Precambrian animal diversity: Putative phosphatized embryos from the Doushantuo Formation of China Jun-Yuan Chen*†‡, Paola Oliveri†§, Chia-Wei Li¶, Gui-Qing Zhou*, Feng Gao*, James W. Hagadornʈ, Kevin J. Peterson§, and Eric H. Davidson‡§ *Nanjing Institute of Geology and Paleontology, Nanjing 210008, China; Divisions of §Biology and ʈGeological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125; and ¶Department of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan, China Contributed by Eric H. Davidson, January 28, 2000 Putative fossil embryos and larvae from the Precambrian phosphorite The terminal Proterozoic follows the most recent phase of the rocks of the Doushantuo Formation in Southwest China have been worldwide Varanger glaciation, and it extends to the Precambrian͞ examined in thin section by bright field and polarized light micros- Cambrian boundary at 543 Ma (5, 30). In Hubei and Guizhou copy. Although we cannot completely exclude a nonbiological or provinces of China, the latest Varanger glaciation event is repre- nonmetazoan origin, we identified what appear to be modern cni- sented by the Nantuo tillites (31). The tillites are believed to have darian developmental stages, including both anthozoan planula lar- an age of 610–590 Ma (32), and a U-Pb radiometric date suggests vae and hydrozoan embryos. Most importantly, the sections contain a greater age for the underlying formation (33). The Doushantuo a variety of small (<200 ␮m) structures that greatly resemble gastrula Fm lies immediately above the Nantuo Fm, representing transgres- stage embryos of modern bilaterian forms. sive deposits that occurred as the result of a rise in sea level because of the melting of continental glaciers. The time gap between the end he prediction that stem-group and perhaps even crown-group of the glaciation and the beginning of transgressive deposition is of Tbilaterians existed in Neoproterozoic times, well before the unknown length, except that it is certainly younger than the 610- to Precambrian boundary, is supported by many kinds of argument. 590-Ma-old Nantuo tillites. The age of the Doushantuo Fm could be as old as 580 Ma (26), and pending direct measurement, its age The most general and important of these arguments is that almost Ϯ all major bilaterian clades already are represented in the Lower must fall within the range 570 20 Ma (27) [Saylor et al. (34) Cambrian, in remarkably preserved deposits such as the Chengjiang argue that it is toward the younger end of this range]. It is fossil lagersta¨tte(1). Fossil remains of diverse bilaterian forms from important to stress, however, that whatever the absolute time horizon represented by the Doushantuo Fm, it is likely to precede the Lower Cambrian have been obtained from many other regions the lowest strata with which bilaterian remains have so far been of the globe as well (2–4). The latest Precambrian also has yielded associated (20). trace fossils of unmistakable bilaterian origin (5–7). These remains The Doushantuo Fm is a marine deposit containing phosphate- indicate that major evolutionary diversification of animals already dolomite sequences. In Beidoushan, in the Weng’an district of had occurred by the onset of the Cambrian, and, therefore, more EVOLUTION central Guizhou Province (Fig. 1), the phosphate deposit is divided remote ancestral forms must have been alive earlier. Cladistic by an erosive surface into two units. The fossils described here are analyses, both morphological and molecular, clearly indicate that from the base of the upper phosphate unit, 0.2–6 m in thickness. the bilaterians are monophyletic, i.e., they derive from a latest This high-resolution fossil bed is about 30% phosphate, present as common ancestor that gave rise only to bilaterians (see ref. 8). How the mineral fluorapatite [Ca5(PO4)3F]. Phosphatic beds within this deep in time were this common ancestor and its offshoots, the deposit are grainstones composed of 1- to 5-mm phosphoclasts. common ancestors of the three great bilaterian clades (ecdysozoans, These derive from a phosphatic surface that formed on the sea lophotrochozoans, deuterostomes; refs. 8–10) is unknown. Molec- floor, in the process recrystallizing existing surface sediments. In ular phylogenies based on sequence comparisons of protein do- addition to replacing carbonate sediments, soft tissues of metazoan SPECIAL FEATURE mains shared by all bilaterians suggest that the time of divergence embryos, larvae, adults, and algae also appear to have been of these proteins may extend back to 600–1,200 million years mineralized (26–29). The phosphatized sediment crust was then (megaannuum; Ma) ago (11–16). In addition, arguments based on broken into small fragments by heavy