Mussentuchit member fossil eggshell light microscope photographs and corresponding metrics

The first fossil eggshell from the Cenomanian-age Mussentuchit Member of the Cedar Mountain Formation was described over fifty years ago. In the half-century since, oodiversity of this rock unit has been limited to a single, taxonomically unstable ootaxon, currently formulated as Macroelongatoolithus carlylei. Recently, there has been a renewed effort to recover and describe the macrofauna of the Mussentuchit; however, these advances are limited to the body fossil record. Here, we examine the range of eggshells present in the Mussentuchit Member and assess the preserved biodiversity they represent. Gross morphological and microstructural inspection reveals a greater diversity of eggshells than previously described. We identify six ootaxa: three Elongatoolithidae oogenera (Macroelongatoolithus, Undulatoolithus, Continuoolithus), eggs laid by oviraptorosaur dinosaurs; two oospecies of Spheroolithus laid by ornithopod dinosaurs; and Mycomorphoolithus kohringi, laid by a crocodylomorph. The diversity of Elongatoolithidae in the Mussentuchit requires a co-occurrence of at least three putative oviraptorosaurs, the oldest such phenomenon in North America. The occurrence of the crocodylomorph oogenus Mycomorphoolithus is the first recognized occurrence outside of Europe, and the youngest yet documented. This new ooassemblage is more representative of the known paleobiodiversity of Cenomanian-age strata of Western North America and complements the body fossil record in improving our understanding of this crucial–yet poorly documented–timeslice within the broader evolution of the Cretaceous Western Interior Basin.

Methods
In this study, we examined all fossilized eggshells recovered from the Mussentuchit Member of the Cedar Mountain Formation and housed at the NCSM (n > 2,500), plus additional loaned material from the OMNH (n > 1,500), FMNH (n = 77), and BYU (n = 8), to extensively analyze ootaxonomic diversity across twenty localities.  NCSM and FMNH fragments from the Mussentuchit Member were collected between 2012 and 2022, and fragments and thin section slides loaned from BYU and the OMNH were collected by Jensen and Cifelli et al., respectively.

We systematically categorized fragments of eggshell into ‘morphotypes’ based on external ornamentation patterns, and selected the highest quality samples (free of additional matrix, appropriately sized) for photography and consumptive sampling. Fragments selected were photographed with a Keyence VHX-7000 Digital Microscope before creating radial thin sections to assess microstructural features such as crystal arrangement, pore networks, the ML/CL boundary, and ML/CL thickness ratios. We encased fragments in epoxy resin, cut the resulting blocks using a Buehler IsoMet 1000 Precision Saw, and polished them using a Buehler MetaServ 250 Grinder/Polisher to a thickness of 80 μm. We analyzed finalized thin sections with a Nikon Eclipse Ci Pol light microscope (with attached DS-FI 2 camera) and the Keyence VHX-7000 Digital Microscope for microstructure. Measurements of TST, ML and CL features were taken digitally using ImageJ. Further analysis of microstructure and ultrastructure, with a focus on crystal splaying across the ML/CL boundary, was undertaken on a Hitachi SU8700 field-emission scanning electron microscope at the NCSU Analytical Instrumentation Facility. We viewed samples at 100x magnification at variable pressure, with a 50 Pa backfill of dry nitrogen and a 20 kV accelerating voltage. These combined analyses were used to determine ootaxonomic assignment.

Photographs included herein are from the microscope cameras and were not included in the manuscript. The images were used in correpsondence with ImageJ to collect quantitative and qualitative eggshell data (the csv file).

首个发现于锡达山组穆森特奇特段森诺曼阶地层的化石蛋壳，已于五十余年前被记述。在此后的半个世纪中，该地层单元的卵化石多样性（oodiversity）仅局限于单个分类学上不稳定的卵分类单元（ootaxon），当前被归为卡莱尔长形蛋（Macroelongatoolithus carlylei）。近期，学界对穆森特奇特段的大型动物群开展了新一轮的发掘与记述工作，但相关进展仅局限于实体化石记录范畴。本研究针对穆森特奇特段产出的各类蛋壳化石展开分析，并评估其所代表的保藏生物多样性。通过宏观形态与微观结构观察，本研究发现该地层的蛋壳化石多样性远超此前的记述结果。本次研究共鉴定出6个卵分类单元：3个长形蛋科（Elongatoolithidae）卵属，分别为长形蛋属（Macroelongatoolithus）、波纹蛋属（Undulatoolithus）、连续蛋属（Continuoolithus），均为窃蛋龙类恐龙所产；2个球形蛋属（Spheroolithus）卵种，由鸟脚类恐龙产下；以及科林吉微形蛋（Mycomorphoolithus kohringi），其产卵者为鳄形类动物。穆森特奇特段产出的长形蛋科化石多样性表明，该区域至少共存有3种疑似窃蛋龙类物种，这是北美地区目前已知最古老的同类共存现象。鳄形类卵属微形蛋属（Mycomorphoolithus）的此次发现，是该属在欧洲以外地区的首次确认记录，同时也是目前已知最年轻的该属化石产出记录。本次发现的全新蛋化石组合，更能代表北美西部森诺曼阶地层的已知古生物多样性特征；其补充了实体化石记录，有助于我们更好地理解白垩纪西部内陆盆地演化过程中这一关键却鲜有记录的时间切片。

Methods
本研究对所有采自锡达山组穆森特奇特段、馆藏于北卡罗来纳州自然科学博物馆（NCSM，标本量>2500件）的化石蛋壳，以及额外借自俄克拉荷马自然历史博物馆（OMNH，>1500件）、菲尔德自然历史博物馆（FMNH，77件）与杨百翰大学（BYU，8件）的馆藏标本展开分析，以全面探究20个化石产地的卵分类单元多样性。穆森特奇特段的NCSM与FMNH馆藏蛋壳碎片采集于2012至2022年间，BYU与OMNH借出的蛋壳碎片及薄片标本则分别由Jensen以及Cifelli等人采集。

研究团队根据蛋壳的外部纹饰模式，将蛋壳碎片系统划分为不同的‘形态型’，并选取质量最优（无多余围岩、尺寸合适）的标本用于摄影与损耗性采样。选取的蛋壳碎片首先使用基恩士（Keyence）VHX-7000数码显微镜进行成像，随后制作径向薄片以分析其微观结构特征，包括晶体排列、孔隙网络、乳突层（ML）-柱状层（CL）边界以及二者厚度比。研究人员将蛋壳碎片包埋于环氧树脂中，使用标乐（Buehler）IsoMet 1000精密锯将包埋块切割成片，再通过标乐MetaServ 250磨抛机将其抛光至80μm厚度。我们使用搭载DS-FI2相机的尼康（Nikon）Eclipse Ci偏光显微镜，以及基恩士VHX-7000数码显微镜对制备完成的薄片进行微观结构分析。利用ImageJ软件数字化测量蛋壳总厚度（TST）、乳突层与柱状层的相关参数。针对微观结构与超微结构的进一步分析（重点关注ML/CL边界处的晶体张开情况），在北卡州立大学分析仪器中心的日立（Hitachi）SU8700场发射扫描电子显微镜上完成。扫描成像时采用100倍放大倍率、可变压力模式，腔室填充50Pa干燥氮气，加速电压为20kV。上述综合分析结果用于确定蛋壳化石的卵分类单元归属。

本文所附照片均来自显微镜成像系统，未纳入正文中。这些图像配合ImageJ软件用于采集蛋壳化石的定量与定性数据（相关CSV文件已附）。