Environmental and diagenetic controls on the morphology and calcification of the Ediacaran metazoan Cloudina

Cloudina is a globally distributed Ediacaran metazoan, with a tubular, funnel-in-funnel form built of thin laminae (ca. 1–10 μm). To what degree local environmental controlled morphology, and whether early diagenesis controlled the degree of calcification of Cloudina, is debated. Here we test these hypotheses by considering assemblages from four, coeval localities from the Upper Omkyk Member, Nama Group, Namibia, from inner ramp to mid-ramp reef across the Zaris Subbasin. We show that sinuosity of the Cloudina tube is variable between sites, as is the relative thickness of the tube wall, suggesting these features were environmentally controlled. Walls are thickest in high-energy reef settings, and thinnest in the low-energy, inner ramp. While local diagenesis controls preservation, all diagenetic expressions are consistent with the presence of weakly calcified, organic-rich laminae, and lamina thicknesses are broadly constant. Finally, internal ‘cements’ within Cloudina are found in all sites, and pre-date skeletal breakage, transport, as well as syn-sedimentary botryoidal cement precipitation. Best preservation shows these to be formed by fine, pseudomorphed aragonitic acicular crystals. Sr concentrations and Mg/Ca show no statistically significant differences between internal Cloudina cements and botryoidal cements, but we infer all internal cements to have precipitated when Cloudina was still in-situ and added considerable mechanical strength, but may have formed post-mortem or in abandoned parts of the skeleton.

Using the Kruskal-Wallis Test, the H-value for these data is 14.96, with a P-value of 0.002, indicating that the sinuosity of the Cloudina tubes varies significantly between localities. Z-tests also confirm that the sinuosity varies between all localities except between Driedoornvlakte and Zwartmodder.
Diagenetic preservation of Cloudina. The form of preservation of Cloudina varies across the Zaris subbasin transect (Table S2).
Cloudina from Zebra River are preserved as calcite surrounded by wackestone (Fig. 1E). The ornamental features of the tube are not preserved and the tube cavity is infilled with sparry calcite. Individuals show brittle and ductile deformation. Paired laminae (Figs. 4K-L) are present, and the inter-lamina space is infilled by acicular crystals (Fig. 4D,G,N) ranging from 9.4 to 15.5 µm in length (mean = 11.7 µm, n = 6) and 2.1 to 4 µm in width (mean = 3.1 µm), forming the inter-lamina cement. In some areas the laminae are undulating, with a prominent inter-funnel cement infill ( Fig. 4C-G,J-L).
Patchily luminescent inter-funnel cement is also present, formed by abundant clusters of acicular cements with blunt terminations which nucleate from the laminae (Fig. 4B,D,E,G). A dull luminescent, pore-lining patchy cement ( Fig. 4C-G) overlies inter-funnel cements. Later tube cavity infill consists of calcite spar with duller luminescence (Fig. 4B). In Cloudina riemkeae, the acicular cements are finer and pore-lining cements are not observed, but inter-funnel cements with dull luminescence are present, followed by more luminescent pseudomorphosed aragonitic botryoids which nucleate from the outer tube walls (Fig. 4H-I). There is variation in the preservation of this inter-funnel cement, which varies even within a single Cloudina tube: inter-funnel cements  The cloudinomorphs of Omkyk are dark in colour and composed of large (0.1-1 mm) calcite spar crystals (Fig. 1F). These also lack ornamentation, but some retain the stacked funnel-in-funnel structure 7 . Individuals show brittle and ductile deformation. CL highlights three generations of cements within the cloudinomorph tube ( Fig. 5A,B,D). First a thin (200 µm) acicular cement generation, which is not continuous throughout the tube cavity, followed by an isopachous cement with patchy or dull-luminescence with limited zonation (Fig. 5D). The tube cavity is infilled by zoned sparry cement, which nucleates from the isopachous cement (Fig. 5D).
Zwartmodder Cloudina are also preserved wholly as coarse calcite spar (Figs. 1G, 6A,C). Fine external features, including external phlanges and the annulated outer wall, are well preserved (Fig. 1G), and Cloudina shows evidence of brittle deformation (Fig. 6C). The Cloudina skeleton is preserved a mould, infilled by a centripetal, sparry calcite cement with dull luminescence that becomes brighter towards the centre of the moulds (Fig. 6A,B). Individual laminae cannot be detected, but inter-lamina and inter-funnel cement are present as sparry calcite (Fig. 6F). Individual or paired laminae are evident through the spalling of the tube walls in areas where interfunnel cement is not present and are instead separated by micrite infill (Fig. 6F). Under CL, the micrite shows three zones of cement growth, dull-luminescent followed by bright luminescent, and a final non-luminescent zone (Fig. 6B,E,F). These cement zones protrude into the sparry calcite infill, which can also be seen under SEM along with small, cube shaped holes (Fig. 6G).
Elemental distribution. Strontium (Sr) concentrations and the Mg/Ca ratios were sampled from Cloudina and associated early cements from Driedoornvlakte and Zebra River using EMPA (See Methods, Supplementary Information; Fig. 7; S1; Tables S3-S6).
The Kruskal-Wallis test shows a statistical difference between the Sr concentration of different cements at Zebra River, between the inter-laminae cements and both laminae (P = 0.016) and inter-funnel cements (P = 0.014), and between the micritic matrix and both inter-laminae cement (P = 0.001) and inter-funnel cement (P = 0.030) (Table S7). There are no statistical differences in Mg/Ca contents of any measured features (Table S8).
Cloudina riemkeae from Driedoornvlakte also shows statistical differences in Sr concentration between the inter-laminae and the inter-funnel cements (P = 0.041) (Table S9), and between inter-laminae and the inter-funnel www.nature.com/scientificreports/ cements compared to the Cloudina wall (P = 0.009 and P = 0.002, respectfully) (Table S9). Sr content of the interlaminae cement and Cloudina wall are statistically different to the inclusion-rich spar (P = 0.023 and P = 0.011, respectfully), but this is not the case for the inter-funnel cement (P = 0.291) which is recrystallised as an inclusionrich spar (Fig. 3E,J; Table S9). There is a significant difference between the Cloudina wall at Driedoornvlakte and the other Cloudina-associated cements, probably as a result of the dolomitisation. Sr content within internal cements associated with Cloudina hartmannae show no statistical differences, but there is a significant difference between the inter-cloudinomorph cement and botryoidal cements (P = 0.043) (Table S10).
There are no statistical differences in Mg/Ca contents of any measured features at Driedoornvlakte apart from the Cloudina wall of the Cloudina riemkeae, due to selective dolomitisation (Tables S11, S12).

