The three-dimensional(3D) morphology, anatomy, and in-situ chemical composition analysis of fossils are crucial for systematic paleontology and determining their phylogenetic positions.Scanning electron microscopy(SEM) coupled with energy-dispersive X-ray spectroscopy(EDS), offers valuable structural and chemical information for the analysis of fossils. However, its primary limitation is the restriction to two-dimensional surface data, which limits the exploration of fossils' 3D complexities. Conversely, 3D X-ray microscopy(3D-XRM), also known as a novel form of micro-computed tomography(micro-CT) facilitates the non-destructive 3D reconstruction of fossil specimens. Nevertheless, it lacks the capability to provide in-situ compositional data. Acknowledging the constraints inherent in these individual techniques, and in response to the evolving requirements of paleontological research, this study introduces an integrated approach that combines 3D-XRM with EDS-coupled focused ion beam scanning electron microscopy(FIB-SEM). This innovative strategy is designed to synergize the advantages of both techniques, thereby addressing challenges that conventional methods cannot. It enables the rapid identification of regions of interest(ROI) within fossil specimens at micrometer resolution. Subsequently, this method collects detailed data on both 3D structures and chemical compositions at the nanometer scale for the identified ROI. This integrated approach represents a significant advancement in paleontological and geological research methodologies, promising to meet the increasing demands of these fields.

The early Ediacaran Weng'an biota (Doushantuo Formation, South China) provides a rare window onto the period of Earth history in which molecular timescales have inferred the initial phase of crown-metazoan diversification. Interpretation of the embryo-like fossils that dominate the biota remains contentious because they are morphologically simple and so difficult to constrain phylogenetically. Spiralicellula from the Weng'an biota is distinguished by spiral internal bodies, allied through development to Megasphaera or Helicoforamina and interpreted variously as metazoan embryos, encysting protists, or chlorophycean green algae. Here we show, using X-ray microtomography, that Spiralicellula has a single-layered outer envelope and no more than 32 internal cells, often preserving a nucleus and yolk granules. There is no correlation between the extent of spiral development and the number of component cells; rather, the spiral developed with each palintomic stage, associated with cell disaggregation and reorientation. Evidence for envelope thinning and cell loss was observed in all developmental stages, reflecting non-deterministic shedding of gametes or amoebae. The developmental biology of Spiralicellula is similar to Megasphaera and Helicoforamina , which otherwise exhibit more rounds of palintomy. We reject a crown-metazoan affinity for Spiralicellula and all other components of the Weng'an biota, diminishing the probability of crown-metazoan diversification before the early Ediacaran.

The Weng'an Biota from the Ediacaran Doushantuo Formation in Guizhou Province, southwestern China, is known for its three-dimensionally phosphatized acritarchs, multicellular algae, and embryo-like animal fossils. Among these diverse microfossils, acanthomorphic acritarchs have played a significant role in the biostratigraphic subdivision and correlation of the lower-middle Ediacaran System. However, most previous studies on the biostratigraphy of the Doushantuo Formation in the Weng'an area have focused on large acanthomorphic acritarchs (LAAs, vesicle diameter >200 mu m), whereas the smaller acanthomorphic acritarchs (SAAs, vesicle diameter <100 mu m) from the Weng'an Biota have been largely overlooked. In this study, we examined >500 thin sections and discovered a large number of well-preserved, small (<100 mu m) and medium-sized acanthomorphic acritarchs (MAAs, vesicle diameter ranging 100-200 mu m). In total, we have identified SAAs in four genera and six species (Tanarium conoideum Kolosova, 1991, emend. Moczyd & lstrok;owska et al., 1993; Tanarium elegans Liu et al., 2014; Mengeosphaera membranifera Shang, Liu, and Moczyd & lstrok;owska, 2019; Mengeosphaera minima Liu et al., 2014; Estrella recta Liu and Moczyd & lstrok;owska, 2019; Variomargosphaeridium gracile Xiao et al., 2014), as well as two types of MAAs (Tanarium tuberosum Moczyd & lstrok;owska, Vidal, and Rudavskaya, 1993, emend. Moczyd & lstrok;owska, 2015; Weissiella cf. W. grandistella Vorob'eva, Sergeev, and Knoll, 2009, emend. Liu and Moczyd & lstrok;owska, 2019). This updated acritarch assemblage of the Weng'an Biota is valuable for correlating the Ediacaran Doushantuo Formation between the Weng'an and Yangtze Gorges areas. It also serves as a tool to test the proposed acritarch biozones in Ediacaran formations of South China and other localities, including Australia, Siberia, and the East European Platform.

