which is too large to enter in calcite lattice sites (Sturchio et al., 1998; Kelly et al., 2003). Uranium concentration in calcites in usually < 10 ppm (Reeder et al., 2001), but in speleothem calcite is typically around 0.2 ppm (Eggins et al., 2005) and rarely above 10 ppm (Reeder et al., 2001; Hellstrom, 2003). In ABS6-C the U average content is 2 ppm with maximum values of 12 ppm (Fig. 3.7) and is incorporated preferentially in the opaque porous layers characterized by fiber-like crystals. The high Uranium content combined with low detrital Th has been also beneficial to reliably date and produce a good age model for Lamalunga coralloids. Examples of continental carbonates consisting exclusively of primary calcite, and having high U concentration (Ortega et al., 2005; Woodhead et al., 2006; Frisia et al., 2017; Wang et al., 2017) show highly elongated columnar fabrics.

Iron is mostly concentrated in the micrite levels at the base of coralloids where it reaches values of up to 20000 ppm in ABS6 and up to 5000 ppm in ABS5 (Fig. 3.4-7). The basal surface consists of a red

sediment of Fe-oxides clots within a matrix of micrite and clay. High fluorescence indicates also the presence of organic material. Iron in addition is also present in correspondence of the hiatus (Ht C) in ABS5. In ABS6 instead its concentration is very low in other parts of the samples. This could possibly lead to link Fe to interruption in calcite deposition.

Y is commonly bound to organic substances mobilized during leaching from the soil and transported into the cave by infiltrating water (Borsato et al., 2007). If this is the case Y transport onto coralloids derives from hydroaerosols generated from the splashing of dripping water (Dredge et al., 2013; Hartland, 2013; Vanghi et al., 2017). From the results of the PCA performed on ABS6-B, Y correlates with Sr and low greyscale values and have opposite sign to Ca. It derives that Sr and Y are possibly associated to organic compounds trapped in the porosity between fiber-like crystals.

Another element, bromine, is abundant in the impurities-rich layers of ABS6-B and C (Fig. 3.6 and 3.7). Br only correlates with U, which means that most likely is bound with organic carbon. Another plausible natural source of the halogen Br, providing 75-95% of the CH3Br and bromide salts (e.g. sodium bromide, NaBr), is sea aerosol. In the lower atmosphere, Br is contained only in small concentrations as gaseous bromine (Wofsy, 1975). Terrestrial environments, which have low salinity, are usually comparatively poor in bromine (Ziegler et al., 2008). Goede and Vogel (1991) and Goede et al. (1998) studied the origins of Sr and Br and their relationship in two Tasmanian speleothems. They concluded that Sr input likely represented an input from terrestrial dust and Br possibly derived from sea-salt aerosols which is common in speleothems from caves in coastal areas like Lamalunga cave which is not far from the Adriatic coast. Because Br is present only in the porous parts of the coralloids, it can be inferred that it was likely to be associated with fluid inclusions, according to the chemical behavior of halogens that strongly partition into the fluid phase (Zhu, 1993; Wei, 2005).

3.6.4 Incorporation and concentration of the elements

The high amount of non-Ca elements found in Lamalunga samples is likely due to evaporative processes that are involved in the coralloids formation and that tend to concentrate elements at the speleothem active surface. This is why most of the impurities are located on the lens-shape convex parts of the coralloid surface. These bands are formed by porous type of fiber-like fabric and, most likely, the impurities are mostly preferentially hosted in the porosity.

The high concentration of strontium in Lamalunga calcite coralloids, suggests that this element is incorporated outside the crystals lattice, in the porosity of fiber-like crystals, as expected by the fact that

rhombohedral carbonates can accommodate large cations only in small quantities (Ortega et al., 2005). On the other hand Mg, which is smaller than Sr, may be accommodated in the lattice (Folk, 1974) weather in compact or open fabrics. This seems also confirmed by the weaker correlation between Sr and Mg in the opaque bands respect to the translucent parts of the sample. Contrary to Mg, Sr extra- lattice incorporation in speleothems does not interfere with the crystals growth. SR-micro XRF maps and greyscale values revealed that Sr has higher concentration in the porous and convex parts of ABS6 samples (Fig. 3.3; 3.5B-7; Fig. 3.9, Table 3.3), characterized by fiber-like elongated crystals arranged in bundles. However, the micro-XRF maps (Fig. 3.8) revealed that the appearance of Sr starts in the translucent band and that a sharp, positive peak coincides with the opaque band characterized by high intercrystalline porosity. On the other hand, Mg trend is slightly different: its increase remains constant and unaffected by the change into fiber-like fabric, and so it is its demise. This may suggest that, contrary to Mg, Sr incorporation is more influenced by fabric changes. Sr incorporation increment seems to be directly proportional to the increase in fabric porosity. However, it seems unlikely that the presence of strontium in the parent fluid is the factor producing the fabric change from compact elongated to porous fiber-like because its presence can be in either the compact or the porous fabric.

In figure 3.3, it is possible to observe that Sr is higher in regions of ABS6-A showing high fluorescence. Therefore, high Sr content in the coralloids is not only due to evaporation, but also to the preferential binding of Sr with organic material from the soil. However, the opposite is not necessary true: namely, where Sr concentration is high the fluorescence (greyscale values) is also high. This is also demonstrated by the weak correlation between the fluorescence intensity and Sr concentration (r = 0.1). De- spite Sr not being necessary associated to the presence of organic material its intake rises during increased transport of impurities (and thus organic compounds) from the soil zone. Conversely, in ABS5 the correlation between Sr and fluorescence is higher (r = 0.4). Fluorescence intensity and Sr are especially high at the micritic levels that represent a reduction or cessation of coralloids growth as confirmed by the U-Th ages (Lari et al., 2015; Vanghi et al., 2017). In this specific case, the incorporation of the elements like Sr, bound to the organic material, could have been deposition onto the surface of dry coralloid by aerosol during relatively arid phases. The chemical signature of the hiatus thus is different from that of the calcite deposited during normal growth (Dredge et al., 2013). If this hypothesis is valid for the strontium present in the ABS5, it can be regarded as an index of local dry periods for the Lam- alunga region. Other non-Ca elements like Y, Br and U, possibly have a similar behavior of Sr as confirmed by the high correlations.

Si, which is the second most represented element in the coralloids, was likely introduced in the cave via infiltration from the soil, bound with organic material. Indirect relationship between this element and the organic material is showed by Si negative correlation with Ca which in turn negatively correlates with the fluorescence (Fig. 3.3). This strong negative correlation with Ca, is not observed for Mg and

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Sr (Fig. 3.11) indicating that Si is principally concentrated where calcium is low and the calcite is less dense, whereas Mg and Sr are not necessary hosted in the porosity of the calcite. Another hypothesis is that Si was transported by dust as particulate suspended in dry aerosol. The micro-XRF map in figure 8 shows that the highest Si concentration occurs in grains up to 100 µm in diameter suggesting that it a possible transport as particulate. Particles of Si deposited on the surface of the coralloid, would have fostered the formation of crystals growing with different orientations and separated by gaps. Figure 5B shows that some of the low-Ca areas do not correspond to pores and are not occupied by either Sr or Mg suggesting that it could potentially host Si. Due to the strong association between Si and intercrystalline porosity, Si might control the opening of the fabric: from compact columnar elongated (Ce) calcite to porous fiber-like. The same does not seem to be verified for strontium or magnesium that most likely only controlled the elongation of the columnar crystals characterizing all the Lamalunga coralloids.

3.6.5 Models of elements incorporation

Following the petrographic observations and the geochemical maps of Lamalunga samples and the internal physical conditions of the cave, three models have been here proposed to explain the source, the transport and the incorporation of the elements into the coralloids. Two of the models are linked to climatic variations and acted on long term temporal scale. The third model does not reflect climatic changes but is connected to discrete short term events like volcanism or increased aeolian transport.

3.6.6 Humid phase (climatic)

During humid climatic phases the infiltration inside Lamalunga cave would have intensified and so would have been the spray and the hydroaerosol circulating through the galleries. Leached material from the soil zone most likely entered in solution but weaker evaporative processes would have not been able to concentrate the elements at the speleothems growth surface. This could explain the precipitation of clean compact elongated columnar (Ce) calcite which is commonly linked to humid conditions (Frisia, 2015) (Fig. 3.3).

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3.6.7 Dry phase (climatic)

When the local climatic conditions changed to relatively drier and the infiltration regime into the cave decreased, evaporative phenomena exceeded at the expenses of hydroaerosol formation. Part of the elements would have been transported in solution by the seepage and incorporated in the calcite. How- ever, particulate from dust would also have been introduced by in-cave dry aerosol and deposited on the speleothem surfaces. Evaporation, enhanced by dry-aerosol (Dredge et al., 2013), would have concentrated the element at the tips of the coralloids forming the lenticular opaque bands rich in non-Ca elements (Fig. 3.4-7). Particulate would have then created an ideal medium for the formation of fiber- like crystals that are characterized by high intercrystalline porosity (Fig. 3.9). Aerosol stream flow progressively decreased moving away from the entrance, being minimal in the deepest parts of the cavern. This could possibly explain why ABS5 differs in its morphology and internal structure from ABS6 coralloid, which is coeval. ABS6 grew in an ample chamber, closer to the entrance and thus more affected by air circulations and evaporation, which lead to the formation of fiber-like crystals. ABS5 instead, was growing in a small chamber almost at the end of the gallery where evaporation was not strong enough to concentrate the film of fluid at the tip of the coralloid and Ce fabric could form. Enhanced evaporation on ABS6 tips explains also why non-Ca elements result here 2 to 4 times more concentrated than in ABS5. In addition, fluorescence in ABS6 is almost twice than in ABS5 possibly suggesting that bacteria favored by dry conditions (Banks et al., 2010) could have colonized ABS6 surface.

3.6.8 Non climatic episodes

Discrete events, like volcanic eruptions, are usually shorter term compared to climatic oscillations and they can both happen during dry or humid periods. Occasionally, allochthonous material like aeolian dust or volcanic ash, accumulated in the soil zone and, being extremely reactive to weathering it was readily transported inside the cave bound to organic colloids in water or particulate suspended in air currents. As for the humid phases, these episodes are visible in the coralloids as dark opaque bands. ABS6-C micro-XRF maps, have shown that Si incorporated in the calcite has a granular appearance. Si is thus allogenic and has been transported inside the cave mostly as solid particulate. The trend of Si curve (Fig. 3.8) with a sharp positive peak and slower decreasing, suggests that it entered in the calcite very readily and then got progressively consumed. This lead to speculate that its origin is possibly linked

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to volcanic events that are instantaneous natural phenomena and can spread Si-rich ashes at great distances.

3.6.9 Greyscale values significance

Given that GV reflects the grade of porosity in the sample and the higher is the porosity and the most likely is the accommodation of the impurities, greyscale values can potentially give a relative estimation of elemental concentration. Said that, it seems that strontium and yttrium, that group with GV in the PCA performed on ABS6-B, were the major fraction of colloidal bulk representing the impurities trapped extra-lattice in the coralloid surface. In Vanghi et al. (2017) we interpreted fiber-like and micrite, signified by low greyscale values, to suggested variable drip rates, high CaCO3 supersaturation and strong evaporation. In contrast, the dense “clean” elongated columnar fabric, which leads to high greyscale levels, reflects a steady water supply and more continuous calcite precipitation under relatively lower CaCO3 supersaturated solutions. Because water was flowing slowly, less soil entrainment occurred and detritus and oxides are almost absent from the speleothems (Ayalon et al., 1999) as showed by the clean compact calcite of the coralloids from Lamalunga. This agrees with a previous study published by Oster et al. (2014) which lead to the same conclusions by comparing reflectance measurements (greyscale values) and petrography to trace element variation and stable isotopes analyses on a stalagmite from western North America.

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Fig. 3.12. Schematic figure that shows all the principal characteristics of Lamalunga coralloids.

3.7 Conclusions

Synchrotron and conventional micro-XRF high resolution maps are a powerful tool to investigate the pattern of distribution of chemical elements in the finely laminated structure of cave coralloids. In Lam- alunga cave this type of speleothem, unlike stalagmites, do not form from waters directly dripping on the surface, but it is the result of hydroaerosols combined to strong evaporative processes (Vanghi et al. 2017) and possible dry aerosol contribution.

In parts of the cave more subjected to air currents and located near to the entrances, strong evaporative processes, organic binding and dry aerosol concentrate non-Ca elements in the axial parts of the coralloids in form of biconvex lenses. In this case coralloids are clustered in complex aggregates with typical cylindrical digitate shape mostly formed by porous fiber-like fabric with finely laminated internal structure (Vanghi et al. 2017).

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In deeper parts of the cave and in areas characterized by more efficient infiltration dry aerosol is absent or negligible, evaporative effects are less pronounced and microbial and organic binding are limited to growth interruptions when the speleothem surface is also characterized by dissolution phenomena.

The elemental incorporation in coralloids is strongly influenced by the growth mechanism as well as the climatic context. During dry phases and in highly evaporative morphologies (ABS6) the elements are incorporated intra-laminae, mimicking the internal microstratigraphy which implies that visual changes also reflect chemical changes. Non-Ca elements (Si, Mg and Sr) are preferentially incorporate between fiber-like crystals in opaque, porous and high-fluorescent layers that form lens-shaped dark bands or in micritic layers (Fig. 3.12). During wet phases and in less evaporative morphologies (ABS5) coralloids are characterized by isopachous bands of compact calcite, formed by elongated columnar crystals, with weak fluorescence and lower content in non-Ca elements.

However, in both cases Lamalunga coralloids are characterized by unusually high non-Ca elemental concentrations respect to stalactites, stalagmites and flowstones from similar environmental and climatic contexts. In particular in high-evaporative ABS6 coralloids Sr and Y are > 3 times more concentrated with respect to coeval layers in ABS5 (Table 3.3). This suggests that the incorporation of elements like Mg, Sr, Br, Y and U although influenced by climate and environmental changes is strongly controlled by the position of the coralloid inside the cave as well as its geometrical evolution during its growth.

In Lamalunga coralloids Si is the most abundant non-Ca element and is accommodated in the intercrystalline porosity sometimes in discrete “grains” suggesting a possible particulate origin. Solid particles at the surface of a speleothem could preclude the formation of compact calcite and foster the formation of crystals with divergent borders. Thus, possibly, Si is more likely responsible for the highly porous fiber-like columnar fabric and contrary to Sr and Mg, is linked to discrete events, most likely volcanic eruptions (Sauro, 1991; Wulf et al., 2004).

In conclusion, growth and elemental incorporation in Lamalunga coralloids are influenced by climate, discrete environmental events, as well as by the hydrophysical characteristics of the cave environment that control the amount of evaporation and dry-aerosol incorporation. Evaporation in speleothems, has to be accounted not only for stable isotopes fractionation but also for the incorporation of non-Ca elements. Petrological observations coupled with fluorescence and grey scale analyses are fundamental and complementary tools that allow the correct interpretation of speleothem growth mechanism and chemical proxy data interpretation.

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3.8 References

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Chapter 4. Neutron scattering pilot study on speleothem fabrics complementary characterization or advance in knowledge of speleothem properties?

4.1 Background

The scope of sedimentary petrology is to investigate sedimentary rocks by microscopy and spectroscopy methods to understand the processes that result in their formation. Speleothems can be considered sedimentary rocks, and their unquestionable value as archives of climate change has, as outlined in the previous chapters, nurtured tremendous advances in the tools used to probe their crystal structures, crystal surfaces, geochemistry and, recently, porosity at the microscopic scale (Higgins, 2006). Much research has focused on how crystals change in size, shape, orientation during the growth of a single speleothems but there is still little knowledge about the role of interconnected porosity in driving early diagenetic processes, such as dissolution-reprecipitation. By favouring infiltration of fluid across speleothem layers, porosity and micro-porosity may result in the post-depositional re-setting of the original signal (Bajo et al., 2016). Interconnected porosity and permeability in speleothems are a function of the textural (fabric) and microstructural (dislocations, stacking faults, twins, vacancies) characteristics of the crystals composing speleothems, and reflect processes related to mechanisms and pathways of crystal growth (Frisia et al., 2000; Frisia et al., 2018).

Speleothem porosity and architecture has been commonly investigated by CT-scan tomography (Mickler P. J., 2004; Vanghi et al., 2015; Walczak, 2015; Chawchai et al., 2018).

It is clear from these studies that there are internal structural features of speleothems which may provide information about their conditions of growth and potentially facilitate the degree of alteration of the primary signal (Frisia et al., 2000; Maire et al., 2009; Martín-García et al., 2009; Perrin et al., 2014; Frisia, 2015; Bajo et al., 2016). What conventional CT scan tomography highlights, however, are cm—scale architectural properties of stalagmites, which are useful to appreciate before any other analytical method is applied if a speleothem is suitable or not for palaeoclimate investigation. The microstructure of pore space and its evolution during reaction with pore-contained fluids is a critically important factor controlling fluid flow properties in speleothems. Size, distribution and connectivity of pores, fractures and grain boundaries, dictate how water migrates into and through the micro-environments and ultimately react with the

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carbonate crystal surfaces. In order to interpret the history of climate from speleothem data, its physical and chemical “fingerprint” preserved in the crystals must be fully explored over widely different length scales.

