A possible billion-year-old holozoan with differentiated multicellularity

SUMMARY Sediments of the Torridonian sequence of the Northwest Scottish Highlands contain a wide array of microfossils, documenting life in a non-marine setting a billion years ago (1 Ga). 1–4 Phosphate nodules from the Diabaig Formation at Loch Torridon preserve microorganisms with cellular-level ﬁdelity, 5,6 allowing for partial reconstruction of the developmental stages of a new organism, Bicellum brasieri gen. et sp. nov. The mature form of Bicellum consists of a solid, spherical ball of tightly packed cells (a stereoblast) of isodiametric cells enclosed in a monolayer of elongated, sausage-shaped cells. However, two populations of naked stereoblasts show mixed cell shapes, which we infer to indicate incipient development of elongated cells that were migrating to the periphery of the cell mass. These simple morphogenetic movements could be ex-plained by differential cell-cell adhesion. 7,8 In fact, the basic morphology of Bicellum is topologically similar to that of experimentally produced cell masses that were shown to spontaneously segregate into two distinct domains based on differential cadherin-based cell adhesion. 9 The lack of rigid cell walls in the stereoblast renders an algal


RESULTS
Butterfield 14 has pointed out that multicellular organisms in pre-Ediacaran age deposits were likely to have left behind ontogenetic stages in the fossil record. We have examined about 50 petrographic thin sections of phosphatic lenses in the Diabaig Formation (ca. 1 Ga) that preserve populations of benthic and planktic organisms trapped in former lake bottom sediments ( Figures 1A-1D). These include unicells and cell clusters of various kinds, some of which have been documented previously. 1,2,6,15 In several thin sections, we observed cell clusters that are composed of aggregations of two distinct cell types, indicating a condition that constitutes a step toward complex multicellularity sensu Knoll. 16 Further investigation revealed a second set of cell clusters that appeared very similar in size and form but that lacked the fully differentiated second cell type. Here, we describe these interesting fossils and show intermediate morphologies that are consistent with an ontogenetic series driven by a differential cell-adhesion model.
The morphology of the new multicellular organism consists of a spheroidal mass of mutually adpressed cells enclosed by a peripheral layer of elongate, sausage-shaped cells. The interior cell mass forms a stereoblast (Figures 2A-2C, 2F, 2H, 2J, and S1-S3) of roughly isodiametric cells that average~2.5 mmi n diameter (Table S1). Exceptional preservation in calcium phosphate (francolite) and authigenic clay minerals 5 ( Figure S3) retains intracellular biological features that, in this case, consist of a single dense, organic ''spot'' (Figures 2A and 2C, arrows). In well-preserved specimens, such inclusions occur in about half of the interior cells. These might represent preserved nuclei, but we consider that, more likely, they are the condensed remains of the entirety of the cytoplasmic cell content. 6 The cells of the stereoblast retain mutually compressed walls, so that the original multicellular topology, including Y-shaped junctions 17 (Figures 2B and 2C, circles), is retained. There is no evidence that these interior cells possessed rigid cell walls, because the shape of each cell is established by mutual compression with adjacent cells. This indicates the likelihood that individual cells were bounded by just a cell membrane or a thin, non-rigid cell wall. A carbon map of a specimen from an ultrathin section ( Figures 2J and S3) also shows very thin interior walls as compared with the exterior cell layer. Although it is not possible to completely rule out that each of the interior cells possessed a thin, flexible cell wall, we found no examples of cells that possessed interior membranes that might have pulled away from any such cell wall. This is not the case for various other isolated cells of different organisms found throughout the Diabaig phosphates in which multiple concentric layers are apparent and in which true cell walls are quite evident. 6 The outermost cell layer consists of thicker walled, sausageshaped (elongate) cells, which form an enclosing layer that is unmistakably distinct from the isodiametric cells found in the interior stereoblast (Figures 2A, 2B, 2D-2K, and S1-S3). The elongate cells that form the peripheral layer are around 1.5 to 2 mm in diameter and generally about 3 to 4 times that in length, although, in some cases, they can be much longer (e.g., Figure 2K). The average width-to-length ratio for a set of 6 specimens was 0.28 (Table S1). The elongate shape is best demonstrated in surficial focus, as seen in Figures 2E-2G, 2I, and 2K (see also Figures S1-S3). Here, these cells crowd together to form what appears to be a rigid, outer spherical shell. The peripheral cells often occur in sets of 4 or more adjacent cells that are positioned parallel to each other ( Figures 2F, 2G, 2I, 2K, and S1-S3), creating a tiled arrangement of sets of parallel cells, or, in some cases, covering the entire surface of the stereoblast in parallel-aligned, elongate cells ( Figure 2K). In medial cross-section, the peripheral cell layer is shown to be one cell in thickness (Figures 2A, 2B, 2D, 2H, 2J, and S1-S3). In light