Embryo-like fossils from the early Ediacaran Weng'an Biota (SW China, 609 myr ago), widely interpreted as holozoans, potentially provide insights into the early evolutionary development of metazoans and the rise of the animal kingdom. However, the biodiversity of the embryo-like fossil assemblage is largely underestimated and its more precise phylogenetic affinities within the holozoan tree are still under debate. We describe a new species of embryo-like fossil, Ostiosphaera rara n. gen. n. sp., from the Ediacaran Weng'an Biota. These 3D, phosphatized specimens exhibit a spherical morphology, a thick ornamented envelope with a circular opening and a membrane-bound, multicellular inner body. In terms of biological characteristics, O. rara shows similarities with a number of extant and fossil analogues, including testate amoebae, unicellular green algae, the cellular slime mould Fonticuida and co-occurring Weng'an embryo-like fossils. Although the phylogenetic affinity of O. rara is difficult to constrain precisely based on the available evidence, it is reasonable to follow the holozoan interpretation for these fossils because they share the same grade complexity with co-occurring embryo-like fossils, such as Megasphaera and Helicoforamina , in terms of the combination of biological features. These new holozoans resemble asexual reproductive gemmules of fossil and living demosponges in size, morphology, circular opening and cellular anatomy. If this similarity reflects biological affinity rather than convergent evolution, this discovery would force us to rethink the evolutionary history of Precambrian sponges. Thematic collection: This article is part of the Advances in the Cambrian Explosion collection available at: https://www.lyellcollection.org/cc/advances-cambrian-explosion

The origin and early evolution of animal development remain among the many deep, unresolved problems in evolutionary biology. As a compelling case for the existence of pre-Cambrian animals, the Ediacaran embryo-like fossils (EELFs) from the Weng'an Biota (approx. 609 Myr old, Doushantuo Formation, South China) have great potential to cast light on the origin and early evolution of animal development. However, their biological implications can be fully realized only when their phylogenetic positions are correctly established, and unfortunately, this is the key problem under debate. As a significant feature of developmental biology, the cell division pattern (CDP) characterized by the dynamic spatial arrangement of cells and associated developmental mechanisms is critical to reassess these hypotheses and evaluate the diversity of the EELFs; however, their phylogenetic implications have not been fully realized. Additionally, the scarcity of fossil specimens representing late developmental stages with cell differentiation accounts for much of this debate too. Here, we reconstructed a large number of EELFs using submicron resolution X-ray tomographic microscopy and focused on the CDPs and associated developmental mechanisms as well as features of cell differentiation. Four types of CDPs and specimens with cell differentiation were identified. Contrary to the prevailing view, our results together with recent studies suggest that the diversity and complexity of developmental mechanisms documented by the EELFs are much higher than is often claimed. The diverse CDPs and associated development features including palintomic cleavage, maternal nutrition, asymmetric cell divisions, symmetry breaking, establishment of polarity or axis, spatial cell migration and differentiation constrain some, if not all, EELFs as total-group metazoans. This article is part of the theme issue ‘The impact of Chinese palaeontology on evolutionary research’.