To date, there is no systematic classification of speleothem porosity as related to the arrangement of crystals within speleothem layers at a scale that is substantially that used for palaeoclimate investigation. The present research has already demonstrated that micro-scale investigation of speleothems is revolutionizing low-temperature geochemistry applied as environmental and climate archives (Chapter 3). Furthermore, the importance of studies of speleothems by combining optical petrographic observations, UV-fluorescence and a novel approach based on high-resolution scanning of the entire samples has been highlighted as one of the best approaches to provide valuable base-line information about speleothems conditions of formation in the natural system.

Speleothems are polycrystalline materials, and the orientation distribution of a textured polycrystalline material has been traditionally determined from a few individual pole figures of lattice planes hkl measured by X-ray, electron or neutron diffraction. To date, most of the textural analysis of speleothems has been carried out by Electron Backscattered Diffraction (Neuser and Richter, 2007). Traditionally, texture analysis has relied on pole figures, which give a statistical directional distribution of poles to a specific lattice plane. The recorded intensity is proportional to the number of lattice planes in that orientation. Given that most research takes for granted that speleothem crystals grow with a preferred c-axis orientation perpendicular to the substrate, it is surprising that textural analyses has been neglected. However, the objective of textural analysis is to determine preferred orientation of polycrystalline samples, which, in the case of speleothems, is related to growth processes, because metamorphism is not an option.

Following high spatially resolved investigations illustrated in the previous chapters, here it was explored the application of neutron tomography and neutron scattering to speleothem fabrics with the aim of answering questions related to the role of particle size and particle orientations on intercrystalline chemical reactions that may result in remobilization of primary signals. Following Wenk et al. (1984), neutron scattering was applied to speleothems because this technique is believed to enable determination of complete pole figures on a single spherical specimen and is advantageous for coarse grained materials. At microscale-level, speleothems are coarse grained materials, thus, neutron scattering seemed to be promising to extract accurate pole figures from speleothems.

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The preliminary study was carried out at the Australia's Open Pool Australian Lightwater (OPAL) reactor at the Australian Nuclear Science and Techonology Organisation (ANSTO) using three different beam-lines: neutron tomography with the DINGO tomographer, small-angle neutron scattering (SANS) and the diffractometry using with QUOKKA and the KOWARI instruments respectively. At ANSTO, neutrons beams which are the source of the different equipments, are created at the OPAL nuclear reactor by fission.

OPAL was accessed through a successful competitive proposals entitled “Neutron tomography and scattering in speleothems: the influence of porosity and texture on the accuracy of palaeoclimate interpretations”. The research proposal was designed in collaboration with the beamline scientists. Experimental costs have been covered by an Australian Institute of Nuclear Science and Engineering postgraduate reseach award (AINSE PGRA 2016-2017). The experiments run for 12 days, between April and May 2017.

4.2 A Brief introduction to neutron science

The scattering of radiation by surfaces or interfaces characterized by roughness has been the object of a large body of materials science research (for a review see Sinha et al. (1988)). X-ray scattering (diffraction) is the interaction between incident X-rays and electrons in a medium, thus, it depends on the density of electrons and their distribution around the atom.

Thus, for biological objects, and light atoms, X-Ray scattering may not resolve structural defects or the nature of interfaces, because the density contrast is very low. This is not true for neutrons, where the variation in contrast becomes a powerful analytical technique.

Neutrons were discovered in 1932 by Chadwick and are electrically neutral particles with mass similar to that of a protons. Two aspects make neutrons advantageous and useful for material analysis. First, they do not have charge, therefore they are not scattered by electrons and can penetrate matter in depth better than other charged particles like in ionizing radiation (e.g. X- rays). Secondly, neutrons interact with the nucleus of an atom and as the nuclear force has shorter range (after 2 fermis it rapidly decreases) compared to the electrical force, solid matter is not very dense for the neutrons (Hammouda, 2008; Pynn, 2009). Therefore, neutrons interact considerably with light materials (e.g., substances containing hydrogen) and penetrate into heavy materials with minimal attenuation (Schwarz D. et al., 2005). This property allows the use of neutron scattering to investigate particles and porosity in mediums composed of light elements that would

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be very difficult to detect using X-ray scattering and also allows contrast matching experiments. Despite this unique advantages, neutron beam tecniques need to be combined to the other conventional tecniques, like X-ray scattering or electron microscopy, to investigate the full range of structural properties of matter (Hammouda, 2008; Pynn, 2009).

The scattering signal is observed when there is contrast between two media at a given Q and it is represented by a power law curve. The power law regions of the curve can be described by:

(i) High Q domain (or Porod’s region), characterized by a constant negative slope (m) and a linear relationship between I and Q. In this region, the contrast is created only at the interface between two different media. Therefore, the information gained is only related to the surface of this interface which is expressed by the negative slopes value (m), also called the Porod exponent; (ii) Low Q domain, or Guinier regime, where the relationship between I and Q is flat and I is asymptotic as Q approaches zero. The observation window is very large and, therefore, a structural order of the media can be obtained; (iii) a transition regime which represents the inflection point of the curve and is related to the z-weighted radius of gyration of the particles (Rg) (Rouquerol et al., 1994) (Fig. 4.1). (iv)

Fig. 4.1 Idealised power law scattering curve showing Guinier, Transition and Porod regimes.

Information about the structure of the examinated specimen is expressed by the power law scattering curve, which is the sum of the scattering contribution from all the multiple structural

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levels within the sample. However, the power law curve is only a featureless representation of each independent structures that posses their own shapes and sizes. The characterization of these structures is obtained through a modelling and the choice of the model is dictated by an a-priori information available for the considered system and the characteristics attempted to find (Rose et al., 2014; Avaro, 2017).

4.3 Brief introduction to neutron tomography

Tomographic techniques (using x-ray or neutrons beam) allow representing three-dimensional structure as a series of two-dimensional images formed from parallel sections (Sutton, 2008). Tomography is a powerful non-destructive imaging tool that allows studying the spatial distribution of porosity by inspecting variations in the density of a material. These tecniques are based on the universal law of attenuation of radiation passing through the matter. Differently to the X-ray analogues (practised in hospitals as a medical diagnostic tool or in airports to identify dangerous items), neutron tomography permits to perform analyses at higher resolution and penetration and yield a different contrast between materials. This because, unlike x-rays whose attenuation coefficients increase with atomic number Z, neutron beam attenuation does not show any dependence with Z and is significantly attenuated by light materials like hydrogen-rich substances (Schwarz D. et al., 2005). Therefore, neutrons allows the detection of hydrated phases and organic matter within the analyzed specimen.

4.4 Materials and method

All material analysed in neutron scattering and tomography pilot study consist of calcium carbonate. Because neutron scattering has the potential to reveal interparticle distances, porosity, presence of organic compounds and particle surfaces, and by considering that this was the first attempt to apply the technique to speleothem science, samples were carefully selected on the basis of their petrographic diversity. Three samples from stalagmite FR16 were selected from the translucent (Tr), low porous (P) and relatively high porous (PP) zones respectively (cf. cap. 5). FR16 is characterized by compact and slightly more porous columnar crystals, as described in chapter 5 and is representative of the most typical columnar calcite fabric. For comparison, and to build a systematic classification of fabrics from a “neutron beam point of view” a laminated

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stalagmite, TM19, from Tam Doum Mai cave (Laos) consisting of couplets of columnar compact and columanr porous calcite was selected. TM19 developed in a monsoonal (wet/dry seasons) context with high seasonal contrast in drip rate, whereas FR16 grew in a relatively mild Mediterranean climate, with at least two periods of infiltration (end of Winter and Autumn). Consequently, the fabrics of TM19 were greatly influenced by an ON/OFF system, with very porous columnar crystals forming in periods of high drips. By contrast, FR16 fabrics show subtle transitions between more and less compact columnar calcite (Chapter 5) reflecting a less contrasted seasonal regime. Regardless to the characteristics of drip rates, both TM19 and FR16 architectural elements consit of columnar calcite crystals with the c-axis perpendicular to the substrate. In the attempt to build a systematic classification of fabrics based on micro-structural and microporosity data, samples that are characterized by diverse orientations and compaction of particles also needed to be assessed. A coralloid speleothem (ABS5) from Lamalunga cave (Southern Italy) were selected because of the “fibre-like” nature of the crystals (see Chapter 2). For each coralloids two regions have been studied: a porous area (P) consisting of fiber-like crystals and a translucent (Tr) region with more elongated columnar crystals. A flowstone (CL9) from Collalto cave (Italy) (Martín-García et al., 2017) provided an exemplary of speleothems consisting of crystals with at least three optically visible orientations of the c-axis and diverse phases (calcite, quartz and iron oxides). As the most “disordered” end member in terms of random orientation of the crystals we utilised a dolomitized stromatolitic bindstone (CS10) from Costalta cave (Italy).

For SANS analyses, the aforementioned samples were complemented by a Triassic microbialite (MIK7), which likely preserves macroscopic relicts of aragonite and organic compounds.

4.4.1 Textural analysis

Textural analysis was carried out at the neutron diffractometer “KOWARI”. The ANSTO KOWARI is commonly used in materials engineering to analyse residual stress in metals and alloys (Lavigne et al., 2018). Its application in speleothem science is therefore quite novel. So far it is the only technique that allows the study of texture in speleothems like deformation and orientation changes of the growth axis.

Six cube-like samples of ca. 10-20 mm3 were used for neutron texture analysis because it eliminates absorption correction and other background effects. The results are visualized as pole figures (Suwas and Ray, 2014). Measurement time was conducted at a wavelength of 1.65 Å that

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provides several advantages in case of calcite samples: (i) At this wavelength the neutron flux is close to maximum. (ii) At this wavelength four CaCO3 diffraction peaks (reflecting the orientation of the planes of the reticular space): (104), (006), (110) and (113) are close together so can be measured at once utilizing 15° coverage of the KOWARI detector positioned at 2θ = 36°. (iii) The pole figures permit to reconstruct the orientation distribution function, provided that the weakest intensity (110) pole figure is resolved. The experimental setup has been used previously on KOWARI to measure calcites with a measurement grid of 5°×5°.

4.4.2 Porosity analysis

Neutron tomographic analysis perfomed at the DINGO beamline under the supervision of the beam-scientist Dr. Floriana Salvemini allows investigating macro-porosity. The technique was applied on two 20 cm long fragments of the stalagmite FR16. For each piece, a window inlcuding the region of interest, have been selected and individually scanned. The DINGO field of view has been set to about 50x50 mm2 6LiF scintillation screen. The pixel resolution is ca. 25 μm and guarantees a spatial resolution sufficient to resolve the structural features of interest inside the samples. To perform the tomographic reconstructions, 360° 1439 angular projections assured a high sampling rate, with acquisition time of around 90 seconds per single projection. High resolution scans were acquired in ca. 24h for both FR16 samples. The data have been processed using the Octopus code for tomographic reconstruction and the obtained slices have been then recomposed using the AVIZO© software.

Micro- porosity (micrometre to sub-micrometre scale) has been investigated by small angle neutron scattering (SANS) at QUOKKA beamline. Samples were cut in thin slices of 15x15 mm and 1 mm thick. Each slice was set on demountable cells between two quarz windows separated by a 1 mm to 4 mm spacer. Data were acquired using an Ordela 21000N High Count-Rate TwoDimensional neutron counter detector. The detector was sequentially positioned at distances of 1.3 m, 12 m and 20 m from the sample and a full scan time was of 40 min per sample. Small- angle neutron scattering analysis is a non-intrusive method for investigating microstructural inhomogeneities within a powder or solid object (Beaucage, 1995). SANS also allows good contrast matching and is particularly useful to study properties of materials formed by phases with different densities, which, for speleothems could be either organic compounds and calcite or water and calcite (Rose et al., 2014; Avaro, 2017).

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4.5 Results and Discussion

4.5.1 Speleothem textural analysis (KOWARI)

Pole figures show the results of our speleothem textural analysis experiments (Fig. 4.2) (Lavigne et al., 2018). The translucent columnar crystals in FR16-Tr show that there are a few contours around the same crystallographic orientation for the poles of the planes perpendicular to the c- axis, that is the (006) planes (Fig. 4.2 top). FR16-P is the porous fabric in stalagmite FR16, still consisting of columnar calcite, but characterized by intercrystalline linear pores elongated in the direction of growth (the c-axis). Critically, there is no difference with FR16-Tr. The planes perpendicular to the c-axis are all clustered around the same crystallographic direction. Similarly, the pole to the planes parallel to the c-axis (110) and those coinciding with the cleavage rhombohedron (104) are also clustered around crystallographic directions consistent with calcite symmetry. FR16-PP is the sample with the highest porosity, which shows smaller crystals when observed by optical microscopy. Critically, FR16-PP is the fabric that is characterized by more dispersed orientations, without a well defined cluster around the same crystallographic direction. FR16-PP is behaving more like an aggregate of crystals with diverse orientations than single crystals. Somehow FR16-PP is “less textured” than FR16-P and FR16-Tr. It seems to be making a transition to a fiber-like crystal aggregate (Bajo et al., 2016).

The CL9 flowstone fabric is apparently compact, as seen by optical petrography, but definitely characterized by at least two distinct crystal orientations. This is very well illustrated by the pole figure (Fig. 4.2 panel CL9), where two distinct cluster planes are perpendicular to c-axes at 180 degrees from each other. The poles of (104) and (110) are on arcs, suggesting that the fabric consists of crystals with a range of orientations and are, therefore, less textured than the columnar fabrics of FR16-Tr, FR16-P and FR16-PP. The TM19 sample shows two distinct pole spots for (006), close to each other, and a radial distribution for (110). TM19 is, therefore, becoming more similar to a fibrous aggregate, because the orientations are random, and not even on an arc as in CL9. The ABS coralloids from Lamalunga cave show a typical “fiber crystal” pole figure. The (006) planes definitely show contours, which are more pronaunced in ABS5-P (porous), whereas in the compact specimen (ABS5-Tr) there is still a preferential, unique crystallographic orientation. The other planes show a radial distribution, typical of spherulitic growth.

In synthesis, the results highlight a progressive decrease in “textured” properties from FR16 to ABS5.

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Fig. 4.2 Pole figures of speleothems from the neutron textural analyses carried out at the ANSTO KOWARI beamline. See text for the details.

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4.5.2 Significance of textural analysis of speleothems

Neutron scattering application to speleothem science is still an unchartered territory. Most of the relevant information on the samples analysed for this pilot study, in fact, could have been already obtained by optical microscopy, without the use of sophisticated techniques. However, the results from KOWARI neutron diffraction experiments allow advancing our understanding on how speleothem grow now that also Transmission Electron Microscopy has gained new momentum (Frisia et al., 2018) and that speleothem fabrics can be explained via multiple nucleation and growth mechanisms. The unicity of neutron scattering is that it is sensitive to small, irregular edges in a sample where the overall appearance is that of a single crystal, but it is, in reality, a composite crystal. The FR16 specimen is the perfect example of the power of neutron scattering. Optical microscopy observations of FR16-Tr reveal a compact, translucent, impurity-free fabric characterized by large crystals with uniform extinction. At atomic scale one would think that each columnar crystals is a single individual resulting from a monomer-by-monomer atom/molecule attachment on advancing steps (cf. Frisia et al., 2000). In an attempt to link this mechanism with environmental or climate parameters, crystallographic continuity maintained along the growth layers indicates that the FR16 stalagmite grew regularly under constant drip rate, from waters with low impurities loading (FR16-Tr end-member). Than, the pole figure of FR16-Tr and FR16- P suggest that the crystals grew according to the classical spiral growth as postulated by Frisia et al. (2000) on the basis of Scanning and Transmission Electron Microscopy observations. The case of FR16-PP is different: under the petrographic microscope the fabric is more micritic, formed by arrays of small crystals, whose orientations, due to their small scale, could not be detected. Neutron scattering not only supports the petrographic observations but also reveals that the crystals have different orientations. Hence, it can be stated with confidence that the micrite fabric in speleothems is a relatively poorly textured aggregate. Micrite formation in speleothems has been related to bio-mediation and presence of organic compounds (Frisia et al., 2012; Frisia, 2015). These are likely to be located at crystal boundaries. Thus, the clear microstructural difference observed by neutron scattering between the columnar fabrics and the micrite in FR16 suggests different mechanisms of formation.

The other stalagmite analysed at KOWARI, TM19, when observed at the optical microscope consists of milky, porous and translucent compact laminae, which appear to consist of columnar porous and columnar compact fabrics. However, neutron scattering reveals that TM19 has pole figures more similar to what would be expected by a fibrous texture. Thus, neutron scattering is detecting a structure of the TM19 calcite that is different from that of FR16. Most likely, the

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crystals composing columnar individual aggregates in TM16 are separated by organic compounds and have a more rugged interface. The presence of organics creates a slight mismatch between the individual crystal units (at micrometre to nanometre scale) that result in the “girdle” observed in the pole figures. Preliminary experiments carried out by Frisia (personal communication, 2018) indicate that TM19 may have crystallized by particle attachment rather than a spiral growth mechanism, and that organic colloids bridge the particles. Thus, in terms of environmental significance, TM19 fabrics reflect influence of organic colloids flushed onto the stalagmite (likely during the monsoon season).