microscopy (LM), the walls of these outer cells appear darker than those of the stereoblast, indicating a thicker cell wall, distinct from those of the interior cells. This is seen in the holotype ( Figure 2A) and in many of the other illustrated specimens (e.g., Figures 2D, 2H, 2J, S1, and S2). This is also somewhat evident in the carbon map in Figure 2J, although in this specimen, the carbon signal from the cells of the epidermal layer is masked somewhat by the carbon signal from the enclosing francolite (see also Figures S3E and S3F). The cells of the peripheral layer never show an interior ''spot'' like many cells of the stereoblast, indicating a persistent taphonomic difference in the two cell types, or perhaps loss of the protoplast at maturity in the epidermal cells, as, under LM, their interiors are more transparent that those of the interior stereoblast. Many specimens show some degree of cell loss within the stereoblast as a whole. This can be seen in medial sections of Figures 2B and 2D, where parts of the interior are missing well-preserved cells (see also Figure S1). Overall, this arrangement of two distinct cell types forming a spherical organism has not been previously described in the fossil record and is formalized here as Bicellum brasieri Strother & Wellman gen. et sp. nov.
The structural details used to describe Bicellum exist because of the unique qualities of cellular and sub-cellular preservation provided by phosphate and authigenic clay mineralization. 5,6 The taxonomic richness that characterizes the Torridonian lake See also related size data in Table S2. deposits, however, is recorded primarily in palynological preparations of fine-grained siliciclastic rocks that yield organic-walled microfossils (OWMs). 1 An OWM comparable to Bicellum was also recovered in palynological strew mounts, but, as documented in Figure 3, its appearance as a flattened, dispersed OWM is somewhat different than its 3D form. Here, the wall is characterized by marginal circular structures (arrows in Figures 3A, 3C, and 3D), which correspond to crosssectional views of the elongated cell peripheral layer. The average diameter (28.5 mm; Table S2) and ovoid ( Figures 3D and 3E) to circular ( Figures 3A-3C) shape are similar to the 3-dimensional form, but the interior stereoblast is not structurally preserved in the dispersed form, nor is the cellular nature of the peripheral layer readily apparent. In spite of these preservational differences, Bicellum, in its dispersed form, has now been recognized from 11 sample localities found throughout the Torridonian sequence (Table S2).
Tightly bound, spherical cell clusters found in association with B. brasieri fall outside the prescribed complex morphology for the species as presented here. These multicellular cell clusters consist of naked stereoblasts without an enclosing wall or cell layer. They are most commonly quite spherical ( Figures 4A-4E), although some larger, ellipsoidal specimens have also been found ( Figure 4F). The cells that compose each mass are tightly adpressed without intervening spaces; indeed, many exhibit straight lines of contact and clear 120 (Y-shaped) junctions where three cells meet. They are generally isodiametric, with diameters of 2 to 3 mm. The closeness of the cells indicates the originally cohesive nature of these cell masses; they appear as if they were tightly pressed together in life. The lack of intervening space also indicates that, in life, these cells did not possess rigid cell walls; if present, the cell walls clearly had a  Table S1 for related cell size data. These free, spherical cell masses ( Figures 4A and 4B) are indistinguishable from the stereoblasts that characterize the interior cells of B. brasieri. After examination, it became apparent that some of these masses occasionally included elongate cells, unlike the enclosed stereoblasts described earlier. The incomplete cell mass in Figure 4C contains only one such elongate cell (indicated by an arrow) in the midst of what are generally isodiametric cells. Figure 4D illustrates another example photographed in median optical section. Here, embedded among generally isodiametric cells, is a single row of elongate cells (between arrows). This specimen also contains a few additional isolated, individual, elongate cells scattered through the cell mass. Another specimen photographed in median section ( Figure 4E), shows at least four elongate cells (indicated by arrows), which are now located at the (E) Here, some elongate cells (arrows) now appear at the periphery of the cell mass, although a clearly distinct epidermal layer has yet to become established. Note that there is no apparent distinction between the cell walls of either cell type. (F) This ellipsoidal specimen, which is cut in a subtangential section, shows substantial alignment of sausage-shaped cell types (arrows). periphery of the largely isodiametric cell mass. Here, these elongate cells appear to constitute part of a single circumferential layer, one cell in thickness, that surrounds the cell mass; however, the individual cells that make up this layer have thinner walls, matching those of all the interior cells of the stereoblast, and not thickened as seen in the peripheral layer of B. brasieri.T h e cell mass in Figure 4F appears somewhat even more organized, with several rows of nascent elongate cells preserved (indicated by arrows).