current activity and then regulatory evolution lead to the proposition that microfaunal redeposited and mixed in with adjacent lime muds. For current bilaterian stem groups must have evolved through several stages of discussion of this fossilization process, see ref. 28. regulatory complexity during Precambrian time (17, 18). But thus far, unequivocal paleontological evidence of bilaterian forms has Methods extended only to the final period of the Neoproterozoic, a few The samples included in the present study were collected from million years before the Cambrian ‘‘explosion’’ (5–7, 19, 20). the Wusi and Baidoushan quarries of the Weng’an phosphate The Lower Cambrian strata of Southwest China overlie phos- mine. Thin sections were prepared by grinding and were phate deposits known as the Meishucun Formation (Fm), which mounted on standard microscope slides for examination. Section contains small shelly fossils mainly composed of mineralized spines thickness was 30–50 ␮m, allowing for some three-dimensional and plates of various types of animal (21, 22). The Late Precam- visualization at different planes of focus. About 2,000 sections brian is represented by a thick sequence of carbonate deposits, the have been examined, and over 4,000 digital images of embryos Dengying Fm. The top of the Dengying Fm contains the tubular and larvae have been recorded. These images were obtained by fossil Cloudina (19). Below these fossils are frondose Ediacaran transmitted light microscopy at ϫ100 or ϫ200 magnification, remains (23), the affinities of which are enigmatic (24, 25). What and many samples also were photographed. Selected samples has been missing is evidence of animals belonging to the bilaterian lineage deeper in time, i.e., from the Lower Dengying, or from Abbreviations: Ma, Megaannuum (i.e., million years); Fm, Formation. earlier deposits anywhere in the world. One such deposit is the † phosphatic Doushantuo Fm, which underlies the Dengying Fm in J.-Y.C. and P.O. contributed equally to this work. ‡ Southwest China. We report here a preliminary exploration of To whom reprint requests should be addressed at: Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125. E-mail: [email protected]; or embryonic and larval animal forms represented in samples of Nanjing Institute of Geology and Paleontology, Nanjing 21008, China. E-mail: Doushantuo phosphorites from one particular location. [email protected]. PNAS ͉ April 25, 2000 ͉ vol. 97 ͉ no. 9 ͉ 4457–4462 Downloaded by guest on September 28, 2021 were examined as well in a polarization microscope using cross-polarized light with a gypsum plate. Apparent Fossil Embryos and Their Affinities with Modern Forms. Each of the forms we report in the following was encountered multiple times. The sections are heterogeneous: some are rich with apparent fossil embryos and͞or microscopic sponges in various stages of development, whereas others are devoid of any specimens of interest. The object of this paper is to report the presence of diverse microscopic structures, which, at high resolution, resemble several modern forms of cnidarian larvae (Fig. 2); and most importantly, gastrula stage embryos that appear bilaterian in their morphological details (Fig. 3). We are aware that microscopic mineral inclusions of nonbiological origin can easily resemble biological forms, including embryos, and we cannot rigidly exclude the possibility that they are not fossils at all. However, we consider two kinds of evidence that point instead to a biogenic origin for these embryo-like structures: first and most important, the remark- able consistency of their morphology and dimensions as observed in multiple independent observations of each of the types that we describe; and second, their appearance under polarized light, which allows direct visualization of the crystal structure. Each individual crystal element, defined by the orientation of its axes with respect to the plane of polarization, is displayed in the polarization micro- scope images by a different color. As we summarize in Discussion, the results of the polarized and bright-field microscopy are consis- tent with the argument that the objects described in Figs. 2 and 3 are indeed fossilized embryos, which originally were composed of epithelial layers of cells surrounding internal cellular structures that arose by invagination. Possible Cnidarian Gastrulae. Fig. 2A shows a form strikingly similar to an early anthozoan planula. The key characteristics, as illustrated in the drawing in Fig. 2B, are its elongate shape, in the fossil about 320 ϫ 220 ␮m; its single cell-thick ectodermal wall, which can be seen clearly in the portion of the fossil at 9 o’clock, and the multipolar endodermal ingressions protruding