The case of CL9 was selected to test the hypothesis that neutron diffraction may reveal when a speleothem suffered diagenetic alteration and isothermal recrystallization processes (Ostwald ripening), where the latter is most likely responsible of the development of crystal rays (Frisia, 1996). Although the answer is not straightforward, there is a clear difference in the pole figures of CL9 and those of FR16 and TM19. CL9 is clearly textured, but (006) poles plot at almost 180 degrees, and there seems to be a rotation of the c-axis, with one family of crystals at 180 degrees from the other. More interesting is the plot of the (104), which is the orientation of the so called “coarse modulated microstructure” observed in diagenetic calcite and reported by Gunderson and Wenk (1981). These authors show a stereogram where coarse (the scale is about 500 nm) modulated structure orientation as derived from diffraction patterns is aligned on arcs, similarly to what recorded by KOWARI. What causes this is, as yet, not known, but has been ascribed to basal stacking disorder (Wenk et al., 1983).

Neutron diffraction data suggest that modulated microstructure may actually be the expression of two dominant directions of the c-axis and of a rotation of the other axial planes possibly induced during dissolution-reprecipitation in an environment where the process is constrained in micropores. All this is speculation, but neutron diffraction may shed light on diagenetic textures.

The ABS5-Tr and ABS5-P are calcite coralloids, whose growth is controlled either by hydroaerosols or evaporative processes rather than being fed by dripping water like in “conventional” stalagmites (Vanghi et al., 2017). At the optical microscope, the coralloids consist of elongated, needle-like crystals arranged in fans with sweeping extinction. Neutron diffraction provides additional information to the macro-scale fabric. The pole figures are typical of materials consisting of fibres at a very small scale. Thus, the visible acicular calcite crystals may consist in bundles of fiber-like crystals with diverse orientations, which result in the “girdle” typical as shown in figure 4.2 (lower two panels). Only (006) shows a highly crystallographic orientation. The environmental process of formation reconstructed from ABS5 (that of hydroaerosol supply

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and evaporative processes), can be complemented by the notion that each single needle-like crystal is itself a composite crystal, whose growth likely occurred by particle attachment, more similar to TM19 than FR16. Eventually, this first attempt at applying neutron scattering to speleothems is promising as it has potential to recognize mechanisms of growth of speleothem crystals, which influence their capture of climate and environmental proxy data (see Frisia et al. (2018)).

4.5.3 Porosity analysis

Two types of porosity have been observed in FR16 stalagmite: (i) empty pores (48.5 % of the total), through where the neutrons beam can pass without being attenuated (leading to black regions in the image) and (ii) pores possibly filled with organic compounds or fluid inclusions (51.4 % of the total) that scatter the beam (leading to bright regions in the image). The first noticeable difference between black pores (empty) and bright pores (organic-rich), is the dimension (Fig. 4.3). Empty (black) pores are usually larger than filled (bright) pores. The second difference is related to the shape. Black pores show more round shapes compared to bright pores that are instead elongated. In the figure the pores are represented in tensor view which does not display their real shape (rendering view). When tensor view is applied, pores are represented as ellipsoids, which allows better characterization of their dimension, orientation and interconnections (Fig. 4.3).

Open columnar fabric is more porous (90.8 % of the total stalagmite porosity) compared to compact columnar (8.9 %). The micritic fabric (FR16-PP), appears very dense (0.4 % of pores). Columnar calcite only hosts micron-sized porosity wherease in open columnar pores range varies significantly (Fig. 4.4). Apparently, some pores seem to be a few mm3. However, these relatively high values likely relate to interconnected porosity (Floriana Salvemini pers. comm., 2017). By contrast, most of the pores in the micrite have a volume ranging from 1 to 2 mm3. No clear distinction is size has been observed between black and bright in the same fabric. The only exception is the columnar fabric where white pores are significantly smaller and more abundant comapred to black pores.

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Fig. 4.3 Tensor view 3D representation of the porosity observed in a laminated portion of FR16.

The results derived from the neutron tomography, combined with petrography highlight a strong correlation between pore distribution and fabrics. As expected, compact columnar fabric is dense compared to open columnar fabric, which accounts for 90% of the total porosity. Fluid inclusions and organic matter, in compact calcite, are mostly concentrated in small pores (< 200 microns) (Fig. 4.4). In open columnar fabric (FR16-P), fluid inclusions and organic matter are present in both small and large pores. Thus, the presence of impurities (organic compounds) possibly impeded a perfect coalescence of crystals. However, organic compounds did not disturb the growth of single crystals by creating rugged interfaces and sub-grain boundaries, which is probably why the neutron scattering results of FR16-Tr and FR16-P are similar. Micritic fabric, which is usually considered porous, because light transmits through it in the same way as in columnar porous fabrics, is revealed to be denser than compact columnar calcite. This contrasts with the thin sections observations and may be a bias due to the detection of DINGO, which is not able to detect pores that measure less than 150 micrometers. Therefore, micrite fabric most likely only contains micropores.

Relative to KOWARI, DINGO has not produced any further improvements with respect to what offered by X-ray computed tomography (XCT) or micro CT scan that has been already used in speleothem science (Bajo et al., 2016). The use of XCT is preferred because is less time consuming and produces higher resolution results. XCT is also more accessible in many univerisities or research centres as it is not sourced by nuclear energy. In a previous work (Vanghi et al., 2015), I applied XCT on a stalagmite with different fabrics to study the distribution of the

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density, using greyscale, that corresponds to the changes in the textures reflecting different environmental conditions of formation. Mapping the internal density variation allowed to represent the internal stratigraphic architecture by applying different coloured masks to different greyvalues ranges. CT scan helped to identify several stratigraphic subunits, especially where macroscopic laminations could hardly be identified. Similar works have applied XCT images to extract density variations and compare them to petrographic observations by Chawchai et al. (2018), Walczak (2015) and Mickler P. J. (2004). Their aim was to idenfy the best plane along which the stalagmite should be sectioned prior to further geochemical analyses. Neutron tomography is by far more time consuming, of more difficult access and the results are not easy to interpret.

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Fig. 4.4 Histograms of the different kinds of porosity in FR16 analysed by neutron tomography. For each fabric observed in FR16 (compact columnar, open columnar), two histograms have been created accordingly to the presence/absence of organic material and fluid inclusions. On the x-axes are reported volumes ranges of the pores in microns squared.

4.5.4 SANS scattering

SANS scattering curves for all the analysed samples only showed the Porod region whereas the Guinier region was not observed at low-Q. For FR16 porous samples (FR16-P and FR16-PP) cross sections, SANS intensity is higher than in the translucent sample (FR16-Tr). FR16-P and FR16-Tr almost overlap from 0.0008 < Q > 0.002 then the two curves separate.

Frasassi samples show an intesity of from 1 to 0.5 orders of magnitude lower than the samples considered as a comparison, CL9, CS10, MIK7 and TM19. MIK7 however, for low Q, shows an intensity very similar to FR16 and then, at about Q = 0.4, its curve deviates.

The scattering vector Q ranges from 10-3 to 1. Data fitting to the Porod model yelded a Porod esponent (m), that describes the slope of the Power Law curve (lnI(Q) vs lnQ) (Porod, 1953). The resulting intensity versus scattering vector plots superimpose perfectly and they follow a power law with an exponent oscillating around the value 3 (see table 4.1). Porod esponent is lower for Frasassi samples compared to the other samples.

At high-Q, the high amplitude peaks indicate the noise created by the background therefore this Q region have not been considered.

Sample m FR16 Tr 3.43 FR16 P 3.15 FR16 PP 3.54 CL9 3.41 CS10 3.66 MIK7 3.28 TM19 3.62

Table 4.1 Power law slope parameters (m) for the analysed carbonate samples.

FR16 samples curves (Fig. 4.5) have lower intensities for the analysed Q-range compared to the other samples which alludes to a lower contrast between different phases. The different phases in

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all the analysed samples is likely due to the interface between pores and calcite crystal faces. Therefore it is possible to infer that FR16 fabrics have a more homogeneous and ordered internal structure and less porosity compared to the other analysed samples. This inference supports what already was observed with neutron scattering data.

Although, the fitting of the curves has still to be improved at high and low Q (Fig. 4.7 and 4.8), slopes (m) between 2 and 3, as shown by these samples (table 4.1), is typical of “mass or surface fractals” such as branched systems (gels) or a clustered networks which usually have rough surfaces (Fig. 4.6) (Porod, 1953; Schmidt, 1988). The concept of the fractal nature of particles seems to agree with the non-classical crystallization model which suggests that crystals may form as a result of aggregation of primary particles specifically by oriented attachment of nano-particles (Besselink et al., 2016). The formation of surface fractal aggregates preceeded by the formation of < 3 nm primary species has been proven by experiments on the crystallization of gypsum under controlled conditions (Stawski et al., 2016).

This roughness however, can also be an artefact due to the using of solid samples, that have been also previously polished with sand-paper, so that they do not have a perfectly flat surface and causing a certain degree of anisotrophy. Thus, it is not possible to have precise quantitative information (Jitendra Mata pers. comm., 2018).

Further analysis and the application of more appropriate models are required to better understand the potentiality of this tecniques applied to speleothem science. Given that this is probably the first study applying neutron diffraction, SANS and tomography to speleothems, having identified that neutron scattering has potential to refine classification of fabrics as related to growth mechanisms it is already an interesting and very promising result. For the other techniques, neutron tomography appears not to be apt for a rapid, user-friendly identification of macroporosity in stalagmites of climatic significance. SANS is very complex in terms of data interpretation, and it would seem that without other techniques, such as Transmission Electron Microscopy (TEM) observations, it may lead to misinterpretation of the speleothem properties. It is here suggested that future work should be conducted on more stalagmites, whose growth mechanisms have been studied by TEM and whose environment of formation has been well monitored (cf. Frisia et al., 2018). At the present state of knowledge, it is recommended that an inventory of stalagmite fabrics should complement textural analysis data.

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4.6 Conclusions

Three different techniques for textural analyses have been applied to calcite samples characterized by different porosity and crystalline habitus. The aim of this work was to study the distribution of the internal network of porosity that favours the movement of fluids across speleothem layers and possibly promoting post-depositional processes. Specifically, dissolution and re-precipitation processes compromise the paleo-climatic and environmental interpretation of the geochemical signal. Furthermore, we investigate particles characteristics like, surface and orientation because they are related to the mechanism of crystal growth. This is crucial, because the classical theory of nucleation, that considers atoms, ions or molecules as the basic building blocks of larger crystals (Hu et al., 2012), has been put into question by new Transmission Electron Microscopy findings. The alternative theory that has been proposed is the non-classical nucleation theory, which includes prenucleation clusters (e.g. nanocrystals) as building blocks for mineral formation (Demichelis et al., 2011).

Neutron tomography (DINGO) permitted to study and quantify the distribution of the porosity inside the samples based on the content of organic material. However, this technique has a relatively lower spatial resolution and does not offer any further improvements with respect to XCT (Trtik et al., 2011) which, at least in speleothem science, remains the preferred method.

Strain scanner (KOWARI) helped to study the crystallographic orientation of the c-axis from different calcite samples. Neutron scattering diffraction data obtained during the present study constitute the first step toward the advancement in understanding speleothem crystals mechanisms of growth. These results support petrographic fabric observations. Specifically, compact columnar fabric shows a single preferential orientation of the c-axis whereas in its porous subtype these orientations are more dispersed (Frasassi samples). The flowstone fabric (CL9), which is apparently compact calcite, shows two family of clustered crystals with the c-axis rotated 180

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degrees from each other. The laminated stalagmite TM19 and the coralloids show a radial (360 degrees) distribution of the c-axes orientations typical of spherulitic growth.

SANS data can be useful to investigate the phase homogeneity and order structure in a crystalline specimen. Speleothems can host fluid inclusions, detrital particles or organic matter inside the inter-crystalline porosity, which creates different interfaces and thus different contrast detected by the machine. Frasassi samples demonstrated to be the cleanest samples among all the samples analyzed confirming the petrographic observations and confirming that these samples are possibly, the best suited for paleoclimate investigation.

However, this is only a pilot study and further analyses are required to better understand the potentiality of all these techniques that are relatively new to speleothem science. So far, only the tomography has been applied to stalagmites and the lack of bibliographic material for comparison, makes the interpretation of the data very difficult. A future objective can potentially be the creation of an inventory of stalagmite fabrics using KOWARI and QUOKKA data. To accomplish this, a wider variety of samples, with different textures, needs to be analysed. This will help to better characterize all fabric types found in speleothems through a very detailed textural point of view. Considering that there is a tight relationship between crystal mechanism of growth, habitus and chemical elements incorporation, a fabric inventory will help future research to better interpret the geochemical signal recorded in speleothems and thus make palaeoclimate and palaoenvironmental reconstructions more accurate and reliable.

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4.7 References

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Chapter 5. Climate variability on the Adriatic seaboard during the last glacial inception: the Frasassi cave case study

-Manuscript Vanghi, V., Frisia, S., Borsato, A., Hellstrom, J., Bajo, P. Climate variability on the Adriatic sea-board during the last glacial inception: the Frasassi cave case. Accepted in Quaternary Science Reviews Journal.

5.1 Abstract

A stalagmite (FR16) from Frasassi cave, located on the Adriatic coast of the Italian Peninsula, offers a continuous 16 ka stable isotope record spanning from 112.8 ±1.5 ka until 96.6 ±1 ka, corresponding to the marine isotope stages MIS 5c and MIS 5d (Lisiecki and Raymo, 2005). The Mediterranean basin is located between the mid-latitudes and the tropic, which combined to its complex orography make the interpretation of its climatic dynamics very challenging. FR16 δ13C records five cycles of quick transitions from humid to dry conditions and at least two of them coincide to the stadial shifts GS24 and GS25 at the onset of the last glacial, at 111 ka and 104.5 ka respectively. The trend of the oxygen is completely different and the GS24 and GS25 are relatively less evident. This unconformity is probably explained because δ13C fluctuations are mostly controlled by regional to local scale processes, like reduced precipitation over the area and thus reduced drip rate in the karstic environment. The trend of oxygen isotope ratios instead, is mostly linked to differential seasonal rainfall dominance that has attenuated the effect of the stadials. Petrographic observations have been introduced to the study to better understand the process acting behind the isotopic records. In FR16, fabrics that are sensible to hydrological changes, vary from open columnar to micrite during dry periods. While FR16 δ13C displays expected average values for its geographical location and karst characteristics, the δ18O profile is ca. 3.5 ‰ more negative compared to other available Italian speleothem records suggesting the interplay of different moisture sources.

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5.2 Introduction

Pleistocene millennial climate variability from the Northern Hemisphere (NH) has been largely investigated in both marine and continental records. Climate dynamics, ruled by feedback systems, are extremely complicated because multiple factors act simultaneously at a different spatial and temporal scale. Therefore, the acquisition of records of climate variability from all around the world is the only way to investigate and understand the fingerprint of these climate changes. This information will be then used by numerical models to test and predict future climate variability (IPCC, 2007) and to understand the teleconnections between different areas of the globe. Throughout the Quaternary, major orbital climate reorganizations took place during the glacial cycles. The important open questions regard the onset, the cause and the role of this millennial variability within such transitions. Despite that lot of attention have been dedicated to study the demise of glacial periods (called terminations), the mechanisms involved in glacial inceptions (transition from interglacial to glacial) are relatively less constrained due to the lack of high resolution and cross dated records at different site (Landais et al., 2006). Past rapid growth of NH continental ice sheets is commonly attributed to reduced summer insolation in boreal latitudes (Milankovitch, 1941; Paillard, 1998; Hayes and Waldbauer, 2006). Considering that we are currently in an interglacial era and that summer insolation is near to its minimum but there is no sign of a new ice age, the challenge relies on studying and characterizing past glacial inceptions to be able to predict the next one (Ganopolski et al., 2016). In this study, the focus is on the transition from the last interglacial- marine isotope stage 5e (MIS5e)- to the last glacial - marine isotope stage 4 (MIS4). MIS5e climate is particularly interesting to us because it may be considered as analogue to the present long-lasting warm interglacial (Kelly et al., 2006). Thus, investigating this period could help to forecast the future climate variability. Nowadays, we are probably experiencing a relatively abrupt global warming (when global temperatures rise of 1 degree in a matter of decades) and from this perspective it is particularly interesting to examine intra-interglacial millennial-scale rapid cooling (stadials) and warming (interstadials). Evidence of abrupt climatic oscillations are well documented in several records from North Atlantic marine sediment cores and Greenland ice cores (North Greenland Ice Core Project, 2004; Oppo et al., 2006). However, the effects of these abrupt climate changes on the European continent, specifically in the Medi- terranean region, are poorly constrained (Galaasen et al., 2014; Govin et al., 2015).