Lamina thickness.
Organic laminae have not been preserved at the studied sites. Laminae are preserved as moulds at Zebra River and Zwartmodder (Figs. 4,6), at Driedoornvlakte they have been dolomitised (Fig. 3), www.nature.com/scientificreports/ whilst at Omkyk laminae are not detectable at all (Fig. 5). Laminae thickness at Zebra River and Driedoornvlakte were measured using both CL and PPL images and using CL at Zwartmodder. Cloudina of Driedoornvlakte and Zebra River have laminae of similar thicknesses, with Driedoornvlakte laminae thicknesses ranging from 2.1 to 9.6 µm (mean = 5 µm, n = 23) and Zebra River Cloudina laminae ranging from 3.3 to 10.8 µm in thickness (mean = 6.5 µm, n = 23), with a T = 1.872, and P-value = 0.034 (Fig. 8A).

Wall thickness.
Cloudina wall thickness is defined as the width between the outer tube wall and the inner tube wall forming the internal cavity ( Fig. 2A). Measurements of wall thickness were taken from bedding surfaces at Driedoornvlakte, Zebra River and Zwartmodder. At Driedoornvlakte, maximum wall thickness for any individual Cloudina tube ranges from 0.25 to 1.88 mm (mean = 0.93 mm, n = 147) (Fig. 8C). Ranges were lower at Zebra River and Zwartmodder, from 0.16 to 1.43 mm (mean = 0.51 mm, n = 79) and 0.12-1.38 mm (mean = 0.42 mm, n = 87), respectively (Fig. 8C).
When comparing maximum wall width: maximum tube width, Cloudina at Driedoornvlakte show the greatest ratios ranging from 0.077 to 0.505 (mean = 0.235), with Zwartmodder ranging from 0.079 to 0.434 (mean = 0.190), and Zebra River showing the smallest range from 0.065 to 0.375 (mean = 0.235) (Fig. 8C). All localities show a weak positive correlation between the thickness of the tube and the maximum wall thickness (Fig. 8D). Ratios are statistically different between Driedoornvlakte and both Zebra River and Zwartmodder (P = 0.031 and P = 5.77 -5 , respectively), but not between Zebra River and Zwartmodder (P = 0.060).

