Multicellularity arose multiple times in the evolutionary history of eukaryotes, and simple multicellularity may have a deep history tracing back to the Paleoproterozoic. However, complex multicellular organisms with cellular and tissue differentiation did not appear in the fossil record until the Mesoproterozoic, and it is not until the Ediacaran Period (635–541 Ma) when diverse assemblages of complex multicellular eukaryotes evolved. In the intervening Tonian Period (ca. 1000–720 Ma), the fossil record of multicellular organisms is poorly documented. To address this knowledge gap, we investigated Chuaria and associated carbonaceous compression fossils from the Tonian Liulaobei Formation in North China. These fossils have been variously interpreted as unicellular or multicellular organisms. Our analysis using backscattered-electron scanning electron microscopy (BSE-SEM) revealed direct evidence for simple multicellularity in some of these fossils and suggests that Chuaria may have had a multicellular vegetative stage in its life cycle. This study demonstrates that BSE-SEM has the potential to unveil the hidden diversity of multicellular organisms in the Tonian Period, thus enriching our knowledge about the multiple origins of multicellularity in this critical geological period before Cryogenian glaciations.
The rise of multicellularity represents one of the major transitions in the evolutionary history of cellular life (Maynard Smith and Szathmary, 1997). However, in notable contrast to the rise of eukaryotes, which is also regarded as a major evolutionary transition, the development of multicellularity is not an evolutionary singularity. According to phylogenetic and paleontological data, the evolutionary march toward multicellularity occurred convergently in many clades and took extraordinarily protracted paths. Simple multicellularity—characterized by clusters, filaments, or sheets of cells that arise via mitotic cell division from a single progenitor and that have limited cell differentiation and cell-to-cell communication (Knoll, 2011)—evolved independently at least 25 times among eukaryotes alone (Grosberg and Strathmann, 2007; Herron et al., 2013). In contrast, complex multicellularity—characterized by intercellular communication and tissue differentiation—has occurred in only a handful of eukaryotic groups (Knoll, 2011). Both the fossil record and molecular clocks indicate that simple multicellularity, as represented by cellularly preserved colonial coccoids and filamentous fossils (Butterfield, 2009), evolved no later than the early Paleoproterozoic (Hofmann, 1976; Schirrmeister et al., 2013). However, complex multicellularity appeared in the fossil record only in the middle Mesoproterozoic (Butterfield, 2000) and did not become ecologically significant until the Ediacaran Period or 635–541 Ma (Yuan et al., 2011; Xiao et al., 2014a).
A literal reading of the fossil record, however, should be taken with caution because of problems about the preservation of soft-bodied organisms, the interpretation of morphologically simple forms, and the incomplete understanding of life cycles. Those problems are conspicuously prominent in Proterozoic carbonaceous compression fossils, which often do not preserve any traces of cellular structures or a complete life cycle. For example, the carbonaceous compression fossil Chuaria, which is diagnosed as submillimeter- to millimeter-sized, thick-walled spherical vesicles and is common in the Proterozoic Eon (particularly in the Tonian Period, ca. 1000–720 Ma), has been variously interpreted as a unicellular or multicellular organism. Chuaria has been regarded as a unicellular/coenocytic eukaryote because of its similarity to acritarchs (Vidal and Ford, 1985), which are often uncritically treated as resting cysts of unicellular eukaryotes. It has also been interpreted as a colonial cyanobacterium based on its putative association with Nostoc-like filaments (Sun, 1987), or as a multicellular eukaryote because of its excystment structures and a potentially complex life cycle (Kumar, 2001; Sharma et al., 2009). These interpretations are difficult to resolve because Chuaria fossils are morphologically simple, apparently lack cellular preservation, and do not record a complete life cycle.
Recently, variable-voltage backscattered-electron scanning electron microscopy (BSE-SEM) has been proven as an effective method with which to study carbonaceous compressions and to bring to light hidden morphological details that are otherwise invisible in reflected light microscopy (RLM) and secondary-electron scanning electron microscopy (SE-SEM). Briefly, by adjusting the accelerating voltage in BSE-SEM and thus allowing the primary electrons to penetrate to different depths, it is possible to illuminate subsurface structures in carbonaceous compressions (Orr et al., 2002; Pang et al., 2013; LoDuca et al., 2015; Muscente and Xiao, 2015). This technique offers an immense opportunity to test whether Chuaria and associated fossils are multicellular organisms, because it has the potential to elucidate cellular structures preserved in carbonaceous compressions.