The Mediterranean is a densely populated region, which has been the cradle of modern western civilization. It is a climatically and morphologically complex region and because of its latitude is

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regarded as a transitional zone influenced by both mid-latitude (like the North Atlantic Oscilla- tion-NAO) and tropical (like the South Asian Monsoon) variability (Lionello et al., 2006; Hurrell and Deser, 2009). Moreover, the Mediterranean Sea itself is a heat reservoir and a source of moisture for the surrounding terrestrial areas. Teleconnections in the Mediterranean basin show both spatial (from synoptic to mesoscale) and time (seasonal cycles modulated by multi-decadal to centennial scale) variability. The Mediterranean has a large meridional gradient where less than 2,000 km separate hot and arid regions from mountainous climates with permanent glaciers (Lionello et al., 2006). Thus, orography and land-sea distribution play an important role in estab- lishing local climate and its teleconnections pattern to the global scale (Lionello et al., 2006).

There is currently a shortage of coupled palaeo-temperature/palaeo-hydrological information from terrestrial archives in particular from the Mediterranean area. However, speleothem research is gradually remedying with studies including stalagmites high resolution records that are considered one of the most reliable and chronologically precise archives of climate change (Fairchild and Baker, 2012). Over the last decade, research addressed its focus on studying the drivers of δ18O and δ13C variability in calcite speleothems, which is particularly difficult for cave located at mid latitudes (Barker et al., 2011). Speleothems from mid-latitudes are less sensible to large scale environmental changes and, in addition, local karst-specific processes, which may be unrelated to paleoclimate, can impact on the climate signal recorded by speleothems. To be sure to give a correct interpretation of the palaeoclimate signal, different approaches, such as using a suite of proxies from a single speleothem should be used (Oster et al., 2010).

In the present study, I investigated the stable isotope variability of a stalagmite (FR16) sampled at Frasassi cave close to Adriatic cost of Italy. The δ18O and δ13C records show contrasting results and, therefore, I combined petrographic observations to better understand the processes controlling the isotopic signal and analyse local to larger scale climate variability. Frasassi location is especially interesting because is at the opposite side of the Appennine respect to Corchia cave, a well-known cave amongst the palaeoclimate community that has been largely studied (Drysdale et al., 2004; Drysdale et al., 2005; Drysdale et al., 2007b; Zanchetta et al., 2007; Bajo et al., 2012; Bajo et al., 2017) and whose record overlaps with Frasassi’s. Comparing Frasassi and Corchia records should help understanding different responses to local circulation changes and the related orographic effects over time in the Mediterranean.

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Fig. 5.1 A) Geographical location of Frasassi cave system (43°24′03″N 12°57′43″E) and of the other sites mentioned in the text. B) Section of the western part of Grotta Grande del Vento and location of where FR16 was collected (Manhattan chamber, V° karstic level). The altitudes of the karstic levels indicated in the section are calculated from the river bed. The section of the cave is simplified versions from the original made by Dottori D., Maccio S., Bocchini A., Coltorti M., Novelli A. and Recchioni R. during the speleological campaigns from 1973 to 1985.

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5.3 Field location and sample

The Frasassi cave system constitutes a famous Italian show cave (around 350,000 visitors per year), located in Central Italy (Marche) on the Eastern side of the Apennines, about 40 km from the Adriatic Sea (43°24′03″N 12°57′43″E) (Fig. 5.1). The climate of the region is this part of the Apennine is subcontinental with a mean annual temperature of 13°C and annual rainfall averaging 1000 mm/year. Most precipitation generally occur in late autumn and winter, with very limited amount of snow- fall during January and February, whereas evaporation exceeds precipitation during the summer months (Galdenzi et al., 1999; Galdenzi and Maruoka, 2003; Galdenzi, 2012).

The vegetation on the ridges of Mt. Frasassi and Mt. Valmontagna between 700 and 1000 m a.s.l. is mainly represented by emicryptophile grass meadows, whereas the along the Sentino River Gorge is dominated by a hop hornbeam forest (Ostyria carpinifolia) (Biondi et al., 2010).

Frasassi caves are cut in the Jurassic Calcare Massiccio Formation, a pure limestone about 1000 m thick and highly fractured and due to its great permeability, identifies the main aquifer of the zone (Fig. 5.2). The Bugarone Formation, a 60 m thick Jurassic limestone, with marly and cherty beds overlies the Calcare Massiccio Fm and is relatively less permeable. This is overlain by the upper Jurassic-Lower Cretaceous Maiolica limestone followed by the Mid-Cretaceous Scaglia Bianca and Marne a Fucoidi Fm and the Upper Cretaceous Scaglia Rossa Fm (upper Cretacic- Eocene). A 2000 m thick evaporitic level, the Upper Triassic Burano Formation, consisting of anhydrite and dolomite, sits at the core of the anticline (Galdenzi and Maruoka, 2003; Mariani et al., 2007; Galdenzi et al., 2008a). The larger cave of the system, Grotta del Fiume – Grotta Grande del Vento, consists of more than 20 km of passages between 200 m (current river bed) and 360 m a.s.l.. However, about 100 caves open along the cliffs of the Sentino River Gorge, a 2 km long canyon cut through the Mt. Frasassi- Mt. Valmontagnana anticline (Galdenzi and Maruoka, 2003; Mariani et al., 2007; Galdenzi et al., 2008a) (Fig. 5.1).

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Seven horizontal galleries at different elevations and connected by vertical shafts, were generated by tectonic uplift, fluvial erosion and aggradation during successive Quaternary glacials and interglacials (Galdenzi and Maruoka, 2003) (Fig. 5.1c). Fluvial sand in the Grotta della Madonna, which represent the oldest level (VI) is dated, by 10Be-26Al series, at 750 ± 260 Ka (Cyr and Granger, 2008). Not considering the active first level, which corresponds to the present topography of the river-bed, the youngest karstic level (II) stands at an elevation of 210 m above sea level. 14C dating on calcite rims and subfossil eels in level II yielded an age of 8.4 ± 0.2 ka (Mariani et al., 2007). Cave development by infiltration of meteoric water seem to have played only a marginal role in the development of this karstic system (Galdenzi and Maruoka, 2003).

The formation of the Frasassi cave system itself is mainly driven by sulfuric acid (H2S) speleogenesis (SAS), which consists on the oxidation of H2S producing the highly corrosive sulphuric acid (H2SO4). Frasassi ground waters are enriched in H2S and salts due to the rise of mineralized waters from the underlying anhydrite that, in the presence of oxygen in the phreatic fresh water zone or in the cave atmosphere, oxidizes. This acidic solution causes then dissolution of the limestone (Galdenzi and Maruoka, 2003). Furthermore, the presence of chemolithoautotrophic microorganisms have been inferred to have played a role in speleogenesis by accelerating oxidation of

H2S from which they gain energy for their metabolic activity, causing sulphuric acid as a by- product (Galdenzi and Maruoka, 2003; Mariani et al., 2007; Jones et al., 2015). When the limestone walls are exposed to H2S exhalations, in fact, calcium carbonate is dissolved and then replaced by microcrystalline gypsum as corrosion residue.

The present-day infiltration water is characterised by low concentrations of calcium (60 ÷ 90 mg/L), low alkalinity values (100 ÷ 250 mg/L), low sulfate (about 3 ÷ 15 mg/L), and chloride (5 ÷ 20 mg/L) concentrations. On the other hand, dissolved oxygen values are high (about 0.32 mmol/L) and waters are supersaturated with respect to calcite (SIcc = 0.1 – 0.7). Furthermore, the Mg/Ca molar ratio of the parent waters range from 0.05 to 0.10 (Tazioli, 1990; Galdenzi et al., 2008a). This is expected from dissolution of a limestone host-rock (cf. Borsato et al. (2016)) and suggests absence of SAS. In fact, the dripwater composition differs substantially from the sulfidic groundwater of the lower aquifer characterised by high sulfate (150 ÷ 210 mg/L), high sulphide (10 ÷ 20 mg/L), and high chloride (500 ÷ 850 mg/L) concentrations (Tazioli, 1990; Galdenzi et al., 2008a). Stable isotope (δ18O, δD), and tritium analyses suggest that the recharge area is located at an altitude ranging from 600 to 1000 m a.s.l., with a mean water residence time ranging from several months to a few years (Tazioli, 1990).

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The levels of cave air pCO2, fluctuates from 400 ppmv in winter up to 1500 ppmv in the peak tourist season (April to September), with daily fluctuations up to 1,100 ppmv directly correlated to the number of visitors (Menichetti, 2013).

Stalagmite FR16 was a naturally broken fossil stalagmite recovered from “Manhattan” chamber located in the fifth karstic level of Frasassi cave at ca. 257 m a.s.l., i.e. about 50 m above the present-day sulfidic groundwater (Galdenzi et al., 2008a). The roof of the chamber is about 20- 30 m high and the bedrock (Calcalre Massiccio fm) above is approximately 400 m thick. The infiltration received in Manhattan chamber comes exclusively from the Calcare Massiccio limestone fm. Several deep shafts, from which sulfuric acid exhalations could arise from underneath, are present in this level and gypsum powder deposits on the floor of the chamber testify to the corrosive action of sulfuric vapours. The cylindrical shaped stalagmite is 814 mm long and was found broken into three pieces at the time of collection. The uppermost fragment does not represent the natural top-end of the sample because it has a marked fracture shape (Fig. 5.2).

5.4 Methods

5.4.1 Petrographic and fluorescence microscopy

Stalagmite FR16 was embedded in epoxy resin to minimize damages during the cutting, then sectioned along its vertical growth axis and polished by using diamond-polishing papers of progressively finer grits. The polished samples were subsequently imaged with an Epson Expression 11000XL digital scanner scanning at 600 dpi, which enables highlighting microstratigraphic changes at a decimetre scale. On these digital images, samples of interest for petrographic observations were identified. Fabrics were identified by using a Leica MZ 16A stereomicroscope and a Zeiss Axioplan microscope in plane polarized (PPL) and cross polarized (XPL) light at the University of Newcastle, Australia. The petrographic log of fabric changes along FR16 was built following Frisia (2015). Fluorescent light images stimulated at blue (488 nm) and green (543 nm) wavelength lasers were obtained by using a Zeiss Axio Imager A1 fluorescence microscope with an LED Colibri controller and Olympus software at the University of Newcastle, Australia. Given that a suitable method for fluorescence standardization is still unavailable, focal plane depth, laser power and exposure time were held constant during the acquisitions in an attempt to normalize image intensities (Orland et al., 2012). A short exposure time ranging from 1 to 1.5 seconds was used in order to avoid images which were too bright and might not reflect the real fluorescence

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intensity (Waters, 2009). The images were obtained by simultaneous excitation using both blue and green wavelength lasers. The classification of the fabrics was performed following the criteria proposed for stalagmites and flowstones in Frisia et al. (2000). Discontinuities within the sample were classified according to Martín-Chivelet et al. (2017).

In addition to standard and fluorescence petrography, greyscale values were used to refine microstratigraphy. The greyscale allows visualizing colour changes in thin sections, which are diagnostic of fabrics (open vs. compact, impurity ridden or impurity free) and type of discontinuities along the vertical growth axis. Each pixel value represents the level of grey intensity of the 16- bit images, ranging from black (value 0) to white (value 255). Grey pixel values were measured on high resolution (600 dpi) RGB (red, green, blue) images of the polished slab observed under transmitted light. Grey values were obtained as average of the intensities of the red, green and blue light (grey level= 0.299R + 0.587G + 0.114B) in each point along the line-scan (Muangsong et al., 2011; Duan et al., 2014). The values were calculated using the ImageJ software along a line-scan at 1 pixel resolution corresponding to 42 μm.

5.4.2 U-Th dating and age model

U-Th dating was carried out on 27 prismatic chips (~200 mg drilled along the entire length of the speleothem and, particularly, close to discontinuities recognised from the petrographic observations.

U-Th dating followed the method described in Hellstrom (2003). The samples were dissolved in nitric acid and then spiked using a mix of 229Th-233U-236U tracer solution. The U and Th were eluted in Eichrom TRU-spec selective ion exchange resin. Dried samples were then dissolved in diluted nitric acid. The measurements were performed using a Nu Instruments Plasma MC- ICPMS at the University of Melbourne. An equilibrium reference material (HU-1) was used to correct for instrumental drift and an additional in-house speleothem standard of known age (YB- 1) was used to check for the reproducibility of results. An initial 230Th/232Th ratio of 1.5 ± 1.5 was used to calculate corrected ages using a Monte Carlo approach finite-growth-rate technique following Hellstrom (2006) and construct age-depth model and growth-rate time series (Drysdale et al., 2005; Scholz et al., 2012).

To test the periodicity of the isotopic signal of FR16 a continuous wavelet transform analysis has been performed the Grinsted et al. (2004) toolbox for MATLAB.

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5.4.3 Stable isotopes analysis

For stable isotopes analyses ~ 1 mg of powder (n=556) was collected with a dental micro-drill using a 1 mm bit along the vertical growth axis of the stalagmites. The sampling interval chosen between consecutive points ranged between 1 mm and 2 mm on fast and slow growing stalagmite’s portions respectively. The calcite powders were then purged with pure He and then acidi- fied at 70° C with 0.05 mL of phosphoric acid (H3PO4). The CO2 gas produced was analysed on an AP2003 continuous-flow mass spectrometer at the laboratories of the Geography department of the University of Melbourne. Internal and international standards were used to quantify measurements uncertainties and to calibrate FR16 powder samples to the V-PDB international reference in ‰ (per mille) notation.

Modern drip waters were collected from stalactites in 8 different sites of the cave system during the August 2016 speleological campaign. Water dripping were collected in plastic containers from which an aliquot for the hydrochemical analysis of δD and δ18O composition was transferred in 7 ml glass vials, using mono-use syringes. Water collection was performed monthly over a period of 8 months at one site to monitor the seasonal changes in the isotopic composition of the drip. Approximately 2 mL of sample water were analysed using the Picarro L2120 cavity ring-down spectrometer (CRDS) of the department of Geography at the University of Melbourne. The so- topic ratios were normalised to the international V-SMOW standard using a four-point calibration using two international (GISP2, VSMOW) and two in house standards (WOOLIES, LAKE) of known composition. The results from the WOOLIES standards were used to determine the analytical 1σ uncertainty: ≤ 0.1‰ for δ18O and ≤ 0.3‰ for δD. In case of bigger uncertainties, the samples were re-analysed.

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Fig. 5.3 Principal fabrics observed in FR16 (photo in the centre of the figure). On the left side, photos at parallel and crossed nicols of some of FR16 thin sections. On the right side, micrographs of the principal fabrics observed under the optical microscope. A) Open columnar (Co), B) compact columnar (C) and micrite and microsparite (Ms/M) separated by the hiatus (thin red arrow). Note the presence of condensed laminae approaching the surface of the hiatus. C) micrite and microsparite and D) alternating compact and open columnar (Co/C). White scale bars in the micrographs correspond to 1 mm. Thin sections short side measures 1.2 cm.

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Fig. 5.4 FR16 stable isotope signal (δ13C and δ18O in black and grey respectively) compared to the greyscale values (blue graph). Greyscale plot is superimposed to the petrographic log (dark line) where C = compact columnar fabric; C/Co = laminated part where compact and open columnar fabric alternate; M/Ms alternation between micrite and microsparite fabric. “Hi” indicates the hiatus visually marked in the polished slab as a change from micrite to laminated fabric. The hiatus is also recorded by the abrupt shift in the isotopic profiles. U-Th dating samples locations are indicated with a red rectangles along the growth axis of the stalagmite.

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5.5 Results

5.5.1 Microstratigraphy and petrology

FR16 shows a complex internal stratigraphic architecture and morphology. The lower part of the sample consist of a cone-shaped edifice overlayed by a cylindrical structure separated by yellow, opaque laminae that prolong coating the stalagmite flanks (Fig. 5.3). Dense and optically dark calcite is predominant in the cone-shaped morphology whereas stacked white and translucent layers of calcite identifies the candle-shape structure and the yellow levels. Critically, both the orientation of the vertical growth axis and the diameter of the speleothem varied through time (Fig. 5.3).

The internal stratigraphy of FR16 has been described by identifying different architectural elements and following the six-fold hierarchy proposed by Martín-Chivelet et al. (2017). In FR16, single growth layers (second order) and fabric (third order) have been used to recognize three different types of architectural elements: non-laminated compact columnar, laminated open columnar and laminated micrite/microsparite (Fig. 5.3). The first and the second order architectural elements reflect genetic changes occurred at short-term time scale (from seasonal up to decadal). The laminated calcite is characterized by an internal porosity, where open columnar shows fewer, but larger pores compared to the micrite/microsparite, which has a high density of micro-pores. Due to different degrees of porosity, and, possibly, diverse crystalline structural defects and/or impurity content, light is reflected differently, thus conferring the diverse coloration to the different type of calcite fabrics.