Although these cell clusters are nearly perfectly spherical in overall shape, the positions of individual cells within the clusters does not appear determinate: these cells do not appear to have retained a geometry based on fixed patterns of cell divisions. Cells capable of moving relative to each other, exhibiting liquid-like behavior, will spontaneously form spheres in response to minimizing overall surface tension. 18 Thus, on strictly morphological grounds, we infer that these fossils were originally aggregates of somewhat cohesive cells exhibiting liquid-like behavior. 19,20 Living cells aggregated in this way form the underlying basis of the differential adhesion hypothesis (DAH), which posits that cohesive cell aggregates are capable of self-differentiation when cell-cell cohesion varies between sets of cells. 8

DISCUSSION
In spite of its simple morphology, the characterization of the Bicellum stereoblast, combined with an understanding of its dynamic assembly into a differentiated condition of two distinct cell aggregations, provides clues as to its systematic placement. It seems reasonable to assume that Bicellum falls within one of the lineages leading to one of the six clades that possess complex multicellularity today: animals, plants, florideophyte algae, brown algae, ascomycete fungi, and basidiomycete fungi. 16 The precise dynamics of how Bicellum attained its initial multicellular state has yet to be determined. In broad terms, this would have been through palintomy, aggregative assembly, or cellularization of a coenobium (syncytium). If the initial multicellular condition of Bicellum occurred through successive mitotic divisions, then we would expect to find cell clusters of similar overall size, exhibiting combinations of 2 n cells, as is the case, for example, in the embryo-like fossils of the Doushantuo phosphates. Although it is the case that many different kinds of cell clusters have been found in the same thin sections that contain Bicellum, no such palintomic sequence has yet been recognized. This indicates that the multicellular condition in Bicellum more likely occurred either through cell aggregation or through the cellularization of either a syncytium or a coenobium. Since these non-palintomic forms of multicellularity are also somewhat limited in their distribution within extant protist groups, they provide a means of limiting the potential placement of Bicellum. Other than in the opisthokonts, aggregative multicellularity occurs in the amoebozan, Dictyostelium, three SAR supergroup genera (Sorogena, Sorodiplophrys, and Guttulinopsis), 21 and also some labyrinthulids 22 and the Excavate Acrasis, 22 none of which are a morphological match with Bicellum. Formation of syncytia and/or coenobia occurs in the Archaeplastida and in all holozoan groups, with the exception of the choanoflagellates 13,22 and the Filasterea, which do show aggregative multicellularity. 23 However, the multicellular condition, as exhibited in the Bicellum stereoblast-that is, Y-shaped cell junctions and lack of fixed, or determinate, cell placement-indicates that these cells probably lacked rigid cell walls. This eliminates both cyanobacteria and the eukaryotic chlorophyte algae as likely homologs, because multicellular form in these taxa is strongly influenced by their possession of rigid cell walls. 17 This is also the case for comparison with the florideophyte red algae, which possess cellulosic cell walls and are fundamentally of filamentous or pseudoparenchymatous thallus organization. In addition, the red algae are predominantly marine in their habitat distribution, 24 as are the laminarian brown algae. 25 Y-shaped junctions in fossilized cell masses have most often been associated with tissue-grade multicellularity that is found in animals, 17,[26][27][28] and this condition has been argued as a reason for considering many of the embryo-like fossils of the Doushantuo Formation to be related to animal (Holozoan) lineages rather than Archeoplastida. 17 Thus, in terms of its multicellular condition, B. brasieri seems to be most closely associated with early-branching holozoan groups, especially Ichthyosporea and the Pluriformea (Corallochytrea) clade. 13,29 These groups are all unicellular protists with a multicellular stage in their life cycle, typically occurring in the form of a spherical cell mass. 30 The Precambrian occurrence of holozoans is documented in the well-known ''embryo-like'' fossils of the Doushantuo Formation, in spite of uncertainty as to their exact phylogenetic placement with respect to the Metazoa. [31][32][33] The Doushantuo fossils are found in considerably younger (ca. 600 Ma) deposits that are entirely marine in origin as compared with the billion-yearold lacustrine settings of the Torridonian. 