comparative topology and structure of and parapandorina cell · chemically-distinct from...

Comparative topology and structure of Thiomargarita and Parapandorina cell

clusters: Both Doushantuo and Thiomargarita diads exhibit an undisturbed division

plane (Fig. 1b, b’). Three-cell clusters, thought to result from the incomplete division of a

two-cell stage are present in both Parapandorina and Thiomargarita (Fig. 1c, c’).

Whether or not the undivided larger cell of the Thiomargarita triads later undergoes

reductive division to produce a tetrad is presently unknown. Both Thiomargarita and

Parapandorina tetrads exhibit a variety of cell configurations. Four-radiate cross-

junctions of division planes in tetragonal Doushantuo tetrads are shown in Figure 1d’, a

configuration common in extant non-mammalian embryos and in Thiomargarita tetrads

(Fig. 1d). Some Doushantuo octads are also observed to exhibit radiate cross-junctions

(Fig. 1c in 1), which undoubtedly resulted from division of tetragonal tetrads. Rhomboidal

tetrads, which are characterized by two opposite bodies in contact, and two opposite

bodies separated by a gap, are also observed in Parapandorina (Fig. 7.5 from 2,

reproduced in Figure 1e’), and in Thiomargarita tetrads (Fig. 1e). Perhaps a more

common topology observed in Doushantuo tetrads are decussate or tetrahedral

configurations, which result from deformation of preexisting cell-division planes3. Such

deformation is thought to require non-rigid cell walls/membranes and produces Y-shaped

triple junctions of division planes (Supplementary Fig. 1). Although bacterial cell walls

were once thought to be rigid structures, they are now known to be quite elastic4.

Deformation of division planes resulting in Y-shaped triple junctions are observed in

multi-planar Thiomargarita tetrads, including partially-deformed tetragonal tetrads

(Supplementary Fig. 2a), decussate tetrads (Supplementary Fig. 2), and tetrahedral tetrads

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(Supplementary Fig. 3). Y-shaped triple junctions are also observed in 8-cell

Thiomargarita and multi-cell Parapandorina specimens (Supplementary Figure 4, 5c,

5d). Although Thiomargarita from off the coast of Namibia are known to form chains5,

Thiomargarita from the Gulf of Mexico, the focus of this study, have not been observed

to form chains.

Thiomargarita cell clusters have yet to be observed containing hundreds of

internal bodies, such as Megaclonophycus, another globular microfossil from the

Doushantuo Formation. However, these microfossils have never been demonstrated to be

part of a developmental continuum with Parapandorina2. Megaclonophycus are loosely-

packed clusters that contain rounded, rather than polyganol, internal bodies2, and do not

exhibit a blastocoel, as would be expected in embryos with similar numbers of cells6, thus

calling into question their metazoan affinities.

Internal bodies: Some Parapandorina specimens include smaller subcellular

structures of non-diagnostic shape6. Such bodies are consistent with diagenetically-

altered inclusions of the type that are common in Thiomargarita, but as with other

hypotheses, their origin is ultimately ambiguous. A few Parapandorina specimens

(n=10) include larger spherical-to-reniform internal structures6. Hagadorn et al.

6 suggest

that these bodies might be organelles, however they also allow for the possibility that the

larger internal bodies resulted from inorganic mineral precipitation or shrunken

cytoplasm as observed in other microfossils e.g.,7

, including algae from the Doushantuo

Formation (Fig. 3H in 6). As a sheathed organism with vacuole contents that are

chemically-distinct from surrounding waters, Thiomargarita cells could also have

undergone cytoplasmic shrinkage or internal diagenetic mineral precipitation resulting in


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intracellular structures. Inclusions in Thiomargarita also occasionally form aggregates

(Supplementary Fig. 6), likely as a result of cytoplasmic degradation, that are similar in

size and shape to the large intracellular structures observed in a small number of

Parapandorina specimens. These aggregates often exhibit approximate symmetry across

division planes (Supplementary Fig. 6b), and can sometimes be found as pairs in each

cell of a multi-cell cluster, as is also observed in at least one Doushantuo tetrad6, though

these aggregates likely result from degradational, rather than physiological, processes.

