Ecological interactions in Cloudina from the Ediacaran of Brazil: implications for the rise of animal biomineralization

At the Ediacaran/Cambrian boundary, ecosystems witnessed an unparalleled biological innovation: the appearance of shelled animals. Here, we report new paleoecological and paleobiological data on Cloudina, which was one of the most abundant shelled animals at the end of the Ediacaran. We report the close association of Cloudina tubes with microbial mat textures as well as organic-rich material, syndepositional calcite and goethite cement between their flanges, thus reinforcing the awareness of metazoan/microorganism interactions at the end of the Ediacaran. The preservation of in situ tubes suggests a great plasticity of substrate utilization, with evidence of different life modes and avoidance behavior. Geochemical analysis revealed walls composed of two secondary laminae and organic sheets. Some walls presented boreholes that are here described as predation marks. Taken together, these data add further information regarding the structuring of shelled animal communities in marine ecosystems.


vendotaenids (Vendotaenia antique, Eoholynia corumbensis), characterize Tamengo
Formation (Zaine andFarichild 1985, 1987;Zaine 1991;Gaucher et al. 2003;Pacheco et al. 2011;Fairchild et al. 2012). More specifically, Tamengo Formation comprises fine limestone with siliciclastic intercalation and intraclastic breccia at the base, changing towards the top to shales, bioclastic grainstones and rudstones with hummocky crossstratification and fine limestone (Supplementary Figure S1). This sequence is interpreted as a shallow marine environment that sometimes displays more deep water character (Spangenberg et al. 2013). The presence of grainstones and rudstones together with hummocky cross-stratification suggests intermittently high-energy events in an environment usually with low-energy (Spangenberg et al. 2013).
The analyzed fossil samples were previously collected in Tamengo Formation (see Zaine 1991;Meira 2011) and are deposited at the Paleontological Collection of the Institute of Geosciences (University of São Paulo -USP). Some thin sections 8/19) showing microbial mats are from limestones from the rhytmite facies (limestone-marls) of Laginha quarry, Corumbá region, supported by a profile with ~70 m thick. The Laginha section was previously described by lithological features and the depositional environment interpretation followed their description (for more detail see Boggiani et al. 2010). The basal section consists of polymictic carbonate breccias interpreted as slope deposits. It is overlaid by massive fine limestone (mudstone and wackestone) and oolitic/bioclastic grainstones, followed by a thick package of rhytmite of limestone-marls representing deposits of deep water to transitional conditions. The uppermost section consists of grainstone with ooids and bioclast defined as shoal shallow water. Cloudina associated with microbial textures were found in mudstones of one outcrop in Ladário city, defined by a profile of ~13 m, composed mainly by mudstone with intercalated levels of 1-2 m thick 4 pelites (Supplementary Figure 1). Lithologically correlated with these, thin sections and hand-samples with fine limestone also presented autochthonous Cloudina (e.g. GP/L1E-41-42; GP/1E-6218).

Supplementary Text 2. Diagnostic characteristics of boring holes
Boring holes in Cloudina were already reported for phosphatized specimens from Dengying, China (Bengston and Zhao, 1992;Hua et al., 2003) and two calcified individuals from Nama Group (Brain, 2001). These structures were considered to represent the oldest evidences of the fossil record for predatory activity in animals. The presence of holes in fossils of Cloudina from Tamengo Formation marks the third occurrence and geographically expands the possible evidences of predation near the Ediacaran/Cambrian boundary.
Circular holes in marine invertebrates, when of predatory origin (i.e. boreholes or drillholes), constitute an important tool for measuring ecological interactions in the fossil record. However, differentiating holes produced by predators from that originated by other mechanisms is not so simple (Kowalewski, 2002 (Kowalewski, 2002;Harper, 2003).
The holes analyzed here meet the criteria 1 to 3, and possibly the criteria 6. Until now, there are no reports of holes on Corumbella, and in the same way for Cloudina and Sinotubullites of Dengying Formation, this could represent selectivity for prey species. While the low 5 sample size of bored specimens from Tamengo Formation precludes statistical analysis to test location and/or size selectivity (criteria 4 and 5), these conditions were observed in Dengying Formation (Bengtson and Zhao, 1992;Hua et al., 2003), together with the presence of incomplete holes (criteria 7).
However, besides predation, there are other explanations for the origin of the Cloudina holes, and these possibilities need to be considered to a better comprehension of these structures. While predation was proposed, others authors also postulated some alternative hypothesis: dissolution of microdolomite crystals, decomposition by microorganisms, microborings, and parasitism (Debrenne & Zhuravlev, 1997;Zhuravlev et al., 2012).
Even if the phosphatized specimens of the Dengying Formation suffered dissolution processes for preparation of the fossil material, both Brazillian and Namibian material are calcified, and an origin by dissolution of microdolomite crystal can be ruled out.
Additionally, the holes in the fossils illustrated by Zhuravlev et al. (2012) interpreted to be formed by dissolution of microdolomite, are usually of a quadrangular outline. This shape is not observed in neither of the other occurrences (Bengtson and Zhao, 1992;Brain, 2001;Hua et al., 2003;this study). Thus, while it is possible that processes of dissolution created the patterns observed by Zhuravlev et al. (2012), the origin of the holes in Tamengo, Dengying and Nama units may rely on another cause. Zhuravlev et al. (2012) also suggested that microbial decomposition was a possible hypothesis for the origin of the holes. In fact, Hof and Briggs (1992) observed circular pits on crustaceans cuticle caused by the activity of bacteria. But these circular holes had a highly variable size (<10 μm e ≥100 μm, based on Fig 2 of Hof and Briggs, 1992) and high density and proximity of the holes (Hof & Briggs, 1992 : Fig 2A). This situation was not seen in the holes of Cloudina, since they commonly occur as single and isolated holes.

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Although there are parameters to identify a predatory boring hole, it is not always possible to satisfactorily distinguish between holes caused by predators from that produced by ectoparasites (Baumiller et al., 1999;Kowalewski et al., 2000, Kowalewski, 2002Harper, 2003). Nevertheless, there are some lines of evidence that can indicate parasitic origin for circular pits, such as fixations scars and various perforations in the same individual (Matsukuma, 1978;Baumiller, 1990;Kowalewski, 2002). Both situations were not found in perforated Cloudina from Brazil, China or Namibia. -2 ) and primary (D-band, disordered C-C, in green) and secondary (G-band, graphitic C-C, in orange) bands of kerogen.