Embryo-like fossils from the early Ediacaran Weng’an biota provide a window of exceptional fossil preservation onto the period of life history in which molecular clocks estimate the fundamental animal lineages to have diverged. However, their diversity and biological affinities have proven controversial, because they are morphologically simple and, consequently, their interpretation lacks phylogenetic constraint. The subcellular structures preserved in these embryo-like fossils might help to understand their cytology, biology, and diversity, but the potential of these structures has not been fully realized, because detailed microscale physical and chemical investigations are lacking. Here, to remedy this deficiency, we performed a comprehensive study to characterize their micro- and ultra-structures as well as in-situ chemical components. Our results reveal three types of subcellular structure that differ in size, shape, and mineral components: (1) relatively small and spheroidal granules in embryo-like fossils with equal cell division pattern; (2) relatively large, spheroidal, or polygonal granules in embryo-like fossils with unequal and asynchronous cell division pattern; and (3) irregular multi-layered rim-bounded granules in embryo-like fossils with unequal and asynchronous cell division pattern. We propose that the three types may be rationalized to a single taphonomic pathway of preferential mineralization of the cell cytoplasm, preserving an external mould of subcellular granules. We followed the previous interpretation that the spheroidal and polygonal granules should be fossilized lipid droplets or yolk platelets. The distinction between these subcellular structures are largely the result of postmortem degradation processes such as autolysis. The widely preserved lipid droplets or yolk platelets within these Ediacaran embryo-like fossils are compatible with the interpretion of large yolky embryos with maternal nourishment and direct development.

The challenge of identifying fossilized organelles has long hampered attempts to interpret the fossil record of early eukaryote evolution. We explore this challenge through experimental taphonomy of nuclei in a living eukaryote and microscale physical and chemical characterization of putative nuclei in embryo-like fossils from the early Ediacaran Weng'an Biota. The fossil nuclei exhibit diverse preservational modes that differ in shape, presence or absence of an inner body and the chemistry of the associated mineralization. The nuclei are not directly fossilized; rather, they manifest as external moulds. Experimental taphonomy of epidermal cells from the common onion (Allium cepa) demonstrates that nuclei are more decay resistant than their host cells, generally maintaining their physical dimensions for weeks to months post-mortem, though under some experimental conditions they exhibit shrinkage and/or become shrouded in microbial biofilms. The fossil and experimental evidence may be rationalized in a single taphonomic pathway of selective mineralization of the cell cytoplasm, preserving an external mould of the nucleus that is itself resistant to both decay and mineral replication. Combined, our results provide both a secure identification of the Weng'an nuclei as well as the potential of a fossil record of organelles that might help arbitrate in long-standing debates over the relative and absolute timing of the evolutionary assembly of eukaryote-grade cells.

The exceptional fossil preservation of the early Ediacaran Weng'an biota provides a unique window on the interval of Earth history in which animal lineages emerged. It preserves a diversity of similarly ornamented encysted developmental stages previously interpreted as different developmental stages of one taxon. Although Helicoforamina wenganica is distinguished from other forms by a helical groove or canal, it has been interpreted as a developmental stage of cooccurring metazoan, nonmetazoan holozoan, or green algal taxa. Using x-ray microtomography, we show that Helicoforamina developed through one-, four-, and eight-cell stages, to hundreds and thousands of cells. Putative hatchlings are artifacts of incompletely preserved cyst walls. Our results preclude inclusion of Helicoforamina into life cycles assembled from other components of the Weng'an biota but support a holozoan affinity. The similarly ornamented encysted forms shared among the diverse Weng'an biota represent parallel adaptations to the temporally and spatially heterogeneous Ediacaran shallow marine environments.