The very dense and organized crystalline structure of the compact columnar fabric appears dark grey when observed under natural light. Compact columnar crystals length to width ratio is < 6:1 and the size of each individual composite crystals (Frisia et al., 2000) can vertically measure up to few centimetres. In FR16 this fabric does not show any visible lamination. Open columnar fabric, with white/light grey shades, is characterized by elongated crystals arranged in a palisade pattern (Genty and Quinif, 1996) periodically interrupted by narrow vertical fissures. Adjacent crystal boundaries are not serrated and pores between crystals, of the order of a millimetre, are also abundant. Optically visible lamination is due to laminae consisting of alternating open and compact columnar calcite fabrics, and the thickness of single laminae ranges from 100 μm to 1 mm. The optically yellowish micrite and microsparite fabrics, characterized by crystal sizes ranging from <2 μm to between 2 and 30 μm in diameter respectively (Frisia, 2015), show micrometre-

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scale, pervasive porosity. Similarly, micrite and microsparite layers show laminae identified by variable degrees of micro-porosity. Porous layers in the laminated open columnar calcite emit fluorescence when observed under the confocal UV-fluorescent microscope. In the yellowish micrite and microsparite intervals, porous calcite emits a brighter fluorescence with respect to the open columnar fabric in the optically light grey portions of the speleothem. The optically dark, compact calcite sectors of the stalagmite did not yield fluorescent signals. Using these three architectural elements, FR16 stalagmite has been divided in 13 different phases of growth where major visible stratigraphic breaks, marked by fabric and colour changes, occurred (see log in Fig. 5.4).

The impact point of the drip remained constant during the first 5 phases of growth (Fig. 5.4). On the other hand, from phase number 6 it changed and the stalagmite started growing over the previous phases. From phases 6 to 13 the point of dripping kept moving and every change corresponds to the starting of a new phase of growth and, thus a different fabric.

The most occurring morphostratigraphic unit in FR16 is the “patchy type” (sensu Martín-Chivelet et al. (2017)) which is produced by layers of calcite, with different widths and length, piled-up together. Each “patch” does not completely cover the stalagmite upper surface. The “patchy type” is especially evident in the sixth phase of growth of FR16. Flat-topped layers grew horizontally widening the diameter of the stalagmite conferring a cylindrical shape, like in phases 2 and 4. Globular type of stacked convex layers are usually well-defined and have lateral continuity, covering both the apex and the flanks of stalagmite like in phases 7,9, 11-13. Phase 5 is the most different phases of growth of FR16 and is characterized by rimmed “flame-like” laminae. These are very irregular layers with a lateral continuity and milky aspect. Flame-like stacked laminae usually concave up in the central part of the stalagmite and tend to be convex at the side forming a lump. Phases 1, 3, 8 and 10 do not show lamination therefore this classification is not applicable.

The visible sharp stratigraphical transition from phase 5 to phase 6 have been considered a hiatus in the growth and this has been confirmed by the abrupt shift in the values of the δ13C profile (please see section 5.5.3).

5.5.2 U-Th series results

The U-series chronological framework for FR16 covers a period of growth of ca. 16 ka, from 112.8±1.5 ka until 96.6±1 ka (Table 5.1) which corresponds to MIS 5c and MIS 5d (Lisiecki and

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Raymo, 2005). A short hiatus of ca. 1.4 ka has been recognized at 45 cm from the top starting at 101.8 ka, therefore the age model has been broken into two parts. The low U content (72 ppb on average) yielded an average 2σ uncertainty of the younger age model is +0.59/-0.53 ka, whereas for the older one is +0.87/-0.84 ka. All the ages are in a correct stratigraphic order within their respective age uncertainties. The average 2σ error is 1.34% corresponding to ~1300 years which is in accordance with the results obtained from other two Italian speleothems covering the same time-span (Drysdale et al., 2007b) (Columbu et al., 2017). Average growth rate for FR16 is ~58.5 µm per year (Fig. 5.5). After the hiatus, the growth rate is more than 4 times faster than below the hiatus. The faster growth rate is during phase 6 (total length 29 cm) that grew in only 2200 years (130 µm/yr). On the other hand, the lowest growth rate is recorded during phase 5, which is long only 4.5 cm and formed during 6000 years (7.5 µm/year).

Table 5.1 Results of Multicollector ICP Mass Spectrometry U-Th analyses on calcites. The isotope analyses are reported as activity ratios (AR), and the errors are reported in brackets as ±2σ. The black bold line indicates the position of the hiatus.

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Figure 5.5 A) Age-depth model for FR16. Red dots represent the U-Th ages (see Table 1). Dark and light- grey lines represent 1σ and 2σ errors, respectively. B) The derived growth-rate time series, in logarithmic scale, where grey lines represent the 2σ errors. Depths are measured from the top of the stalagmite.

5.5.3 Stable C and O isotopes ratio

The δ13C and δ18O variations in FR16 show two distinct trends above and below the hiatus at 45 cm from the top (from 101.8 to 103.2 ka). Opposite trends for the carbon and the oxygen characterize the older portion of FR16 with a general progressive increase in δ13C values and a general negative trend in δ18O values. The layers surrounding the hiatus are characterised by a sharp negative shift in the d13C values and a positive shift in the δ18O values followed by a trend towards more positive values for both stable isotopes. By excluding the transition between phases 5 and 6, other clear high magnitude (≥1‰) variations in both isotopic signals have not been recognized even where petrographic and microstratigraphic changes are visible (Fig. 5.4). Therefore, only the petrographic discontinuity observed between phase 5 and 6 can be ascribed with confidence to a period of ceased.

The average value of the carbon isotope ratio below the hiatus is -7.8 ‰, whereas for the oxygen is -7.7 ‰. Above the hiatus, δ13C average value is -8 ‰, which is slightly more negative (-0.2 ‰)

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than the pre-hiatus part, and the δ18O average value is -6.9 ‰ , corresponding to an increase of about 0.8 ‰ relative to pre-hiatus values.

To test the isotopic equilibrium of calcite deposition, FR16 δ18O and δ13C records have been correlated between each other. Correlation index is very low (R2=0.02) which can prove that kinetic fractionation, due to evaporation and/or fast CO2 degassing, has not substantially affected the isotopic signals (Hendy, 1971; McDermott, 2004).

5.5.4 Grey values

The greyscale values in the thin sections reflect changes in the way the light is transmitted through the sample according to porosity and impurity content. When greyscale values are extracted from the image of the polished surface of the stalagmite, compact elongated columnar (dark) has lower greyscale levels compared to the open columnar and the micrite/microsparite (white) values. Grayscale values FR16 vary between 94 (dark translucent calcite) and 235 (opaque porous calcite) (Fig. 5.4). For compact columnar fabric the average greyscale value is 122, whereas for the open columnar is 164 and for the micrite/microsparite is 205.

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Fig. 5.6 Comparison of δ13C and δ18O records from FR16 and stalagmite CC28 from Corchia Cave (Drysdale et al., 2007a) during MIS5c and MIS5d. The hiatus in FR16 is indicated with “Hi”. Numbers correspond to the phases of growth. Red arrows represent maximum positive δ13C peaks reflecting arid events. GS24 and GS25 cold stadials documented in Greenland ice core (NGRIP) are indicated in CC28 and shaded in yellow. Summer insolation at 43°N (Laskar et al., 2011) and FR16 ages with respective 2σ- uncertanties are also shown. The blue graph represents the reflectance of the stalagmite surface in greyscale values whereas the bold black line is FR16 growth rate.

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5.6 Discussion

5.6.1 Environmental interpretation of calcite fabric and FR16 δ13C

Spelean calcite δ18O is hypothesized to reflect the δ18O of the meteoric precipitation, and its δ13C should be a proxy of soil processes and/or local hydrological changes. Commonly, in speleothems where the amount effect is believed to be predominant, the two signals should correlate, with more negative δ18O (higher meteoric precipitations) coinciding with more negative δ13C (higher soil efficiency under humid conditions) (Lachniet, 2009). In the Frasassi record, the δ13C and δ18O trends do not show covariance, and, therefore, some mechanism other than the amount effect must be acting. A likely possibility is that the oxygen isotope ratio signal of the moisture source and the air-mass trajectories controlled the rainfall, and thus, the speleothem calcite δ18O signal, while local surface temperature and soil microbial activity influenced its δ13C values (Johnston et al., 2013; Borsato et al., 2015).

Speleothem δ13C is considered a proxy of hydrologic and temperature changes over time that controls the soil CO2 production (Genty et al., 2003; McDermott, 2004; Frisia et al., 2011; Rudzka et al., 2011; Johnston et al., 2013; Bajo et al., 2017). Nevertheless, it is difficult to discriminate between the effect of moisture and that of temperature on the C isotope signal and, therefore, it is best to use complementary proxies to test the climate interpretation (Belli et al., 2013). Sources of carbon in cave drip-water are multiple, including atmospheric CO2, soil CO2, carbon derived from the dissolution of the carbonate host-rock and the CO2 derived from the oxidation of old organic matter (McDermott, 2004; Mattey et al., 2016). All of these sources contribute to the signal of the dissolved inorganic carbon (DIC) pool in the percolating water. Therefore, in ab- sences of SAS, and with limited in-cave kinetic fractionation, due to evaporation and/or fast CO2 degassing, long term trend in spelean calcite δ13C should fluctuate between a “pure soil” signal (i.e. around – 11.5‰) and a pure “host-rock signal” (around + 2.0‰ for the Jurassic Calcare Massiccio see Morettini et al.,2002) depending on the ratio between the DIC derived from the host rock and that derived from the soil (Bajo et al., 2017). This ratio is further influenced by open or closed system conditions in the aquifer. Under open-system conditions, the δ13C signal in spe-

13 leothems mostly reflects the soil CO2 (negative end-member of δ C values). Conversely, during closed-system conditions, the signal is strongly influenced by bedrock dissolution (the positive end-member of δ13C values) (Bajo et al., 2017). Moreover, in temperate climate settings the soil

13 δ C DIC composition is inversely correlated to the soil pCO2, which, in turn, depends on the mean annual surface temperature (Johnston et al., 2013). On the other hand, in semi-arid settings,

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soil δ13C DIC is controlled by the water availability in the epikarst (Bar-Matthews et al., 2003; McDermott, 2004). Therefore, in the lower montane Frasassi cave settings, negative δ13C should reflect warm and wet climate phases, which boost vegetation density and soil efficiency (Johnston et al., 2013; Borsato et al., 2015).

FR16 δ13C signal varies from -6.5 ‰ to -9.5 ‰, which is here interpreted as reflecting sparsely vegetated, lower montane, temperate climate environment and prevailing open-system conditions in the epikarst (see Bajo et al., 2017) (Fig. 5.4). During the first 10 ka in FR16 formation (from ~113 ka to ~102 ka), the δ13C trend reflects environmental conditions plunging into aridity, but punctuated by four smaller scale cyclical variations. These cycles, from wetter toward relatively more arid conditions marked by a more positive peak, coincide with the termination of phases 1 (110.4 ka), the mid-point of phase 3 (108.1 ka), the end of phases 4 (106.2 ka) and 5 (101.8 ka) respectively. The end of phase 5 actually coincides with the major hiatus in FR16. However, it cannot be excluded that all the changes between a fabric to another are actually marked by non- deposition. Changes in the isotopic ratio trends are also reflected by changes in the crystalline fabric confirming the link between carbon signal and water availability inside the cave. These positive peaks in the carbon also correspond to phases of slow growth rate of the stalagmite (Fig. 5.6), which possibly resulted into enhanced 13C enrichment in the parent fluid caused by prolonged CO2 degassing (Mickler et al., 2004). The first cycles (that lasted approximately 2600 years), is characterized by columnar fabric and the second (lasted ~2300 years) by laminated (open/compact columnar) phase 2 and the translucent compact columnar phase 3. The third cycle (1900 years) consists of alternating open and closed columnar calcite fabrics and the fourth cycles (lasted ~4400 years) of micrite and microsparite.

Typically, columnar fabric has been interpreted as forming under slow but constant drip rate from a relatively more diluted (low calcite supersaturation state) and low impurities loaded infiltration water (Frisia, 2015) (Fig. 5.3). Open columnar fabric has been observed as forming under relatively more irregular drip rate, where irregular flushes of infiltrating water are more recharged with diluted particulate (organic and inorganic) that can be incorporated on surface of growth of the speleothems. This explains why fluorescent is emitted when calcite rich in impurities is observed under blue and green lights. The lamination visible in phase 2, 4 and 6 indicates that these two conditions: periods of constant availability of water forming a thin film of fluid on the tip of the stalagmite and relatively dry periods with pulses of impurity-rich flushes, alternated through time. Micrite and microsparite conditions of growth reflect similar, but relatively more severe condition than open columnar fabric (Frisia, 2015). Therefore, here we infer that phase 5 probably

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coincided with the driest period of the entire record that lasted for almost 4500 years and culmi- nated with the interruption of calcite deposition for 1400 years (hiatus). This correlates with the slowest growth rate recorded in the stalagmite and a positive peak in NH insolation curve. There- fore, by considering that the annual rainfall average is 1000 mm/year, an increase in the NH insolation and, thus, in temperature, could have likely caused an enhanced evapotranspiration and reduced infiltration in the area. This also agrees with the observed change in the fabrics. Thus, the transition from columnar to open columnar to micrite fabrics in the interval between 113 to 102 ka and increasing trend of δ13C values strongly suggest increased aridity over time.

After the hiatus at 101.8 ka, phase 6 (2200 years) shows the fastest growth rate of the whole stalagmite, which would suggest high drip, yet the diameter of the stalagmite decreases and the δ13C increases, which would be expected by low drip (Kaufmann, 2003; Muñoz-García et al., 2016). This discrepancy could be due to a change in the calcite supersaturation state (SIcc) of the parent waters, which depend on soil efficiency and water/rock interaction. For example, 1) an

2+ increase in soil pCO2 due to enhanced soil efficiency and 2) higher Ca content in the infiltration water (high SIcc) due to consequent enhanced dissolution of the host rock (Kaufmann, 2003) would be a likely explanation. On the other hand, the fact that after the hiatus the drip point shifted (Fig. 5.3 and 5.4) implies that the route of the feeding system may have changed. Therefore, an alternate hypothesis that could explain the contrasting combination of fast growth rate, narrow speleothem diameter and negative δ13C could have been the resetting of the δ13C signal due to a local hydrology, rather than regional climate change.

Fig. 5.7 Analysis of the isotopic composition (oxygen and deuterium) of modern drip waters collected in Frasassi cave during the period 2014-2015, compared to the water lines of isotopic composition of modern

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precipitation from all the Mediterranean region (Giustini et al., 2016b). The Italian meteoric water line is also showed as well as the local Sicilian water line.

5.6.2 Moisture provenance and interpretation of Frasassi δ18O signal

Ideally, the δ 18O of cave drip-water should record the weighted mean δ18O of the meteoric water falling on the surface above the cave site assuming that near-surfaces evaporation has not occurred (McDermott, 2004). On centennial to millennial timescales, the isotopic signal in precipitations is mainly controlled by air-moisture transport rather than by local factors such as surface temperature or precipitation amount (Rozanski et al., 1982; McDermott, 2004; Sodemann and Zubler, 2010). It is therefore important to evaluate the contribution of moisture source modification to the oxygen isotope signal of the infiltration water at a timescale of a glacial cycle like MIS5-MIS4, which is the interest of this paper. FR16 record which is about 16 ka long, however, is not long enough to clearly evaluate the impact of moisture source changes on the oxygen isotope variation of the rainfall and thus of the percolating water, because usually, it is more evident on longer time-scale. In addition, the record does not include a termination or glacial maximum that would have clearly given a large signature imprint on the isotopic oxygen record of the precipitation due to significant ice volume build-up/reduction. Despite this, it is still possible to finger-print the moisture source of the meteoric water falling over the cave site, hinting for example to the possible origin of storm tracks.

As it was not possible to collect the drip water coeval to FR16, we measured the oxygen and deuterium signature of modern drip waters in Manhattan chamber (where FR16 was collected) and compared that to the isotopic signature of modern precipitation in Italy (Longinelli et al., 2006; Giustini et al., 2016a). This information can then be applied to speleothem isotopic records from the same cave site that are used for paleoclimatic reconstructions aiming to gain information about the composition of the meteoric water coeval to the stalagmite and possibly the provenance of the moisture.

The results (Fig. 5.7) suggest that Frasassi modern dripwater O and H isotope ratio signals fall between the Southern Italian and the Sicily water line (Giustini et al., 2016a), suggesting that modern precipitations at the studies site are mostly sourced from the Southern Mediterranean. However, the deuterium excess (d = 17.1) for Frasassi site is intermediate between values for air masses derived from the Mediterranean Sea (d = +22‰, Gat and Carmi (1970)) and those of Atlantic provenance (d = +10‰, Craig ,1961), suggesting a contribution from both sources.

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Modern dripwater δ18O value at Frasassi site is -8.6 ±0.1‰, similar to the signal of modern precipitations from the Adriatic side of Apennines (Giustini et al., 2016a), and corresponding to an infiltration elevation altitude of ca. 950 ±150 m a.s.l. if we use the local altitudinal gradient calculated for the Marche-Umbria region (Tazioli, 2007).

