Making Eggshell Visible in the Archaeological Record John Stewart A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy University of York Archaeology October 2013 Abstract Despite its presence in many types of deposit, eggshell has long been a neglected archaeological resource. The difficulty of recovering the material, combined with analytical constraints on subsequent taxonomic identification, has led to systematic underuse. This thesis will begin to address this shortcoming by pursuing two main lines of research. First, a novel identification technique based on ZooMS (zooarchaeology by mass spectrometry) and peptide mass fingerprinting is developed for eggshell. The new technique is then applied to archaeological eggshell assemblages from Anglo-Scandinavian York and Norse-era sites in the northern and western isles of Scotland. This provides new insights into egg use in these locations during this period, and raises a range of new questions regarding the use of domestic and wild resources. Second, an investigation into patterns of diagenesis in the eggshell proteins which form the basis of the technique is conducted. The principal aims of this analysis are to explore the prevalent diagenetic processes affecting eggshell proteins and amino acids, and thus to test whether it is possible to produce an estimate of the expected temporal span of the technique based on high-temperature diagenesis, and to establish the potential usefulness of the material for IcPD (intra-crystalline protein diagenesis) dating. The main outcomes of this study are (i) the development and successful application of a new taxonomic identification technique for archaeological eggshell; (ii) enhanced understanding of egg use during the Norse era in Scotland and Anglo-Scandinavian York; (iii) the observation that high-temperature diagenesis cannot be used to accurately predict peptide survival at archaeological deposition temperatures; and (iv) that avian eggshell is not a viable substrate for absolute dating using IcPD (although it may still prove useful for relative dating). 2 Table of Contents List of Figures ..................................................................................................... 15 List of Tables ...................................................................................................... 20 List of Additional Material ................................................................................. 22 Acknowledgements ........................................................................................... 22 Declaration ........................................................................................................ 25 Chapter 1: Introduction ..................................................................................... 26 Section 1: Brief History of Egg Use ................................................................ 26 1.1.1: Introduction .................................................................................... 26 1.1.1.1: Overview ................................................................................. 26 1.1.1.2: Why do people use bird eggs? ................................................ 26 1.1.2: Collection and use of wild eggs in the past .................................... 27 1.1.3: Bird domestication and dispersal ................................................... 31 1.1.3.1: Overview ................................................................................. 31 1.1.3.2: Chicken domestication and dispersal ..................................... 31 1.1.3.3: Goose domestication and dispersal ........................................ 34 1.1.3.4: Duck domestication and dispersal .......................................... 36 1.1.4: Eggs in the archaeological record ................................................... 38 1.1.4.1: Preservation and recovery of eggshell.................................... 38 1.1.4.2: Identification of archaeological eggshell ................................ 39 1.1.4.3: Other applications of archaeological eggshell ........................ 41 1.1.4.4: Eggs in the archaeological record: future applications ........... 42 3 1.1.5: Conclusions on this section ............................................................ 43 Section 2: Eggshell Structure ........................................................................ 44 1.2.1: Overview: the amniotic egg............................................................ 44 1.2.2: Eggshell structure ........................................................................... 44 1.2.2.1: Basic structure ........................................................................ 44 1.2.2.2: Shell membrane ...................................................................... 45 1.2.2.3: Mammillary layer .................................................................... 46 1.2.2.4: Palisade layer, vertical crystal layer & cuticle ......................... 47 1.2.3: Conclusions on section 2 ................................................................ 48 Section 3: Biomineralisation & Eggshell Deposition ..................................... 49 1.3.1: Overview ......................................................................................... 49 1.3.2: Initial stages of crystallisation ........................................................ 51 1.3.2.1: The ACC phase ........................................................................ 51 1.3.2.2: Metastable vs. transiently stable ACC: roles of organic molecules and hydration ................................................................................... 52 1.3.3: Crystal nucleation ........................................................................... 53 1.3.3.1: Nucleation theory ................................................................... 53 1.3.3.2: Intra-crystalline proteins ......................................................... 54 1.3.4: Mineral deposition in eggshell ....................................................... 55 1.3.4.1: Overview ................................................................................. 55 1.3.4.2: Calcite deposition: roles of the organic fraction ..................... 56 4 1.3.4.3: Differences between eggshell and other mineralising systems ........................................................................................................................... 57 1.3.5: Conclusions on biomineralisation and eggshell deposition ........... 59 Section 4: Eggshell Proteins: Structure, Function & Diversity ...................... 60 1.4.1: Overview ......................................................................................... 60 1.4.2: Diversity of chicken eggshell proteins ............................................ 60 1.4.3: Eggshell proteins ............................................................................ 61 1.4.3.1: Proteins expressed in diverse bodily tissues .......................... 61 1.4.3.2: Egg-white and eggshell proteins ............................................. 62 1.4.3.3: Eggshell-specific proteins ........................................................ 63 1.4.3.4.1: C-type lectin proteins ........................................................... 65 1.4.3.4.2: Sequence homology and inter-relationships of CTL proteins ........................................................................................................................... 66 1.4.3.4.3: Roles of CTL proteins in eggshell deposition ....................... 68 1.4.3.5: Effects of sugars on protein form and function ...................... 69 1.4.4: Effects of eggshell proteins on calcite crystal morphology ............ 70 1.4.5: Conclusions ..................................................................................... 71 1.4.5.1: Conclusions on section 4 ......................................................... 71 1.4.5.2: Conclusions on chapter 1 ........................................................ 72 Chapter 2: Early experimental work .................................................................. 73 Section 1: Isolating an intra-crystalline protein fraction in eggshell ............ 73 2.1.1: Overview ......................................................................................... 73 5 2.1.1.1: Introduction ............................................................................ 73 2.1.1.2: Closed biomineral systems: ‘intra-crystalline’ proteins .......... 73 2.1.1.3: The importance of analysing a closed system ........................ 75 2.1.2: Methods ......................................................................................... 76 2.1.2.1: Overview ................................................................................. 76 2.1.2.2: Sample preparation: bleaching test ........................................ 77 2.1.2.3: Sample preparation: leaching test .......................................... 77 2.1.2.4: Demineralisation & rehydration ............................................. 78 2.1.2.5: Analysis by reverse-phase HPLC ............................................. 80 2.1.3: Results ............................................................................................ 83 2.1.3.1: THAA concentration ................................................................ 83 2.1.3.2: FAA concentration .................................................................. 85 2.1.3.3: THAA composition .................................................................. 85