The organic phase in dinosaur eggshell: analysis, models of preservation, and implications for the evolution of bird reproduction

长期以来，人们一直认为，鸡蛋和蛋壳除了形状外，还保留了它们的组织结构，这就产生了蛋壳古组织学领域。蛋壳是一种生物复合材料，就像骨头一样，但它的矿化程度要高得多。此外，蛋壳在保存内源生物分子方面比骨骼更有前景，因为它在石化过程中受溶解-再矿化过程的影响较小。这种稳定的结晶基质含有少量的有机成分，有机相(OP)，它在胚胎发育、蛋壳强化和鸡蛋着色中发挥作用。众所周知，生物矿物和有机成分的复合系统形成了当地的化学环境，使其有机成分在地质年代中几乎不受影响地得到保存。申请者在恐龙蛋化石中的蛋壳颜色色素上测试了这一假设。基于大量的蛋壳化石收藏和斯坦曼研究所卓有成效的中国合作，我们首次成功地检测到了大量的化石鸡蛋色素。这些发现的关键是从生物基质中提取天然产物和代谢物(Christa Müler教授的实验室)和有机化学高端分析技术(Marianne Engeser博士的实验室)领域的共享专业知识，例如高效液相色谱(HPLC)ESI Q-TOF质谱仪。具体地说，我们在蛋壳代谢物胆绿素和原卟啉中检测到了卵突形目恐龙和元古龙的卵。这些化合物使鸟蛋变色，用于检测寄生虫和隐蔽禽蛋。由于这种色素的子宫从头合成及其进入蛋壳的过程被认为仅限于现存的鸟类，因此这项研究具有重要的进化意义，包括感觉和行为生态学的方面。因此，拟议项目的总体目标是了解恐龙和中生代鸟类蛋壳的物质性质。重点将放在有机相上(与矿物相相反，有机相在很大程度上仍未被研究)，探索在蛋壳中保存生物分子的范围。从理论上讲，蛋壳为生物分子的保存提供了一个理想的基质，我们的初步工作证实了这一点。此外，蛋壳色素是环状或线状四吡咯，形成一种稳定的分子类别。我们不仅打算检测、定位和鉴定保存下来的色素的降解产物，而且有可能保存构成蛋壳中鳞状支架的蛋白质化合物。我们的研究是在鸟类繁殖性状进化的背景下进行的，对化石鸡蛋记录的精细理解对此至关重要。

It has long been recognized that in addition to their shape, eggs and eggshells also preserve their histological structure, giving rise to the field of eggshell paleohistology. Eggshell is a biocomposite material just like bone, but it is mineralized to a much higher degree. In addition, eggshell represents a more promising matrix for the preservation of endogenous biomolecules than bone because it is less affected by dissolution-remineralization processes during fossilization. This stable, crystalline matrix contains minor amounts of organic constituents, the organic phase (OP), which plays a role in embryo development, strengthening of the eggshell, and egg coloration. Composite systems of biominerals and organic constituents are known to form a local chemical environment that allows the almost unmodified preservation of its organic constituents over geological time. The applicants tested this assumption on eggshell color pigments in fossilized dinosaur eggs. Based on the extensive fossil eggshell collections and fruitful Chinese collaborations of the Steinmann Institute, we detected fossil egg pigments successfully and in significant amounts for the first time. Key to these discoveries were the shared expertise from the fields of natural product and metabolite extraction from biological matrices (lab of Prof. Christa Müller) and organochemical high-end analytical techniques (lab of Dr. Marianne Engeser), e.g., liquid chromatography (HPLC) ESI Q-TOF mass spectrometry. Specifically, we detected the eggshell metabolites biliverdin and protoporphyrin in eggs of the oviraptorid dinosaur Heyuanni huangi. These compounds color bird eggs, serving in detection of parasitism and in egg crypsis. Because the uterine de novo synthesis of such pigments and their incorporation into eggshell was believed to be restricted to extant birds, this research has important evolutionary implications, including aspects of sensory and behavioral ecology. The general goal of the proposed project is thus to understand the material nature of dinosaur and Mesozoic bird eggshell. The focus will be on the organic phase (which remains largely unstudied as opposed to the mineral phase), exploring the range of biomolecular preservation in eggshell. From a theoretical point of view, eggshell offers an ideal matrix for biomolecular preservation, corroborated by our preliminary work. In addition, the eggshell pigments are cyclic or linear tetrapyrroles that form a stable class of molecules. We intend not only to detect, locate, and identify degradation products of preserved pigments, but there is potential for the preservation of the protein compounds that build up the squamatic scaffold in eggshell. Our research is set against the background of the evolution of avian reproductive traits for which a refined understanding of the fossil egg record is crucial.