FR16 δ18O trend resembles a Gaussian-like curve reaching the most negative values at the top. A mechanism commonly appointed to interpret oxygen oscillations in arid and semi-arid regions, is the “rainfall amount effect” that produces depleted δ18O in the meteoric waters with increased precipitations (Dansgaard, 1964). This has been also regarded as the principal mechanism driving rainfall δ18O variations in the Mediterranean region, like for example in Corchia cave at the same latitude (Bar-Matthews et al., 2000; Bard et al., 2002; Drysdale et al., 2005; Drysdale et al., 2007b), in both glacials and interglacials (Regattieri et al., 2014). A composite record of speleothems from Peqin Cave in Israel, Eastern Mediterranean also shows a similar pattern to FR16 δ18O, which has been interpreted as a response of a dominant amount effect (Bar-Matthews et al., 2003). Similar conclusions have been drawn by Bard et al. (2002) and by Regattieri et al. (2014) for stalagmites from Argentarola cave, in the Tyrrenian coast and a speleothem from Renella cave (close to Corchia cave), for the penultimate glacial/interglacial cycle. Thus, if this were the case, FR16 record would register increasing humidity until ~102 ka (hiatus), which is in disagreement with the δ13C and the low growth rate signals that point to aridity. After the hiatus, the oxygen values start progressively increasing indicating relatively less humid conditions which also contrasts with the interpretation of the carbon signal and the open columnar fabric that mostly characterizes phase 6 to 13.

Conversely, FR16 lowest values of the oxygen correspond to its lowest growth rate and to the micritic phase 5 that suggests minimum water availability (Frisia, 2015). This apparent discrepancy could be due to a differential predominance of winter vs summer precipitations. Winter precipitations indeed have lower δ18O values compared to summer precipitations (Lionello et al., 2006). Thus, lower δ18O values combined to slow growth rate could just indicate a predominance of winter over summer precipitations.

In addition, it is also important to notice that the hiatus at ~102 ka does not seem to have affected the trend of FR16 oxygen which does not show any abrupt shifts in the values contrary to δ13C (Fig. 5.4-6). A possible implication is that the composition of the drip water feeding FR16 neither was significantly affected by in-cave changes such as aquifer hydrology and/or network evolution.

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5.6.3 Regional climatic context of δ18O during the glacial inception

The signals of the carbon and the oxygen have led to contrasting interpretations for FR16 hydrological setting. Comparing Frasassi record with the interpretation of other published climatic archives from nearby locations encompassing the same time frame, can be helpful to understand how the two isotope signals reflected a climatic variation (Fig. 5.6). It has been demonstrated that North Atlantic temperatures and ocean circulation are strongly connected to Mediterranean hydrology because of the correspondence between carbonate δ18O records from the central Mediter- ranean, the records from North Atlantic marine sediment cores and from Greenland ice cores, especially during major climatic shifts like deglaciations (Drysdale et al., 2009; Zanchetta et al., 2016; Regattieri et al., 2017).

Corchia cave record is particularly interesting because is situated almost at the same latitude of Frasassi cave but on the west side of the Apennine, thus we tested the relationship between the two isotopic records and whether they recorded similar climatic changes. CC28 stalagmite from Corchia cave (Drysdale et al., 2007b) covers the same temporal frame of FR16 growing between 96 and 118 ka. CC28 stalagmite, like FR16, recorded millennial scale climate oscillations (abrupt shifts from warm to cool conditions) known as Dasnagaard-Oeschger (DO) events. The glacial inception, after the Eemian interglacial, is characterized by a slow trend to cooler conditions and a sequence of DO events preceding the full glacial onset. Warm events are referred as glacial interstadials (GI or DO) and cold events as glacial stadials (GS) (North Greenland Ice Core Project, 2004). Corchia cave δ18O and δ13C registered two cold and dry events (from 112 to 108 ka and 105 to 102 ka) coinciding with Greenland stadials GS24 and GS25 (North Greenland Ice Core Project, 2004; Landais et al., 2006) and to C25-C23 marine events in the Iberian margin cores (Sánchez Goñi et al., 2005; Martrat et al., 2007) separated by the warm and wet phase DO24. In FR16 climatic records, GS25 and GS24 possibly match with two δ13C abrupt positive shifts at the end of phase 1 (from 111 to 110 ka) and at phase 5, starting at 106 ka and ending with the hiatus at 101.8 ka, which likely record arid spells (Fig. 5.5). Accordingly, they also match with the lowest growth rates of the entire record reflecting conditions of reduced infiltrations and low temperatures, that is: inefficient soil activity above the cave. As the chronologies of both records have similar age uncertainties (> 0.7 ka and >0.9 ka), it is possible to confirm the synchroneity of the two stadials in both speleothems.

GS24 and GS25 cold and dry events are also well represented in CC28 δ18O from Corchia cave. On the other hand, in FR16, GS25 is recorded just by a minor increase of δ18O (< 0.5 ‰) at the

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end of phase 1 at ca. 110.7 ka (Fig. 5.6). GS24 is likely represented by the low growth phase 5, where the micrite fabric, typical of dry conditions, has also been observed and, similarly to GS25, δ18O values only show a small increase. After the hiatus at ~102 ka, both FR16 and Corchia records show an increase in the oxygen, as for the previous two stadials, but this time the trend is gradual and not abrupt.

During DO24, recorded between ca. 110 and 106 ka by FR16, Mediterranean pollen variation (Brauer et al., 2007; Allen and Huntley, 2009) and negative oxygen shift in lacustrine carbonate from Sulmona lake (in the Appenine) (Regattieri et al., 2015; Regattieri et al., 2017), indicate strong seasonality with more abundant winter precipitations and longer summer droughts, suggesting a more Mediterranean character of the climate. In FR16 this interpretation seems to be confirmed by negative values of the oxygen isotope ratios and the presence of laminated fabric (Fig. 5.4 and 5.6): phases 2 to 4. Lamination is visible until the end of the record, excluding brief periods coinciding with phases 8 and 10, characterized by compact columnar fabric. FR16 record thus is mostly entirely characterized by periodic variation in fabrics (laminae0 that can potentially be annual, and record seasonal contrast.

In addition, compared with other Western Mediterranen stalagmite records available for this time frame (Drysdale et al., 2005; Drysdale et al., 2007b; Columbu et al., 2017), FR16 shows a relatively more negative (ca. 3.5 ‰) average oxygen isotope ratio signal (-7.4 ‰) (Fig. 5.6). Frasassi δ18O record is also more negative than the coeval stalagmites from Peqiin and Soreq cave speleothems from Israel (Eastern Mediterranean), whose oxygen isotope ratio records have also been explained as mainly controlled by the amount effect (Bar-Matthews et al., 2003). This suggests that Frasassi site was probably receiving moisture that crossed the Apennine and thus became depleted in O18, but also hints at the possibility of air masses with different provenance. An important factor, that can affect δ18O of continental carbonates on millennial scale, is a change in the relative proportion of Mediterranean (high δ18O) vs North Atlantic (low δ18O) precipitations, which is controlled by the weakening/enhancing of the Atlantic Meridional Overturning Circula- tion (AMOC) and related shifts of the Intertropical Convergence Zone (ITCZ). The mean latitudinal position of the ITCZ moves accordingly to insolation fluctuations. During phases of insolation maxima (like in summer or during stadials), the ITCZ extends northward and vice versa (Harding et al. 2009). This shift would be especially evident in mountainous regions, like the Apennine, due to the orographic effect that may enhance rainfall on the side of the range facing moisture provenance (Regattieri et al., 2015; Regattieri et al., 2017) and create barriers for some tracks.

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This interpretation is supported by the Late Glacial to Holocene stalagmite δ18O record from Savi cave at the northern end of the Adriatic Sea (average δ18O values from -6.6 ‰ to -7.1 ‰) that has been interpreted as a combination of amount effect and atmospheric circulation reorganization following the Last Glacial Maximum (Belli et al., 2013; Belli et al., 2017). Whilst the amount effect dominated during the mild Holocene condition, the influence of atmospheric circulation reorganization was especially evident during abrupt climate changes such as the beginning and the end of the Younger Dryas (YD), which can be considered an analogue of Greenland Stadials. Unexpected negative δ18O values during increased dryness has been interpreted as due to the dominance of storminess coming from the Atlantic, because of the southward expansion of North Atlantic sea ice plus rather than from Mediterranean sources.

To confirm a millennial scale periodicity in Frasassi record, a wavelet analysis has been performed (Fig. 5.8). The millennial periodicity (1 to 4 ka) is particularly evident between 106 and 105 ka in the carbon profile and between 111 and 105 ka for the oxygen which coincides with the DO events GS24 and GS25.

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5.6.4 Possible factors influencing Frasassi δ18O signal

We propose an interpretation for FR16 δ18O record that does not account exclusively for the amount effect as the principal controlling factor but rather by a combination with air-mass provenance and rainfall seasonality.

The first hypothesis for FR16 oxygen signal is linked to the provenance of the air masses that deliver the moisture over the Mediterranean regions, the so called “source effect” (Charles et al., 1994). Changing in the moisture source causes changes in the δ18O signal of the precipitation falling in a certain region (Dansgaard, 1964). Different storm tracks can deliver moisture to this part of the Mediterranean basin. The Northern Adriatic Sea is a strongly cyclogenic areas and most of the cyclones derive from Atlantic. Intense cyclones on the southern side of the Alps are originated by large low-pressure systems above Northern Europe ant take a southward path (Trigo et al., 2002), although their effects may be shielded by the Alpine and Apennine mountain chains (Trigo et al., 1999). Today, the Frasassi area receives rainfall from Genoa-type cyclones, which may cross the central Apennines and reach the Adriatic, as well as non-Genoa cyclones generated in Iberia, the Alboran Sea, the Central Atlantic and the Mediterranean (Horvath et al., 2008). For example, intense rain events within the cold season (September to May) on the Central-Northern Adriatic coast of Italy have been related to the arrival of moisture from the tropical Atlantic and have been associated with formation of hurricanes over the Atlantic Ocean linked to intense convergence of moist air in the Tropics (Krichak et al., 2016). Rainfall originated from the Atlantic tropics and fallen on Frasassi area would be 18O depleted due to Rayleigh distillation (Dansgaard, 1964). In addition, modern FR16 drip water analysis revealed that the local meteoric water composition is related to the southern Italy precipitations. On the opposite side of the Italian peninsula, Corchia is directly under the influence of Genoa-type cyclones and moisture evaporated from the western Mediterranean basin (Longinelli and Selmo, 2003). If the hypothesis of different storm track is correct, Corchia and Frasassi speleothem δ18O range values should not coincide, because they moisture has diverse provenance and, thus, we would expect different isotopic signals.

The second possible explanation of the relatively more negative oxygen values recorded in Frasassi speleothem can be linked to the orographic effect. Because the Apennine ridge acts as a partial orographic barrier to moisture coming from the Tyrrhenian Basin (Genoa-cyclones), the Frasassi area receives most of its moisture from trajectories that cross over the Mediterranean coming from the tropical Atlantic. The distribution of 18O values throughout the eastern central

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Italian coast, which is over 1000 km long, changes only slightly from -7 to -8 ‰ (Longinelli and Selmo, 2003; Giustini et al., 2016a). Mean 18O and D recorded at the station nearest to Frasassi (Fano) from 1995-1999 yielded values of -7.33 ‰ and -47.8 ‰ respectively (Longinelli and Selmo, 2003). Adriatic meteoric waters are ca. 2 ‰ depleted respect to those of the Tyrrenian margin chosen at the same latitude which is explained by Giustini et al. (2016a) as due to the Apennine chain barrier. At the windward side of the relief (the Tyrrhenian side), the air, that has relatively more moisture and heavier signature, precipitates before reaching the top of the mountains and becomes progressively more depleted in 18O. At the leeward side of the Apennine (Adri- atic side), the air is relatively drier and lighter (more negative δ18O). Yet, the more depleted δ18O values in FR16 seem to follow NH insolation maxima. However, the NH insolation maxima, at this time coincides with a Greenland Stadial (GS24), when the ITCZ is expected to have migrated to the south under the effect of an increase in sea ice (Broecker, 1998). Thus, trajectories may have been different from the West to the East side of the Apennines, with a moisture source likely located in the warm tropical Atlantic pool.

There is another possible explanation for the f Frasassi δ18O variation: a different dominance of winter vs. summer precipitations on the stalagmite δ18O signal. This hypothesis is supported by the δ18O trend of FR16, which is almost in phase with the NH summer insolation curve at 43 degrees N (although FR16 δ18O negative peak precedes of ca. 2000 years the highest insolation peak) (Fig. 5.6). NH summer Insolation positive peak at ca. 104 ka that corresponds to the cold phase GS24 (more negative δ18O), could reflects dominant isotopically light winter precipitations (Longinelli et al., 2006) recharging the aquifer. On the other hand, isotopically heavier summer precipitations, due to high insolation rate, would have scantily recharged the aquifer as it is reasonable to assume that evapotranspiration exceeded infiltration. This phenomenon does not nec- essarily indicate that a climatic deterioration occurred in the Frasassi region during a Greenland Stadial. However, when compared to the Corchia speleothem record, by assuming that the Fras- sassi age model is correct, Frasassi stalagmite δ18O values do not seem to follow precisely the timing and trends of GS and GI, which suggests that its δ18O variability most likely reflects the isotopic composition of rainfall, rather than a rainfall amount.

All these hypothesized processes could have led to an attenuation of the effects of the large scale climatic variations represented by the two stadials GS24 and GS25. This would explain why in FR16 record, oxygen isotope ratio values shifts have a lower amplitude than in coeval Corchia speleothems. Corchia cave mostly captures the signal coming from the North Atlantic (Drysdale et al., 2005; Drysdale et al., 2007b; Zanchetta et al., 2007; Regattieri et al., 2014) which is very sensible to abrupt climate changes like stadial and interstadial transitions. Oxygen isotope

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changes of Corchia cave area archives have been indeed interpreted as controlled by chances in the advection of moisture coming from the Atlantic in response to variations in North Atlantic Meridional Overturning Circulation (AMOC) strength.

Furthermore, it seems that the oxygen isotope ratio signal does not follow changes in fabric, whereas the carbon isotope ratio does. Specifically, the hiatus did not coincide in any sharp change in the plotted values of the δ18O. Therefore, drip rate reduction that controls the growth of the speleothem, have not had a substantial effect on δ18O FR16 record.

In conclusion, whilst the range of FR16 δ18O values is clearly linked to the signal of the source moisture that can be enhanced by the orographic effect. Frasassi oxygen trend is most likely linked to global temperature fluctuations as suggested by the good correlation with the norther hemisphere insolation. Therefore, local environmental changes such a water availability reduction/increasing in the karst-cave system did not significantly influence the signal.

5.7 Conclusions

The isotopic and petrographic record of a stalagmite from Frasassi cave, grown during the last glacial inception, has been discussed and interpreted (Fig. 5.6). The petrographic observations have helped to read the FR16 isotopic fluctuations over time decoupling between the effects of hydrology and temperature. Isotopic proxies can be influenced by many different factors, which made their interpretation, sometimes, very complex. While FR16 δ13C trend can be linked to local to regional scale climate conditions such as drip rate changes and temperature related supersaturation fluctuations, the δ18O trend has been interpreted as mainly influenced by the predominance of winter vs summer precipitation, which is linked to the NH insolation. The δ18O signal (values range) likely reflects the composition of the source moisture and thus its provenance and trajectories that have then attenuated the effect of longer-scale climatic variations, such as stadial events, on the isotopic trend.

FR16 δ13C records four cycles of quick transitions from humid to dry conditions and at least two of them coincide to the DO shifts GS24 and GS25 at the onset of the last glacial, at 111 ka and 104.5 ka respectively (Fig. 5.6). The first half of FR16 is characterized by slow growth rate. After a hiatus of 1400 years, at ca. 102 ka BP, the stalagmite re-started its growth relatively faster. The observed petrographic changes are consistent with these cycles toward less water availability

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characterized by open columnar or micrite fabrics. However, after the hiatus, the drip point changed and possibly the supersaturation of the feeding fluid as well, which explains the apparent discrepancy between fabric, growth rate and the Carbon isotope ratio signal observed in this part of the sample.

FR16 δ18O does not follow a similar pattern like δ13C which means that the signal was not significantly affected by kinetic fractionation. Similar cycles, like the ones recorded by the carbon, are not observable in the oxygen of FR16. Only major large-scale shifts, like GS24 and GS25, are apparently recorded by δ18O but to a lesser extent (< 0.5 ‰) than by other stalagmites from the same latitude (Fig. 6). Clearly, the trend of the signal looks attenuated by factor/s contrasting the effect of the cold and arid stadials that here has been recognized as related to NH insolation rate.