MATERIALS AND METHODS
Fossils were collected from the Tonian Liulaobei Formation at the Diangeda-Baieshan section (32°37′51.52″N, 116°46′0.19″E) near Shouxian, Anhui Province, North China (Fig. 1). The geologic and stratigraphic setting of the Liulaobei Formation has been described elsewhere (Dong et al., 2008). The fossiliferous strata were deposited in offshore ferruginous environments (Guilbaud et al., 2015). The age of the Liulaobei Formation is loosely constrained by K-Ar and Rb-Sr radiometric dates to be 900–750 Ma (summarized in Dong et al., 2008), consistent with chemostratigraphic and biostratigraphic correlations (Tang et al., 2013; Xiao et al., 2014b).
Fossils analyzed in this study are carbonaceous compressions preserved on the bedding surface of shales and mudstones. A subset of these carbonaceous compressions represents submillimeter- to millimeter-sized, thick-walled, spherical vesicles that fit the diagnosis of and have been described as Chuaria (Sun, 1987). Other fossils have multicellular structures and are referred to as multicellular aggregates. Both Chuaria fossils and multicellular aggregates were imaged using RLM, SE-SEM, and BSE-SEM. All illustrated specimens were deposited at the Virginia Polytechnic Institute Geosciences Museum (VPIGM, Blacksburg, Virginia, USA).
Under RLM, Chuaria specimens from the Liulaobei Formation conform to the diagnosis of Chuaria circularis (Walcott, 1899). They are spherical vesicles compressed into two-dimensional discoidal to subdiscoidal structures, often with concentric compressional folds (Fig. 2A), V-shaped ruptures (Figs. 2A and 2B), and radial splits (Fig. 2C). Their vesicle size varies from 0.5 mm to 5 mm. They are often randomly disseminated on bedding planes (Figs. 2D and 3A).
In total, 103 discoidal fossils among ∼578 examined specimens were found to preserve multicellular structures (Figs. 2E, 2F, and 3); sometimes, the multicellular nature of the fossils was visible under BSE-SEM but invisible under RLM or SE-SEM (Fig. 3B), suggesting that the multicellular structures are preserved within the compressed fossils. Twenty of them were observed to have a modest number of loosely packed cells (mostly 50–300 μm in cell diameter, with rare outliers) within a common envelope (0.8–5.7 mm in diameter) that often displayed concentric compressional folds (Figs. 2E and 2F); hereafter, these are referred to as enveloped cell aggregates. The others were observed to have tightly packed cell aggregates without a distinct common envelope (Fig. 3), and they are hereafter referred to as naked cell aggregates. The tightly packed cells in naked aggregates became increasingly visible as the accelerating voltage increased from 5 keV to 25 keV, again suggesting that these cells are embedded within the carbonaceous compression, which is estimated to be 2–3 μm in thickness based on Monte Carlo simulations (Muscente and Xiao, 2015). Cells in the periphery were often better discernible than those in the center, where they were obscured (Fig. 3C). Cells were found to be 70–110 μm in diameter (average = 85 μm; n = 934), and their size was relatively constant regardless of aggregate size (Fig. DR1 in the GSA Data Repository1).
INTERPRETATION AND DISCUSSION
The naked aggregates with tightly packed cells are interpreted as spherical multicellular colonies (i.e., simple multicellularity). The variable-voltage BSE-SEM observations indicate that the cellular structures are embedded within the carbonaceous compression. The fact that they are most discernible in the periphery of the aggregate, but are obscured toward the center, suggests that the colonies were three-dimensional structures filled with a solid mass of cells. Thus, when compressed, the central part of a colony became more obscured than the periphery due to greater compaction. The naked aggregates may each contain 36–10,232 cells, assuming close packing, which has a packing density of ∼74%, or 32–8849 cells, assuming random packing with a packing density of ∼64% (Schiffbauer et al., 2012). The lack of morphological differentiation among the cells suggests that the aggregates represent colonies, and the lack of a common envelope indicates that these colonies represent vegetative growth stages.
The enveloped aggregates with loosely packed cells (Figs. 2E and 2F), on the other hand, may represent a stage entering into or exiting from an encystment stage. The presence of concentric folds indicates that the envelopes were thick walled and resistant, a likely sign for dormant cysts or hypnospores (Coleman, 1983). The more variable cell sizes (Fig. 2E), in comparison with cells in naked cell aggregates, may be related to the more dynamic cytoplasm growth or condensation during encystment or excystment. Alternatively, such variations in cell size may in part be a taphonomic artifact. Similarly, it is uncertain whether the sparse numbers of cells in most enveloped aggregates resulted from a taphonomic loss of cells or a biologically programmed reduction of cell numbers.
The compressed vesicles of C. circularis from the Liulaobei Formation are interpreted as relatively thick cyst walls. The lack of cellular content within the cysts could be due to taphonomic loss or biological excystment; indeed, some C. circularis specimens show well-defined V-shaped ruptures (Figs. 2A and 2B; Butterfield et al., 1994, their figure 13F; Sharma et al., 2009, their figures 6k, 6l, and 6n) that may represent excystment openings through which cells were released from germinating cysts.