The early Ediacaran Weng' an Biota (ca. 609 Ma) of the Doushantuo Formation (Guizhou Province, China) encompasses an abundant and exquisitely preserved assemblage of phosphatic microfossils that have provided unique insight into the origin and early evolution of multicellular eukaryotes. However, the affinities of these early organisms are far from certain, including the tubular microfossils Crassitubulus, Quadratitubus, Ramitubulus, and Sinocyclocylcicus. These taxa have been widely accepted as stem-cnidarians or, alternatively, interpreted as filamentous cyanobacteria, or multicellular algae. We use high-resolution X-ray tomographic microscopy to analyse the structure and development of the four taxa. Our data and analysis allow us to conclude that these four taxa were not biomineralized. Crassitubulus, Quadratitubus, and Sinocyclocylcicus, may be grouped on the basis that they exhibit alternating complete and incomplete cross walls, and bipolar growth; which makes them favourably comparable to filamentous cyanobacteria. In contrast, Ramitubulus exhibits only complete cross walls, unipolar growth and dichotomous branching. These features are difficult to reconcile with a cyanobacterial interpretation. They are, instead, more indicative of multicellular algae-like Cambrian Epiphyton. Thus, the Weng'an tubular microfossils constitute a disparate assemblage of cyanobacteria and algae, but none represents early Ediacaran animals. (C) 2019 Elsevier Ireland Ltd Elsevier B.V. and Nanjing Institute of Geology and Palaeontology, CAS. Published by Elsevier B.V. All rights reserved.

The Ediacaran Weng’an Biota provides a unique window on marine diversity during the interval in which the fundamental animal body plans were being established. Here we describe a previously unreported component of the assemblage, millimeter-scale encysted spheres that exhibit a characteristic but simple slit-shaped excystment mechanism (Sporosphaera guizhouensis n. gen. n. sp.), reminiscent of acritarchs. The cysts contain a large inner body or numerous small discrete membrane-bounded bodies. It is possible that the inner bodies represent disaggregated cells of a multicellular body, like an embryo, but there is no evidence to support this interpretation and the occurrence of the excystment structure is not readily compatible with an embryo interpretation. Rather, we interpret the encysted organisms as multicellular stages within the lifecycle of otherwise probably unicellular eukaryotes. The developmental mode exhibited by Sporosphaera, incorporating a resting stage, implies an adaptation to adverse environmental conditions. This parallels the appearance of Large Ornamented Ediacaran Microfossils (LOEMs) which have been interpreted as diapause stages in the embryology of early animals. Sporosphaera is distinct from LOEMs by ornamentation instead of size, which may implicate that not all LOEMs are animal embryos, if any.

在古生物学研究中,以X射线断层成像(Computed Tomography)为代表的三维无损成像技术可以在不破坏化石标本的前提下,同时获得标本外观形态和内部结构的信息,相比传统的可见光成像手段有着明显优势.为推动化石三维无损成像技术在国内古生物学领域的发展,本文系统介绍一种新型显微CT技术——三维X射线显微术(Three-Dimensional X-ray Microscopy).与基于几何放大和吸收衬度成像的传统显微CT技术相比,该技术有若干优势:(1)将同步辐射X射线显微断层成像的光学成像系统引入基于实验室X射线源的显微CT系统中,在几何放大的基础上增加了光学放大,优化了传统显微CT的系统架构,弥补了传统显微CT单纯依靠几何放大的不足,提高了空间分辨率;(2)采用可移动的X射线源和优化的光学成像系统,实现了低能X射线相位衬度成像,可以三维重构传统显微CT技术无法有效探测的、低吸收衬度的化石标本;(3)基于新的成像架构和成像算法,实现了厘米-分米级较大标本内部“感兴趣区域”(Region of Interest)精确导航和局部高分辨率(微米-亚微米空间分辨)成像;(4)可以实现小型扁平标本(宽厚比＞4,宽＜10 cm)高效率、高分辨率成像和长条形微体标本长轴方向自动分段无缝拼接的微米至亚微米级高分辨率重建,弥补了传统工业显微CT针对小型扁平标本和长条形微体标本高分辨成像效果不佳的缺陷.这些优势使得基于实验室X射线源的显微CT成像技术可以获得接近同步辐射X射线源的成像质量,从而有效推动化石生物学研究.