Fossil localities and geological setting

The Proterozoic succession in the Yanshan region (Fig. 1) consists of essentially undeformed sedimentary rocks about 10 km thick, dated by means of zircon U-Pb ages from tuffs, flows, sills and dykes21,22. Macrofossils were discovered at two localities in Qianxi and Kuancheng counties (Figs 1c and 2) within calcareous shales of the Gaoyuzhuang Formation22. The fossiliferous mudstones are rich in organic carbon (total organic carbon of 0.7–4.3%; mean=1.2%; see (ref. 23) and are thought to represent distal turbidites or tempestites deposited near storm wave base. The fossiliferous shales lie below an ash bed dated by U-Pb zircon analysis at 1,560±5 Myr and postdate 1,625±6.2 Myr flows in the unconformably underlying Dahangyu Formation24,25 (Figs 1d and 2).

Figure 1: Locality maps and stratigraphy. (a) Location of the North China, Yangtze and Tarim cratons. (b) Proterozoic outcrops within the North China craton—black box marks the Yanshan area. (c) Regional map of the Proterozoic outcrop within the Yanshan area, showing fossil localities. (d) Generalized Proterozoic stratigraphy in the Yanshan area (on the basis of the stratotype sections in Jixian), showing lithofacies, zircon U-Pb ages (on the basis of (refs 20, 21, 23, 24) and references therein), and fossiliferous horizon. Full size image

Figure 2: The Gaoyuzhuang Formation in the Yanshan area. (a) Generalized stratigraphic log of the Gaoyuzhuang Formation in the Yanshan area, showing variation of lithofacies and fossil horizon. (b,c) Field photographs showing fossiliferous calcareous shales in the middle Gaoyuzhuang Formation near Yamenzi Village, Hebei. Full size image

Description and interpretation of carbonaceous compressions

The fossils are preserved as carbonaceous compressions within mm- to cm-thick mudstone layers; they vary in size, orientation and degree of twisting or folding and, in general, appear to be allochthonous remains, deposited along with encompassing muds transported over short distances (Fig. 3; Supplementary Figs 1,2). Small fragments with irregular shapes are common in the mudstone, but abundant, well-preserved compressions of large size and regular shape have been found in only a few horizons, particularly in the Qianxi area. We measured 167 individuals with a dimension greater than 15 × 3 mm based on all the collected specimens (Supplementary Data 1 and Supplementary Table 1). In all, 53 individuals with regular shapes can be assigned to four morphotypes (Figs 3, 4; Supplementary Figs 2,3): linear (elongate with parallel sides, truncated at both ends), cuneate (distinct taper on one end; other end truncated), oblong (rounded on one end) or tongue-shaped (round end, but without parallel sides). Unlike mat fragments, these compressions have sharp margins on at least two sides. Each shape class shows a more or less normal size frequency distribution (Fig. 5).

Figure 3: Macroscopic fossils from the Gaoyuzhuang Formation. (a) Linear fossil without preservation of either end (a(1)) and fragment of tongue-shaped fossil (a(2)), Qg98017. (b) Linear fossil without preservation of either end (b(1)) and tongue-shaped fossil with longitudinal striations (b(2)), Qg20011; (c,d) Cuneate fossils, 07kg1332 (c), Qg20017 (d). (e) Oblong fossil with possible holdfast, 07 kg1331. (f) Cuneate fossil preserved with differentiated holdfast, Qg98021; (g) linear fossil without preservation of either end. Scale bars, 5 cm (in a,b,g), 20 mm (in c), 40 mm (in d) and 5 mm (in e,f). Full size image

Figure 4: Relative abundance of morphotypes within the Gaoyuzhuang assemblage. Individuals (total n=53) with regular shapes consist of four morphotypes. Blue=linear (26 individuals, 49% of total number); orange=cuneate (16 individuals, 30% of total number); grey=tongue-shaped (8 individuals, 15% of total number); yellow=oblong (3 individuals, 6% of total number). Full size image

Figure 5: Shape distribution of the Gaoyuzhuang macroscopic fossils. (a) Histogram showing the size frequency distribution of maximum widths within and among morphotypes: oblong (red), tongue-shaped (blue), cuneate (green), and linear (orange). (b) Plot of length versus maximum width for all measured fossil individuals from the Gaoyuzhuang assemblage. Red triangle=oblong, blue triangle=tongue-shaped, green diamond=cuneate, orange diamond=linear, and black circle=fragments. Full size image