Supplementary Figure 1: Thiomargarita tetrad exhibiting deformation of the

preceding division planes with Y-shaped triple junctions; (see arrows) as observed in

a cartoon modified after 3 showing an intermediate stage in the deformation thought to

result in many Doushantuo Parapandorina tetrads. Scale bar = 100 μm.



Supplementary Figure 2: Thiomargarita tetrad in an approximately decussate

geometric configuration, with two cell pairs approximately at right angles to one

another, similar to the geometry observed in some Parapandorina tetrads (cartoon

modified after 3). The dark material surrounding the cluster is composed primarily of

small filamentous and spherical cells, with sizes and morphologies similar to the

filamentous and spherical structures commonly found on surfaces of Doushantuo

microfossils. Scale bar = 100 μm.

Supplementary Figure 3: Thiomargarita tetrad exhibiting an approximately

tetrahedral geometry akin to another geometry commonly observed in microfossil

tetrads from the Doushantuo Formation. Scale bar = 100 μm.



Supplementary Figure 4: Thiomargarita octads exhibit Y-shaped junctions (white

arrows), similar to those observed in multi-cell Parapandorina clusters (after 2, Fig.

8.8). Scale bar =100 μm.

Supplementary Figure 5: Thiomargarita cell clusters (a: 3-cell cluster, b: 4-cell

cluster, c, d: probable 8-cell clusters) show a variety of geometries that indicate

deformation of the previous division plane, as observed in Parapandorina clusters.

All scale bars = 100 μm.

Supplementary Figure 6: Aggregates of internal inclusions, presumably resulting

from cytoplasmic degradation, appear as large spheroidal to reniform intracellular

bodies in a small number of a) solitary Thiomargarita cells, and b,c) multi-cell

clusters. All scale bars = 70 μm.

Comparative morphology of Tianzhushiana, Megasphaera inornata, and

Thiomargarita: The Doushantuo Formation contains abundant large spherical



microfossils that exhibit a range of morphological features. Abundant indistinct smooth

spheres of uncertain affinities, generally categorized as sphaeromorphic acritarchs, are

often thought to represent algal resting cysts. Some Doushantuo spherical bodies are

encased in an envelope (Megasphaera8). It has been recognized that some of these

envelopes possess external surface ornamentation (Megasphaera ornata), while others

lack ornamentation (Megasphaera inornata)2. This ornamentation has been central to the

argument for a metazoan egg interpretation of Megasphaera2,9. Recently, it was proposed

that Megasphaera results from taphonomic alteration of the acanthomorphic acritarch

Tianzhushiana tuberifera10, which is characterized by external processes that penetrate a

multi-lamellate outer wall11,12. Similarities between the middle wall of T. tuberifera and

the outer wall of M. ornata, and the finding of an additional outer wall on a few M.

ornata specimens lead to the hypothesis that they represent the same species. Based on

the hypothesis that unornamented globular fossils may have once had an ornamented

outer wall that was not preserved, the unornamented M. inornata was also proposed to be

synonymous with Tianzhushania10. Given the great abundance of unornamented globular

microfossils in the Doushantuo phosphorites, we find this explanation insufficient to

explain most unornamented specimens. Therefore, we retain the use of M. inornata,

which we interpret as possible fossilized giant sulfur bacteria based on its abundance in

phosphorites. Deflated envelopes lacking internal bodies are also observed in the

Doushantuo microbiota2 (Supplementary Fig. 7a'), and similar deflation is commonly

exhibited in Thiomargarita cells with ruptured internal vacuoles (Supplementary Fig. 7a).

However, modern animal eggs and phosphatized Doushantuo acritarchs are also observed



to exhibit deflation and collapse2. Ultimately, unornamented globular fossils are

taxonomically ambiguous and could represent more than one organism.

Supplementary Figure 7: Deflated Megasphaera (a’) (after 2) may indicate an

initially hollow interior, such as the large vacuole in Thiomargarita, which is

occasionally observed to rupture resulting in a deflated cell (a). Scale bar = 100 μm.