In addition, FR16 δ18O average values are relatively more negatively shifted than the δ18O average values of the Italian stalagmites from the Tyrrhenian coast (Drysdale et al., 2005; Drysdale et al., 2007b; Columbu et al., 2017) and from the Eastern Mediterranean (Bar-Matthews et al., 2003). A possible explanation can be a different storm tracks history from these sites. Many different moisture sources can bring precipitations over Frasassi location and nowadays Frasassi rainfalls composition is more linked to the precipitations from the south of the Mediterranean rather than to the eastern or the western sides. This could have been also the case during MIS 5c and 5d, during when FR16 was growing. Moisture coming from the South, possibly originated in the tropical Atlantic regions, due to Rayleigh condensation would have delivered relatively lighter rainfall over Frasassi cave. The “shadow effect” created by an orographic barrier like the Apen- nine could have also lead to even lighter δ18O rainfall over the Adriatic side of the mountains range respect to the Tyrrhenian side where Corchia cave is located. Alternatively, a substantial reduction of 18O enriched summer precipitations over the area could have enhanced the influence of lighter winter precipitations on the oxygen signal recorded by FR16 stalagmite.

Another key-point of this work is the importance of petrography as necessary tool to be combined with the geochemical analysis in order to give a more accurate climate interpretation of the data. It is common to obtain incongruent isotopic results, because they are influenced by the interplay of a myriad of different factors, whose impact is sometimes difficult to discern. It is thus important to calibrate modern proxies and extrapolate back through time assuming that instrumental data represent past conditions, or to use a suite of different proxies from a single speleothem to reconstruct palaeoclimate.

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Sánchez Goñi, M.F., Loutre, M.F., Crucifix, M., Zanchetta, G., Regattieri, E., Giaccio, B., Peyron, O., Santos, L., Duprat, J., Malaizé, B., Wagner, B., Sulpizio, R., Francke, A., Vogel, H., Turon, J.L., Peypouquet, J.P., 2005. Increasing Sadori, L., Masi, A., Sinopoli, G., Lacey, J., vegetation and climate gradient in Western Leng, M., Leicher, N., 2016. Aligning and

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synchronization of MIS5 proxy records from Mediterranean archives: Implications for DEEP Lake Ohrid (FYROM) with independently dated core chronology. Biogeosciences 13, 2757-2768.

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Chapter 6. Conclusions

This thesis main goal has been advancing knowledge on physical aspects of speleothems as support to their climate and environmental significance. This goal has been accomplished by using conventional optical petrography as well as non-conventional methods such as high-resolution imaging, Synchrotron micro-XRF (SR-µXRF) and Neutron scattering.

The following conclusions respond to the research questions and objectives introduced in Chapter 1:

- Objective 1: Benchmarking palaeoenvironmental and climate study of Lamalunga cave and testing the validity of coralloid speleothems as archives of climate changes by implementing petrography and microstratigraphy and high resolution elemental mapping to study the distribution of chemical elements and organic compound

The attention given to the “Altamura man”, a Neanderthal skeleton found in 1993 in Lamalunga cave (South Italy), for almost 20 years after its discovery, has been mostly devoted to its conser- vation and to the endeavour of displaying it in a museum. Only recently, this attention has also been devolved to achieve more scientific goals like its dating, morphometric, and palaeogenetic characterization (see, in this regard, Lari et al. (2015)). Following this line, in this thesis, I set the foundations of a palaeoclimatic and environmental study that have been presented in chapters 2 and 3. Normally, in cave environment, this kind of study are carried out using stalagmites. Since there were not stalagmites associated to the skeleton, the only alternative was to consider coralloids, a type of speleothem that, due to their small dimensions, were commonly deemed unsuitable for palaeoclimate reconstruction. I aimed at proving that coralloids are good archives of palaeoclimate proxy data even though in the existing literature they have been rarely used. I was also aware that the state of the art about their ontogenesis presented different theories, which seemed all valid, depending on the cave context. Thus, my research started from the necessity of understanding the physical mechanisms involved in Lamalunga coralloids formation as an essential requirement before interpreting chemical proxy extracted from their successive growth layers. It was, in fact, previously demonstrated that the modality of CaCO3 deposition in coralloids can alter the original climate-related δ18O and δ13C signal of the percolating water, before being transferred in the speleothem carbonate layers. Another issue related to carbonate formation in coralloids is the uncertainty of the primary phase, whether this is aragonite, calcite or another mineral.

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Thanks to petrographic tools, not only I have been able to comprehend the mechanisms of formation, but also to prove that they were originally formed by calcite and that they are thus apt for being dated with the U-series method because post-depositional alteration has not been detected. I could demonstrate that at Lamalunga cave, hydroaerosol transport and evaporation of the fluid resulting from myriads of droplets transported to the coralloid surface are the main processes controlling the development of coralloids. Hydroaerosol transport and evaporation influence the growth and the incorporation of chemical elements and of organic o detrital particles. Specifically, the rate of evaporation, depending on humid or arid periods, would have regulated the development of compact elongated or porous fiber-like fabric respectively. This is consistent with what documented by the SR-µXRF elemental maps: an unusually high (ca. three times more) concentration of non-Ca species (like Mg, Sr and Si) between the fiber-like crystals in porous and high- fluorescent convex layers with a finely laminated internal structure. This allows hypothesizing an ascending movement of fluid during evaporation, with impurities adsorption on rugged, inter- crystal surfaces. During humid periods, evaporation is limited and consequently, the non-Ca elements and particulates are less concentrated. From a thin film of fluid uniformly distributed on the active growth surface and with low impurity concentration, compact columnar calcite precipitated, forming isopachous bands. Microstratigraphy has been also applied in support to U-Th dating, to correlate similar phases of growth where, due to their reduced dimensions, the sampling for U-Th dating is problematic. The results presented in this thesis suggest that it is possible to use such non-conventional type of speleothems to date archeological and palaeoanthropological findings as well as to extract palaoenvironmental information.

I could, thus, demonstrate, the hydroclimate significance of coralloids fabrics, microstratigraphy and overall morphology.

- Objective 2: characterizing the internal structure of speleothems, and recognize features that may indicate post-depositional changes and re-setting of significant climate signals by employing materials science techniques

Here, I investigated some of the many possible pathway of crystallization in speleothems, which may give rise to bizarre fabrics. This line of research follows the petrographic study of coralloids in that it explores the potential of fabrics as stand-alone tools of palaeoclimate investigation. This has consequences on the accuracy of the preservation of the original chemical and physical properties, because it influences the stability of their constituent phases and the potential for post-

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depositional changes. Neutron scattering, a common material science technique, allowed investigating speleothem crystals at sub-micrometre scale and, specifically, small angle neutron scattering applied to speleothem samples representative of diverse fabrics, highlights that some fabrics may consist of nanocrystal aggregates or, at least, are defect ridden. The results of this study are still preliminary, but they pave the way to a re-evaluation of speleothem incorporation of geochemical data when non-classical pathways of nucleation influenced speleothem nucleation and growth.

- Objective 3: Benchmarking palaeoenvironmental and climate study of Frasassi cave speleothems by combining petrography and geochemistry.

After having demonstrated that petrography is a valid stand-alone tool for the reconstruction of hydroclimate changes, I then combined petrography and stable isotope geochemistry to stalagmites the most common speleothem morphology utilised as palaeo-climate archive. The speleothems where this investigation has been carried out comes from Frasassi cave. The choice of this speleothems was dictated by its location, which is interesting for two reasons: (i) the isotopic signature of modern rainwater in Frasassi and Lamalunga regions coincides and (ii) Frasassi is at the same latitude as Corchia cave, the most studied cave in Central Italy, but at the opposite side of the Apennine mountain range. This gave me the opportunity to test the significance of Frasassi stalagmite O isotopes ratio record. Furthermore, as Frasassi and Lamalunga modern precipitation δ18O signal is comparable, I hope in the future to test the interpretation of the stable isotopes signal extracted from the Lamalunga coralloids by using as benchmark that from stalagmites from a cave that has been possibly influenced by the same climatic dynamics.

In chapter 5, I presented the interpretation of Frasassi stalagmite δ18O signal tied to microstratigraphy and fabrics, and then the comparison between Frasassi stalagmite and the δ18O records of coeval speleothems from the Tyrrhenian coast (like Corchia cave), during the glacial inception MIS5c-5d. The comparison suggests a latitudinal gradient for the isotopic composition of the precipitations. Frasassi average δ18O signal is relatively more negative (c.a. 3.5 ‰) than the Tyr- rhenian coast speleothems δ18O, which I explained as due to different storm track histories and moisture source origin. Such interpretation differs from that reported in literature for the Tyrrhe- nian stalagmites, where the δ18O signal is ascribed to an “amount effect”. I hypothesized that the Apennine belt acts as a topographic barrier for air mass movements from the Atlantic. When

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reaching the Adriatic side, rainfall δ18O signal would have become relatively lighter (more negative δ18O) with respect to the Tyrrhenian side, which is the first encountered by the moisture trajectories. Alternatively, there is the possibility of different moisture source and provenance: in this case Frasassi area would have not received the moisture from Genoa-type cyclones, as it is the case for the western Mediterranean and Tyrrhenian coast, but from the tropical Atlantic, similarly to what observed for NE Italy. The long-term δ18O modulation seems to agree with Northern Hemisphere insolation, which influences the composition of the rainfall depending on winter vs. summer precipitations dominance.

All these hypothesized climate processes could have combined toward an attenuation of the effects of large scale climatic variations, such as stadial events, that in Frasassi are less evident when compared to other Mediterranean speleothems.

The δ13C signal in Frasassi stalagmite reflect local/regional scale humid/arid cycles, and coincide well with changes in petrography. It is noteworthy that, by contrast, the oxygen isotope ratio signal does not follow changes in fabric and, thus, does not reflect the drip rate variability that controls the growth of the crystals. This is further evidence that, whilst fabrics respond immediately to infiltration, which is to rainfall, the δ18O of some speleothems may reflect more a signal stored in the aquifer and, therefore, “homogenized” through the years, rather than the immediate response to recharge.

The ultimate conclusion is that petrographic tools, both the optical and the elemental mapping, provide a versatile and analytically powerful method of investigation of geologic archives of climate significance. The methodologies proposed in this Thesis will, hopefully, contribute to encourage the use of petrography in speleothem-based palaeoclimate research and, most important of all, to petrography as a stand-alone source of valuable proxy data.

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Chapter 7. Future directions

1. Lamalunga cave coralloids and climate context of a unique, complete Neanderthal skeleton.

The present Thesis has set the basis for the continuation of speleothem research in Lam- alunga cave. By continuing the studying of the coralloid formation, we will gain important information concerning the climate and the morphological evolution of the cave, through time, before and after Neanderthals occupation. Over the next few years, as part of a new project, further field work is planned with the intent to commence the archaeological excavation that will answer the question: How did the Neanderthal human enter the cave? Archaeologists would like to remove some skeletal portions of the Altamura man, which are necessary to learn more about early Neanderthals. The overarching goal of the new project is to continue the ongoing scientific work on the cave using new sophisticated technologies that will allow studying the remains in a safe and efficient way. There is the intent to make this unique “product of the past” more attractive and more accessible for cultural tourism. In this way, Altamura’s past heritage will become economically sustainable for its long-term survival and will contribute to the education, research and pride for the local community and the whole country. Moreover, considering the financial problems of the southern Italian region, with high unemployment amongst young people, the “Altamura man” can boost and upgrade its cultural offer attracting consequently more tourism.

In this context, it has been already envisaged advancing the climatic investigation based on Lamalunga coralloids. Considering that the physical mechanisms controlling the formation of the coralloids and related fabrics have been now identified, the next step will be to build a robust age model and a stable isotopes/trace elements record time series for selected Lamalunga cave coralloids by sampling at high resolution using Secondary Ion Mass Spectrometry (SIMS). These results will be then compared to Frassassi and other Mediterranean records to study decadal to centennial climate variability at a regional scale. More ages from coralloids sampled in direct contact with the bones of the Altamura man are needed, because, so far, we only have available the ages from one coralloid directly associated to the skeleton (see Lari et al. 2015). Critically, because coralloids are present below and above the skeleton, to gain the climate scenario during the lifespan of the Altamura man and of the Neanderthal dwelling in the region, the stalagmites from

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Frasassi, which cover almost entirely the interval between 380 and 53 ka, can be utilised. As both coralloids and Frasassi stalagmites show well-defined microstratigraphy re- sponding to infiltration, I believe that one new direction of the present research will be to attempt correlations between equivalent, radiometrically dated intervals by using similar climate-sensitive fabrics.

2. Palaeoclimate study of the Adriatic region from Frasassi speleothems.

Stalagmites from Frasassi cave offer a good opportunity to obtain a long palaeoclimate reconstruction from the Adriatic seaboard, paralleling the palaeoclimate reconstruction from Corchia cave on the Thyrrenian coast. So far, three stalagmites have been dated with a good density of age points per stalagmite, and the results are mentioned in appendixes 1 and 2. However, their age models need to be improved with more ages if critical climate events are to be framed with accuracy. For stalagmite FR11 I have built an age-model from 33 sample points with the record spanning from ca. 354 ka to 266 ka. FR12 stalagmite age-model was produced from 24 sample points and the record spans from ca. 273 ka to 196 ka. Other three stalagmites (FR22, 20 and 19) from the same karstic level (5th), have been dated, but the density of ages is not good for today’s standards. Their growths overlap with FR11, 12 and 16 (see table 6.1).

Sample Growth age (ka)

FR20 From 380 to 308

FR11 From 354 to 266

FR19 From 337 to 293

FR12 From 273 to 196

FR16 From 112 to 96

FR22 From 101 to 53

Table 6.1 List of all the samples collected in Frasassi cave and corresponding time span of growth.

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With improved age models and additional data from FR19, 20 and 22 I will be capable to expand the study presented in appendix 1, which aims to investigate the interaction between climate, tectonic uplift and speleothem growth.

In appendix 2, I presented a work that combined the interpretation of δ13C, greyscale values and fabric changes for stalagmites FR11 and FR12 that represent the oldest records so far analysed in Frasassi cave. For these two samples, in addition to robust age models, I also have a high-resolution isotopic record for both oxygen and carbon: 589 and 260 sample points for FR11 and FR12 respectively. From these data, I expect to produce a high-resolution record of climatic variation that occurred in the Mediterranean area for the late Middle to Upper Pleistocene, spanning from 345 ka until 196 ka and from 112 ka to 96 ka. This would give me the possibility to reconstruct climate for periods before and after the Neanderthalian death and fossilization.

3. Advances in speleothem petrography

Whilst data interpretation for speleothem textural analysis performed by neutron scattering is relatively simple, data reduction and interpretation of SANS results is complex and, therefore, the present pilot study may not suffice to understand its potentiality in the application to speleothem science. The preliminary results of chapter 4 suggest that crystal nucleation in speleothem may follow non-classical pathways, which elegantly explains the development of fabrics such as dendritic and microcrystalline types. The most recent research on speleothem crystallization pathways has indeed revealed that non-classical nucleation occurs in caves, with the nucleation and growth of amorphous phases or aggregation of nanocrystals. This brings about the importance of investigating crystallization pathways and their influence on both fabrics and capture of climate-significant chemical signals. It can be also useful to improve our knowledge behind the processes producing early diagenetic alteration. The future of such research would be coupling Trans- mission Electron Microscopy (TEM), which allows investigating crystals at nano- and lattice scale, Electron Backscattered Diffraction (EBSD), which allows characterization of crystal orientation in polycrystalline materials at the Scanning Electron Microscope (SEM) and neutron scattering with in-situ precipitation experiments towards the full understanding about what parameters control each nucleation pathway. In part, this is already been carried out at the University of Newcastle, but it would be advisable if a net-

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work of researchers with similar interest on continental carbonates crystallization pathways would participate in a common effort to advance insight on the relationships between speleothem crystallization and their capture of climate information.

In addition, a future objective is the creation of an inventory of stalagmite fabrics and textures by combining data from neutron scattering at beamlines such as KOWARI (textural analysis) and QUOKKA (Small Angle Neutron Scattering) of ANSTO to identify “at a glance” which specimens may be best suited for investigating the geochemical signals and thus obtain robust palaeoclimate and palaoenvironmental reconstructions. To accomplish this, a wider variety of samples, with different textures, whose environments of deposition have been carefully monitored needs to be analysed. This type of research has just commenced, but it opens a novel branch in speleothem-based paleoclimate investigation.

Final comments

The research results presented in this PhD Thesis stress the importance of petrophysical proxies in speleothem-based investigation. The common approach in speleothem science still regards petrography as unimportant, and thus not grounding chemical data in their physical context. This is not acceptable any more, considering that crystallization pathways may influence chemical data. This research reveals that conventional petrographic tools, that are easy to acquire and economically viable, hold instead useful information to complement geochemical data. By advancing petrography, it is possible to make palaeoclimate investigation from speleothems available also to researchers with limited funding resources.

Petrography is a relatively humble part of speleothem sciences, and does not make head- lines in high impact factor journals. It is also difficult to quantify and, at times, subjective. This makes it less palatable than high-resolution geochemical techniques. Nevertheless, in this work, I believe I have unequivocally shown that it is, in itself, a powerful hydroclimate archive and it explains phenomena that, at times, are referred to as the results of unspecified “kinetic effects”.