Cuneate fossils are up to 18 cm long with various divergence angles (10–25°) characterizing the tapering end and a ribbon-like (up to 4.2 cm wide) distal part with sharp and nearly parallel margins (Fig. 3c,d). No holdfast was preserved in large specimens, but a rod-like basal stipe can be seen in one small cuneate specimen (Fig. 3f). The linear fossils are up 22.9 cm long and 4.5 cm wide with ragged ends, suggesting they are fragments of larger individuals. The linear specimens (Fig. 3a(1)) resemble the distal ends of cuneate compressions, and size frequency distributions for the two morphotypes overlap strongly (Fig. 5). Although slightly bent or twisted forms can be observed (Fig. 3g), most linear fossils are straight and have a smooth, slightly convex surface (Fig. 3a(1)), suggesting that the thallus was originally composed of mechanically strong organic materials.

Morphometric data suggest that the linear and cuneate remains are drawn from the same source population, an interpretation further supported by statistical data on tapering ratios for all individuals of both forms (Fig. 6). If this inference is correct, the source thalli could have reached lengths of >30 cm (Supplementary Fig. 3). Oblong fossils could also be drawn from this source, but one complete thallus (Fig. 3e) exhibits a narrow ribbon-like base with a terminal spheroidal expansion that may represent a distinct holdfast (Supplementary Fig. 3).

Figure 6: Comparison of tapering ratios and overall shape among the morphotypes. Tapering ratios (T=W max −W min /L’; see the ‘Methods’ section) of cuneate and linear morphotypes with straight margins show in one line without overlap. Tapering ratios of tongue-shaped and oblong morphotypes with curved margins overlap each other. Full size image

Tongue-shaped remains are distinct in size as well as shape, suggesting that they record another distinctive thallus type (Figs 3b(2) and 5; Supplementary Fig. 3). The tongue-shaped fossils are the largest remains in the assemblage (Figs 3a(2)), with dimensions up to 28.6 cm by 7.6 cm (Fig. 3b(2)). Blades clearly had a rounded distal end (Fig. 3b(2)), but the basal morphology remains unclear. Regular longitudinal striations run parallel to the long axis of the tongue-shaped fossils, providing further evidence of complex organization (Fig. 3b(2)). These broad fossils are commonly folded or otherwise deformed (Supplementary Fig. 1), suggesting a composition distinct from that of linear/conical thalli.

Morphometric analysis (Figs 4, 5, 6) further supports the hypothesis that Gaoyuzhuang carbonaceous compressions preserve at least two and possibly three populations of large macroscopic thalli (Supplementary Fig. 3) that grew according to simple but regular developmental programs, and rules out their interpretation as colonial prokaryotes or irregular mat fragments. The regular elongated shapes (and much larger size) of the Gaoyuzhuang fossils clearly differentiate them from the irregular carbonaceous compressions reported from the older Tuanshanzi Formation in the same area15. Moreover, the size and shape distribution of the Gaoyuzhuang compressions (Fig. 5) essentially rule out interpretation as ripped up and redeposited microbial mat fragments. The only plausible alterative is that the Gaoyuzhuang compressions represent decimetre-scale macroscopic, multicellular eukaryotes, and that is how we interpret them here.

Syngenetic multicellular fragments

The inference of organized multicellularity is reinforced by well-preserved cell sheets in organic fragments extracted from fossiliferous samples by acid maceration (Fig. 7). The fragments (up to 1 mm in diameter) consist of polyhedral cells 6–18 μm in diameter (mean=10 μm), closely packed to form a continuous surface. There is little evidence for cell files or other types of tissue organization. Neither is there evidence for cell differentiation within preserved sheets. The cellular fragments are translucent, with various optical densities under light microscopy, indicating they are made of thermally altered organic matter. This composition is corroborated by Raman first-order spectra, which are dominated by D and G bands characteristic of disordered carbonaceous material (kerogen; Fig. 7a; Supplementary Fig. 4). Raman first-order spectra of in situ macrofossils (see Supplementary Fig. 5) are similar, supporting the syngeneity of cellular remains within the host rock. The cellular fragments can easily be differentiated from the reticulate vesicles of contemporaneous acritarchs (for example, Dictyosphaera and Shuiyousphaeridium) in both size and ornamentation26,27,28. We cannot unambiguously demonstrate that the cell sheets originated within the thalli, but the absence of other microscopic fossils or other evident sources for these fragments, and indeed the absence of comparable cell sheets with dimensions >1 mm in any known Mesoproterozoic deposit, makes attribution of the cell sheets to the macroscopic thalli reasonable. We emphasize that the interpretation of the thalli as macroscopic eukaryotes is strong regardless of how one evaluates the cell sheets.