One may choose to name the naked aggregates, enveloped aggregates, and Chuaria vesicles as distinct taxa. However, based on their coexistence and overlapping size range, we propose that they represent different stages of the same organism, and when considered together, they can illuminate the life cycle of Chuaria. The enveloped aggregates provide a key morphological intermediate between the naked aggregates and Chuaria vesicles. If these forms do indeed represent different stages of the same organism, we speculate that Chuaria had a biphasic (planktonic-benthic) life cycle involving (1) a benthic encystment stage and (2) a planktonic colonial stage that grew out of spores released during excystment (Fig. DR2). The planktonic phase was likely vegetative, characterized by rapid cell growth and division, which could involve fast cell cycles with rapid alternation of S (synthesis) and M (mitosis) phases, or rapid hypotrophic growth and karyokinesis to form multinucleate cells that subsequently underwent palintomic cytokinesis (Cross and Umen, 2015); many modern green algae such as Chlamydomonas, Scenedesmus, and Chlorella share these types of cell growth and division to develop a simple multicellular stage (Bišová and Zachleder, 2014). The benthic phase was largely dormant and enclosed within thick vesicles, much like resting cysts of modern green algae (Coleman, 1983). From a taphonomic point of view, it is not surprising that the benthic phase with thick-walled cysts would leave a much better fossil record. Thus, Chuaria is often represented by empty vesicles only, and the planktonic multicellular vegetative stages are rarely preserved (e.g., in the Liulaobei Formation). On the other hand, it is also possible that the multicellular stages have been neglected because BSE-SEM has not been widely applied to investigate all Chuaria assemblages, as our study shows that RLM and SE-SEM are inadequate techniques with which to visualize the hidden multicellularity in some specimens.
It is intriguing that the cyst-forming Chuaria commonly occurs in predominantly anoxic Proterozoic basins, including the Liulaobei Formation (Guilbaud et al., 2015), Chuar Group (Johnston et al., 2010), and the Lantian Formation (Yuan et al., 2001; Guan et al., 2014). It is worth exploring in the future whether anoxic conditions led to fluctuating environments (e.g., nutrient availability) that facilitated the prevalence of encystment in Chuaria and other Proterozoic organisms.
Regardless, Chuaria from the Liulaobei Formation is suggested to be a simple multicellular organism, and possibly a multicellular eukaryote, considering the combination of characters such as large vesicle size, thick vesicle wall, possible encystment, and a biphasic life cycle. Although it is possible that fossils described as Chuaria may encompass a wide range of organisms, including bacteria and unicellular organisms (Steiner, 1997), the present study suggests that the Proterozoic diversity of multicellular organisms is probably underestimated and that a systematic investigation of Proterozoic carbonaceous compressions using BSE-SEM can help to rectify this.
The evolution of multicellularity endowed organisms with critical advantages and greater evolutionary potential, allowing greater size, greater complexity, circumvention of diffusion limits, and many other evolutionary benefits (Knoll, 2011). The discovery of simple multicellular forms in association with Chuaria vesicles from the Tonian Liulaobei Formation allows us to critically evaluate the various hypotheses about Chuaria. Available evidence favors the hypothesis that Chuaria was a simple multicellular organism (i.e., a colonial organism without cell differentiation), possibly a eukaryote with a multicellular vegetative stage in its life cycle. This raises an important question about its relationship with much smaller cell aggregates such as Symplassosphaeridium and Synsphaeridium that are often found in Tonian rocks (Tang et al., 2013, 2015). Although these fossils are characterized by simple multicellularity, they represent an evolutionary stage that underlies the greater diversity and complexity as manifested by the many multicellular organisms that came afterwards in the Cryogenian and Ediacaran Periods (Butterfield, 2007; Ye et al., 2015). More systematic investigation of Chuaria and other carbonaceous compressions using BSE-SEM holds promise to further illuminate the evolution of multicellularity in the Tonian Period.
This research was supported by the U.S. National Science Foundation (grant EAR-1528553), the National Natural Science Foundation of China (grants 41272011, 41602007), the Geological Society of American, Sigma Xi, the Chinese Ministry of Science and Technology (grant 2013CB835000), the Chinese Academy of Sciences (grant KZZD-EW-02, KZCX2-YW-153), the Science Foundation of Jiangsu Province of China (grant BK20161090), and the State Key Laboratory of Palaeobiology and Stratigraphy (grant 20162109). We thank Bin Wan, Jinlong Wang, Lei Chen, and Zhiji Ou for field assistance.
- Received 3 July 2016.
- Revision received 18 October 2016.
- Accepted 18 October 2016.
- © 2016 Geological Society of America