34 Early-branching holozoan clades are not exclusively marine, however. Phylotypes of Ichthyosporea are found in freshwater and terrestrial settings today, 35 and the recently described filasterean, Pigoraptor, was isolated from nonmarine settings, as was Syssomonas, one of two genera in the recently proposed holozoan clade, Pluriformea. 13 Other Ichthyosporea and Filasterea, including species previously considered to be exclusively marine, have been detected in freshwater fluvial settings using environmental metabarcoding. 36 As to their antiquity, the existence of fossil holozoans by 1.0 Ga is perhaps not unexpected, given a reasonable estimate of the branch point between an ichthyosporean plus filasterean clade and the rest of the holozoan lineage at around 1,100 Ma, 37 although there is considerable uncertainty as to this date, based on molecular clock data. 38 Phylogenomic investigations of metazoan origins have begun to assemble a picture of protistan gene regulatory networks that were later re-purposed during the evolution of the first Metazoa. 12,39,40 Intriguingly, various genes associated with cell-cell adhesion appear to be quite ancient, including the discovery that three cadherin families, (lefftyrin, coherin, and hedgling) were present in the last common ancestor between the choanoflagellates and the Metazoa. 41 Some core components of the integrin-mediated complex may predate even the initial evolutionary divergence in the Opisthokonta. 42 Steinberg's differential adhesion hypothesis proposes that, for cases in which cell aggregates display liquid-like behavior, it is the strength of cell-cell adhesions that determines the overall form of the structure. 7,8 The dynamics of cell-cell segregation seen in Bicellum are compatible with that of a differential adhesion model in which the isodiametric cells adhere more strongly to each other than they adhere to the nascent elongate cells. This difference in cell-cell adhesion strengths is expected to give rise to a structure with an inner ball of isodiametric cells surrounded by a layer of elongated cells. 9,43,44 This mature stage then functioned as a cyst, which manifests as the more widely distributed form seen in Figure 3.
Although we are uncertain as to the molecular structure of the cell attachment apparatus in Bicellum, the reasons for differential adhesion are potentially simple. Foty and Steinberg 9 manipulated cadherin levels in cultured cells, showing that changing the expression level of that single protein was able to produce structures with an inner cell ball of strongly adhering cells surrounded by a cortex of more weakly adhering ones. However, even without a change in protein expression, an increase in surface area of the elongate cells, compared with that of the isodiametric cells, would reduce the surface density of adhesion molecules, leading to weaker adhesion between elongate and isodiametric cells. Whatever the mechanism, Bicellum does show that differentiation and morphogenesis occurred in the life cycles of freshwater protists as long as a billion years ago. This early example of complex multicellularity adds to a nascent body of evidence indicating the importance of selection in terrestrial settings during late Mesoproterozoic to early Neoproterozoic time. 4,[45][46][47] Indeed, if Bicellum does belong to the clade Holozoa, as we suspect, it would provide support for recent models proposing Mesoproterozoic eukaryotic crown group origins, 48,49 and it could prove to be a key fossil clue in an ongoing ll OPEN ACCESS debate on the importance of oxygen in the origin and rise of animals. 50

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We acknowledge the facilities and scientific and technical assistance of the Microscopy Australia research facilities at UWA and UNSW. These facilities are funded by the universities and state and commonwealth governments. C. Kong is thanked for his help with electron microscopy and mass spectrometry work. We thank C.T. Baldwin and C. Lenk for field assistance at Loch Torridon and on Eilean Fladday. This resesearch was supported by NERC grant NE/R001324/1 to C.H.W. and P.K.S. and by NASA grant 06-EXOB06-0037 and National Geographic Society grant 8882-11 to P.K.S. and C.H.W. D.W. acknowledges funding from the Australian Research Council via the Future Fellowship Scheme (grant FT140100321).

AUTHOR CONTRIBUTIONS
Original conceptualization was by P.K.S. and M.D.B., with input from C.H.W.; fieldwork was conducted by P.K.S., C.H.W., and M.D.B.; data acquisition and analysis were performed by D.W., M.S., and L.T.; light photomicrography was conducted by P.K.S.; and P.K.S. and C.H.W. wrote the paper, with contributions from D.W. and L.T.