I have been fortunate enough to be capable to conduct speleothem crystallographic research by using also sophisticated techniques, which are not commonly accessible, such

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as Synchrotron radiation-based fluorescence and neutron scattering, and thus enlarging the frontiers of speleothem petrography. I understand this is not available to all researchers. Yet, a simple microscope and a high-resolution scanner have been crucial in my research, and the final comment is that I hope I will be able to encourage other students to use these tools and tell their own palaeoclimate stories.

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Appendix I Speleothem chronology from Frasassi cave system (Central Italy): timing of speleogenesis, uplift rate and paleaoclimate evolution. i. Aim of the research

The over 25 km long cave maze system of Frasassi caves, located on the Eastern side of the Apennines, 40 km from the Adriatic Sea, is an exemplary case of sulphuric acid hypogenic speleogenesis (Galdenzi and Menichetti, 1995). Groundwater is enriched in H2S after dissolution of anhydrite underlying Jurassic limestone at the core of an anticline structure. Quasi horizontal galleries developed at different elevation in the cave system as a result of the interaction between tectonic uplift, and the erosion and aggradation processes marking Quaternary glacial and interglacial phases. Each karst level developed when groundwater remained at the same elevation for a prolonged timespan.

The present research aims at dating the period(s) when development of the different karstic level occurred, reconstruct the geomorphological evolution of the area in the Quaternary, and, subsequently reconstruct climate evolution from stalagmites collected in the 5th karst level where field observations suggested that speleothems should contain at least three glacial cycles.

ii. Materials and methods

U-Th analyses were performed by multi-collector inductively coupled plasma-mass spectrometry at the University of Melbourne. A total of 57 samples were analysed in the three stalagmites. Uranium content typically ranges from 40 to 120 ppb, and 230Th/232Th ratios > 1000 allow a robust age model for each stalagmite.

iii. Discussion

Hypogenic karst processes

During warm and wet interglacials, the river incised what today is the Sentino gorge. At the same time infiltration of meteoric water enriched in soil CO2 enhanced dissolution of the overlying limestone favouring extensive speleothem formation in the galleries (Fig. A1.1).

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By contrast, during cold and dry periods, the reduced infiltration of meteoric water hindered the formation of calcite speleothems and favoured exhalation of groundwater H2S vapours through karst chimney (Fig. A1.2), thus causing corrosion of the cave walls and producing gypsum deposits (Fig. A1.2) (Galdenzi and Maruoka, 2003).

Fig. A1.1 Interpretation of the evolution of Frasassi system from an original, modified from an unpublished drawing by A. Montanari.

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Fig. A1.2 H2S vapours chimney (photo on the left side). Gypsum deposit (photo on the right side).

Frasassi karstic levels

The tectonic evolution of the area is characterized by regional uplifting, which started in the Mi- ocene, coupled with eastward migration of the compressional belt. At present, due to the rollback effect, extension is affecting the main Apennines ridge, whilst compression, driven by subduction, is localized foreland along the Adriatic coast. The Frasassi area is located at the eastern border of this inner extensional zone. River incision in the Sentino valley and consequent water table lowering in the cave system are a response of the regional uplifting (Mariani et al., 2007).

The age of the stalagmites, coupled with published radiometric ages from other speleothems and cave deposits, allow determine the minimum age of the karst levels (Table AI). The data documented 3 phases characterised by the following uplift rates (Fig. A1.3):

From 750±260 kyr to 130±15 kyr the uplift rate was relatively low (0.1 to 0.16 mm/yr) as a response to the progressive eastward migration of the compressional belt coupled with middle Pleis- tocene glacial/interglacial erosional pattern.

From 130±15 to the early Holocene the uplift rate progressively increased, most likely as a response to increased erosion, which characterised the last glacial/interglacial cycles (MIS6 to MIS2).

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From the 8.4±0.2 kyr to the Present the uplift rate accelerated up to 0.6 mm/yr and the region experienced tilting measured in 0.21° dip toward N60°E (Mariani et al., 2007), following the reactivation of Holocene tectonic activity.

Elevation Ele- (m above Uplift Uplift va- Age Error Uplift Location Level present phase mean Dating method tion kyr (kyr) mm/yr river mm/yr mm/yr (m) level)

Present to- 1 0.600 0.600 0.547 pography 205 0.6 (D'Anastasio et al., 2006)

II "Lago 14C on calcite rims and sub- Grotta del delle 8.4 0.2 0.554 0.554 0.243 fossil eels (Mariani et al., Fiume Anguille" 210 4.65 2007)

OSL on quartz from slackwater deposits 111 17 0.092 Grotta del III "Sala Vento 200" 234 29.6 (Peterson et al., 2013)

Grotta del III "Sala U/Th on stalagmite 130 15 0.228 0.205 0.141 Vento Infinito" 235 29.6 (Taddeucci et al., 1992)

Grotta del V "Man- U/Th on stalagmite (present 354.8 21 0.147 0.100 0.164 Vento hattan" 257 52 study)

Grotta della 10Be-26Al on quartz from Beata Ver- 750 260 0.156 0.164 slackwater deposits (Cyr and gine VI level 117 Granger, 2008)

Table A1 Uplift rate of the different karst levels.

Fig. A1.3 Uplift rate for the different karstic levels graph.

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Fig. A1.4 Age models and main fabrics observed for the samples.

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Stalagmite growth rates vs δ18O global records: preliminary hypothesis

In Fig A1.5 the growth rates of Frasassi stalagmites are compared with NGRIP ice core δ18O record and LR04 marine benthic δ18O record .

FR11 (355-241 kyr): The growth rate decreased coinciding with more positive peaks in the LR04 δ18O (+ warm). Given that the stalagmite was growing when the sulphuric groundwater table was

th close to 5 level, we infer that the exhalation of H2S vapours hindered the formation of calcite speleothems and caused decrease or cessation (hiatuses) of their growth rate. Growth rate in this period is, thus, interpreted as influenced by hypogene speleogenesis rather than reflecting a climatic signal.

FR12 (273-196 kyr): Water table lowered and the 5th level raised >20 m above the sulphuric groundwater table. Stalagmite growth rate increased during warmer events in MIS 7, possibly reflecting increased water infiltration.

FR16 (113-96 kyr): During the first 10 kyr, speleothem growth rate increased coinciding with warmer phases of MIS 5d and 5c. In the last 10 kyr the situation reverted: growth rate increased during a cooling trend. This can be explained by in-cave forcing factors, such as increased cave ventilation in response to the progressive lowering of the water table. This process is suggested by stalagmite fabric changes: from translucent compact columnar to open columnar with alternating layers of porous and compact calcite (Fig. A1.5). Couplets of porous and compact calcite are commonly associated to seasonal changes in drip rate and ventilation. In the case of Frasassi, ventilation can be caused by the opening of new entrances connecting cave and surface.

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Frasassi is a complex karst system where tectonics, hydrogeology, geomorphology and climate influenced both speleogenesis of the galleries and growth of the speleothems. Therefore, when interpreting speleothem growth rates and geochemical data all these mechanisms must be taken into account.

Our preliminary data suggest that the 5th level of the cave developed 52 m above the present-day river elevation, was air-filled by 355±21 kyr. During the last 355 kyr, speleothem growth was influenced by climate (net infiltration from the surface) and both dilution and relative elevation of the sulphuric groundwater table.

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Future geochemical and petrographic data will be crucial in the final interpretation of the tectonic and climate history of the cave system.

v. References distributed benthic δ18O records. Cyr, A.J., Granger, D.E., 2008. Dynamic Paleoceanography 20, PA1003. equilibrium among erosion, river incision, and coastal uplift in the northern and Mariani, S., Mainiero, M., Barchi, M., van central Apennines, Italy. Geology 36, 103- der Borg, K., Vonhof, H., Montanari, A., 106. 2007. Use of speleologic data to evaluate Holocene uplifting and tilting: An example D'Anastasio, E., De Martini, P.M., from the Frasassi anticline (northeastern Selvaggi, G., Pantosti, D., Marchioni, A., Apennines, Italy). Earth and Planetary Maseroli, R., 2006. Short-term vertical Science Letters 257, 313-328. velocity field in the Apennines (Italy) revealed by geodetic levelling data. Peterson, D.E., Finger, K.L., Iepure, S., Tectonophysics 418, 219-234. Mariani, S., Montanari, A., Namiotko, T., 2013. Ostracod assemblages in the Frasassi Galdenzi, S., Menichetti, M., 1995. Caves and adjacent sulfidic spring and Occurrence of hypogenic caves in a karst Sentino River in the northeastern region: Examples from central Italy. Apennines of Italy. Journal of Cave and Environmental Geology 26, 39-47. Karst Studies 75, 11-27.

Galdenzi, S., Maruoka, T., 2003. Gypsum Taddeucci, A., Tuccimei, P., Voltaggio, deposits in the Frasassi caves, Central Italy. M., 1992. Studio geocronologico del Journal of Cave and Karst Studies 65, 111- complesso carsico "Grotta del Fiume- 125. Grotta Grande del Vento" (Gola di Frasassi, Ancona) e implicazioni Lisiecki, L.E., Raymo, M.E., 2005. A paleoambientali. Il Quaternario, Italian Pliocene-Pleistocene stack of 57 globally Journal of Quanternary Science 5, 213-222.

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Appendix II Composite δ13C and petrographic 196-355 ka record from Frasassi cave (central Italy) stalagmites: investigating drivers of calcite carbon isotope signals i. Introduction: C isotope ratio in speleothems

The δ13C time-series from two precisely U-Th dated Frasassi stalagmites (FR11 and FR12) (Fig. A1.4) spanning from 195 ka to 354 ka (MIS 7-10) are interpreted on the basis of sequence of fabrics, as these respond to hydrology and CO2 degassing. Fabrics allow the varying contribution of these processes and atmospheric C to be distinguished in the δ13C signal registered in the calcite. Compact columnar fabrics δ13C shifts through time reflect precipitation changes from relatively constant to slow drip rate from “clean” water. Open columnar fabrics suggest higher drip rate and influx of foreign material, like organic molecules, due to increased soil productivity which then leads to more negative δ13C calcite.

ii. Materials and methods

A total of 54 U-Th dates were obtained using a MC-ICP-MS at the University of Melbourne. The mass spectrometer C-isotopes analyses on 865 calcite powders were performed at the University of Melbourne. The petrographic observations were carried out on thin sections using a Leica MZ16A stereomicroscope at the University of Newcastle. The grey scale profile data were extracted from the axial part of the stalagmites using high resolution images and ImageJ software. Some features, like cleavage or fractures visible on the scanned surface, lead to false grey peaks (usually high values). These artefacts has to be excluded from the interpretation by carefully checking the high resolution photo of the sample to see if the increased values actually correspond to the changes in the fabric.

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iii. Results

The Frasassi record covers the interval from 354 ± 20.7 ka to 196.7 ± 4.6 ka ka encompassing two terminations: TIII and TIV and overlapping each other from 275 ka to 266 ka. Both samples are characterized by columnar fabric with two different subtypes: compact (C) and open (Co) (Frisia, 2015) (Fig. A2.1). FR12 is mostly formed by compact columnar intercalated by episodes characterized by open columnar at: 244.4 ± 7.5 ka, 235 ± 7.1 ka, 232.8 ± 6.1 ka, from 216.9 ± 6.4 ka to 215.4 ± 5.2 ka and from 211.7 ± 5.6 ka to 196.8 ± 4.4 ka. FR11, similarly to FR12, is mostly composed by compact translucent columnar crystals but from 317.6 ± 12.7 to 303.3 ± 8.1 ka a lamination formed by alternating laminae of open columnar and compact columnar calcite, is visible. Compact columnar fabric is translucent and yields low grey values (Fig. A2.1). The porous open columnar fabrics appear white and yield high grey values (Fig. A2.1).

Average δ13C values are -4.7 ‰ for MIS 10, -5.3 ‰ for MIS 9, -4.9 ‰ for MIS 8 and -5.3 ‰ for MIS7 (Fig. A2.2). During interglacials, thus, the mean carbon isotope ratio values are more positive than expected if the dissolved CO2 in the parent waters evolved solely from efficient soil respiration (< -7 ‰).

The grey values and the δ13C curves of Frasassi samples co-vary: where the fabric is porous and grey values are high, the δ13C is more negative (Fig. A2.2).

FR11 and FR12 δ13C time series matches with both global reference curves of benthic foraminif-

18 era δ O (Lisiecki and Raymo, 2005) and the Antarctic atmospheric CO2 record, however, FR11 is delayed by 5-10 k years (Fig. A2.2). At TIII terminations Frasassi δ13C record shows a shift of 5 ‰ which, by using the equation: MAT (°C) = −1.68*δ13C (cf. Johnston et al. (2013) and Borsato et al. (2015)), is equivalent to a ~ 8°C degrees shift in the temperature. TIV instead, reveals a shift of 3 ‰ in the carbon curve corresponding to a change of ~ 5°C.

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Fig. A2.1 FR11 and FR12 grey scale profiles. High values (>110) correspond to the porous parts of the stalagmites characterized by open and/or porous columnar fabrics. Translucent zones are formed by compact calcite which is indicated by low grey values (<110).

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Fig. A2.2 Frasassi δ13C record and grey values profile compared to LR04 marine benthic δ18O record

(Lisiecki and Raymo, 2005), Antarctic atmospheric CO2 record (Petit, 1999) and summer insolation curve (Laskar et al., 2004). Marine isotope stages (MIS) 7 to 10 and terminations III and IV are also indicated. Blue dots represent the U-Th dates performed on FR11 and FR12 with 2σ error bars. Green boxes highlight interglacials and interstadials.

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iv. Discussion

Significance of δ13C changes in Frasassi record

Columnar fabric forms under slow drip rate which favours kinetic fractionation through rapid CO2 degassing that can impact δ13C and alter the environmental signal (Dreybrodt and Scholz, 2011; Frisia, 2015). This likely explains the constant more-positive-than-expected δ13C signal found in C fabric in Frasassi samples (Fig. A2.2).

Conversely, higher drip rate does not promote kinetic fractionation and Frasassi δ13C values are indeed relatively more negative in Co sections. Variable/fast drip rate hinders the coalescence of crystalline boundaries and under these conditions open columnar crystals form. During high discharge periods inside the cave, impurities can be deposited on top of the stalagmites which favours the formation of intra laminae porosity (porous columnar) (Frisia, 2015). Open columnar fabric is thus indicative of relatively warmer and more humid periods characterized by high soil respiration rates leading to more negative δ13C in the percolation-water.

A lag in the Frasassi climate record with the global isotopic signal is only observed in FR11 (MIS 10 - MIS 8) which can be explained by different infiltration pathways.

In addition, the shifts in the carbon ratio of Frasassi stalagmites during the terminations are larger compared to the globally-averaged speleothem δ13C record obtained by Breecker (2017): -4 ‰ vs. -1.9 ‰. This can be due, again, to the interference of in-cave processes, which alters the atmospheric climatic signal.

v. Conclusions

Paleoclimatic interpretations can be complemented by grey scale profiles from high resolution images of stalagmites coupled with petrographic observations. This approach has been applied in the present work to interpret the δ13C climatic signal recorded in stalagmite growth layers. Re- cently, Breecker (2017) suggested to subtract the atmospheric certain pCO2 effect on speleothem δ13C records in order to interpret the calcite δ13C values in the light of other factors controlling the carbon signal. Here we propose to also combine petrography and geochemical properties to distinguish between global to local drivers of δ13C anomalies in stalagmites.

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vi. References

Borsato, A., Frisia, S., Miorandi, R., 2015. Carbon Johnston, V.E., Borsato, A., Spötl, C., Frisia, S., dioxide concentration in temperate climate caves Miorandi, R., 2013. Stable isotopes in caves over and parent soils over an altitudinal gradient and its altitudinal gradients: Fractionation behaviour and influence on speleothem growth and fabrics. Earth inferences for speleothem sensitivity to climate Surface Processes and Landforms 40, 1158-1170. change. Climate of the Past 9, 99-118.

Breecker, D.O., 2017. Atmospheric pCO2 control on Laskar, J., Robutel, P., Joutel, F., Gastineau, M., speleothem stable carbon isotope compositions. Correia, A.C.M., Levrard, B., 2004. A long-term Earth and Planetary Science Letters 458, 58-68. numerical solution for the insolation quantities of the Earth. Astronomy & Astrophysics 428, 261- Dreybrodt, W., Scholz, D., 2011. Climatic 285. dependence of stable carbon and oxygen isotope signals recorded in speleothems: From soil water to Lisiecki, L.E., Raymo, M.E., 2005. A Pliocene- speleothem calcite. Geochimica et Cosmochimica Pleistocene stack of 57 globally distributed benthic Acta 75, 734-752. δ18O records. Paleoceanography 20, PA1003.

Frisia, S., 2015. Microstratigraphic logging of Petit, J.R., 1999. Climate and atmospheric history of calcite fabrics in speleothems as tool for the past 420,000 years from the Vostok ice core, palaeoclimate studies. International Journal of Antarctica. Nature 399, 429-436. Climatology 44, 1-16.

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