Abundance of Doushantuo microfossils: The unusual abundance of globular

microfossils in the Doushantuo Formation has long been considered problematic for the

animal embryo interpretation13; concentration via sedimentary processes has been

proposed as a possible solution14. Such a circumstance does fall within the realm of

possibility. However, the globular microfossils generally occur as grains within larger

reworked clasts 2,15,16. Literature on this topic suggests that the fossils were phosphatized,

size sorted by currents, cemented together, ripped-up, and re-deposited elsewhere within

the larger clasts15,16. Each stage of cementation followed by reworking and re-deposition

loses the memory of the former clast size. Such multiple reworking would more likely

dilute, not concentrate, the microfossils in the deposit. Thus, we do not find concentration

by sedimentary processes to be the most likely solution.

The interpretation of the microfossils as bacteria more easily explains their

abundance, as Thiomargarita is known to occur in great abundance (up to 200 g m-2),



which would translate to approximately 107 cells per m-2. In the Gulf of Mexico,

abundance is somewhat lower at ~ 105 cells per m-2 (see Supplementary Fig. 8).

Supplementary Figure 8: Hydrocarbon-seep sediments covered with very abundant

Thiomargarita cells. The width of the pH microelectrode tip at center is ~500 μM.

1 Dornbos, S. Q. et al., Precambrian animal life: Taphonomy of phosphatizedmetazoan embryos from southwest China. Lethaia 38, 101-109 (2005).

2 Xiao, S. and Knoll, A. H., Phosphatized animal embryos from the NeoproterozoicDoushantuo Formation at Weng'an, Guizhou, South China. J. Paleontol. 74, 767-788 (2000).

3 Xiao, S., Mitotic topologies and mechanics of Neoproterozoic algae and animalembryos. Paleobiology 28, 244-250 (2002).

4 Doyle, R. J. and Marquis, R. E., Elastic, flexible peptidoglycan and bacterial cellwall properties. Trends Microb. 2, 57-60 (1994).

5 Schulz, H. N. et al., Dense populations of a giant sulfur bacterium in Namibianshelf sediments. Science 284, 493-495 (1999).

6 Hagadorn, J. W. et al., Cellular and subcellular structure of Neoproterozoicanimal embryos. Science 314, 291-294 (2006).

7 Knoll, A. H. and Barghoorn, E. S., Precambrian Eukaryotic Organisms: AReassessment of the Evidence. Science 190, 52-54 (1975).

8 Chen, M. and Liu, K., The geological significance of newly discoveredmicrofossils from the upper Sinian (Doushantuo age) phosphorites. Scientia Geol.Sinica 4, 317-324 (1986).

9 Xiao, S., Zhang, Y., and Knoll, A. H., Three-dimensional preservation of algaeand animal embryos in a Neoproterozoic phosphorite. Nature 391, 553-558(1998).



10 Yin, C., Bengston, S., and Yue, Z., Silicified and phosphatized Tianzhushania,spheroidal microfossils of possible animal origin from the Neoproterozoic ofSouth China. Acta Palaeo. Polo. 49, 1-12 (2004).

11 Yin, L. and Li, Z., Precambrian microfloras of southwest China with reference totheir stratigraphic significance. Mem. Nanjing Inst. Geol. Palaeo., Acad. Sinica.10, 41-108 (1978).

12 Zhang, Y., Yin, L., Xiao, S., and Knoll, A. H., Permineralized fossils from theTerminal Proterozoic Doushantuo Formation, South China. Paleo. Soc. Mem. 50,52 p. (1998).

13 Xue, Y. S., Tang, T. F., and Yu, C. L., "Animal embryos," a misinterpretation ofNeoproterozoic microfossils. Acta Micropalaeo. Sinica 16, 1-4 (1999).

14 Xiao, S. and Knoll, A. H., Embryos or algae? A reply. Acta Micropalaeo. Sinica16, 313-323 (1999).

15 Xiao, S. and Knoll, A. H., Fossil preservation in the Neoproterozoic Doushantuophosphorite Lagerstätte, South China. Lethaia 32, 219-240 (1999).

16 Dornbos, S.Q. et al., Environmental controls on the taphonomy of phosphatizedanimal and animal embryos from the Neoproterozoic Doushantuo Formation,Southwest China. Palaios 21, 3-14 (2006).



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