Sinuosity of Cloudina. Cloudina at Driedoornvlakte and Zwartmodder show the lowest sinuosity, and
Zebra River the highest (Fig. 2B,C). This systematic variation (Table S2; Fig. 8E) suggests that this feature is in some way environmentally dictated, and that mineralisation of Cloudina occurred via a flexible organic templates that allowed adaptation to local conditions. Very little is known as to the controls on sinuosity in modern benthos, but orientation to maximise feeding efficiency in ambient currents and local sedimentation regime is thought to be a major control in calcareous tubed polychaetes (serpulids) 28,29 . Creating hypotheses as to what advantage might be conferred by increased sinuosity is therefore problematic, but sinuosity may be governed by diverse factors such as substrate type and morphology, vertical or horizontal growth, competition for space, nutrient regime, water depth, and response to hydrodynamic energy and water flow.
We note that sinuosity measurements are based on 2D bedding plane measurements, and further insight might be gained by 3D analysis for both more accurate sinuosity quantification, as well as information about curvature in the third dimension.  (Table S2). We have not observed the micritic microstructure described for the walls of Cloudina, but these modes of preservation are consistent with laminae being organic-rich but calcified. Moldic preservation at Omkyk and Zwartmodder has resulted in the absence of preserved skeletal walls. Early acicular isopachous cement generations are present at Omkyk, which grew from the cloudinomorph walls, followed by an infilling well-zoned clear sparry burial calcite 7 (Fig. 5). Cloudina are preserved only via a sparry infill of moulds at Zwartmodder, which represent dissolved paired laminae together with inter-lamina cement and inter-funnel cement (Fig. 6). Dissolution pre-dated cement growth around the grains of the surrounding sediment as this cement grew into Cloudina moulds (Fig. 6E).
The extent of dissolution at these shallow, inner ramp localities suggest the influence of freshwater via early meteoric diagenesis, perhaps associated with the degradation of the organic material, which would preferentially remove aragonite. By contrast, the mid-ramp sites Driedoornvlakte and Zebra River, preserve very early botryoidal pseudomorphed aragonitic cements formed in cavities and pores within the marine phreatic zone.
Zebra River Cloudina show undulose laminae, with some evidence of both brittle fracturing and ductile deformation (Fig. 4G), in contrast to Driedoornvlakte where there is only evidence of brittle deformation (Table S2). Compaction also caused breakage of Cloudina tubes at Zebra River and Zwartmodder, similar to that of a spalling ooid, where the resultant area was infilled by sediment (Figs. 4G, 6F). Inter-lamina and inter-funnel cements formed prior to the breakage of the Cloudina tubes, and that of the laminae, where moulds were later filled by a burial cement (Fig. 4M,N). The formation of the dull luminescent inter-funnel cement, seen most clearly at Zebra River, occurred before compaction and lithification of the surrounding dolomitised sediment as evidenced by www.nature.com/scientificreports/ the sharp fracture of the inter-funnel cement where sediment has encroached into the tube (Fig. 4J-L). All these cements also formed prior to the precipitation of pseudomorphed aragonitic botryoidal cements both inside and outside the tubes (Figs. 3B, 4B). Botryoids external to Cloudina tube have brighter luminescence than those within the tube, suggesting some degree of diagenetic compartmentalisation. At Zebra River, the pseudomorphed aragonitic acicular crystals are not preserved in the outermost inter-lamina cements, but rather were replaced by the non-luminescent neomorphic cements later in diagenesis.
Inter-lamina, inter-funnel, intra-cloudinomorph, and inter-cloudinomorph cements are all composed of fine, acicular crystal bundles (mean width = ca. 3-5 µm, length = ca. 11 µm) that nucleated on both Cloudina laminae and from the outer wall of the tube. Crystal terminations are blunt and are inferred to be pseudomorphed aragonite. All precipitated prior to transport and breakage of the tubes, and also pre-dated the cement botryoids, so can be inferred to be very early syn-sedimentary. The sparry calcite noted previously 15 is either neomorphic or burial spar that formed after the replacement or dissolution of these original cements.
Inter-funnel cements, first described by Grant 3 have also been documented in Cloudina from Brazil 14,30 , Paraguay 9 , and Spain 31 , suggesting that such cements are a widespread feature of Cloudina present irrespective of early diagenetic setting, mineralogy, or palaeogeographic region. These cements probably formed when Cloudina was in-situ and provided mechanical strength and rigidity to the tube.
These cements are similar to those described from the skeleton in the extant sphinctozoan sponge Vaceletia, suggested to have a basal mode of biomineralisation 32 . Here, the skeleton is secreted upon a non-collagenous organic template, which becomes substituted by crystalline aragonite deposited as tangled crystal bundles of aragonite. The organic framework consists of proteins and polysaccharides rich in galactose, glucose and fucose, the latter suggesting that bacterial EPS (exopolymeric substances) may be involved in calcification 33 . In most cases, the basal parts of the skeleton, which is free from living tissue, is infilled by a micritic granular secondary deposit. The presence of organic matter has led to the suggestion of biofilm or microbial involvement in such cement precipitation for Cloudina 14 . Similar secondary deposition can occur where aragonite crystals continue www.nature.com/scientificreports/ to grow after soft tissue has vacated a region of the skeleton. This is known in taxa as diverse as scleractinian corals 18  Elemental signatures. Elemental signatures of similar cements cannot be compared directly between localities because of differing diagenetic histories, but statistically significant differences between phases can be determined for each locality. First, we note statistical differences in Sr concentration between various Cloudinaassociated cements and botryoidal cements. This potentially indicates that the Cloudina-associated cements were of a different origin. However, the botryoidal cement used for this comparison is located within Cloudina tubes, adjacent to intra-cloudinomorph cements and here there is no significant difference in Sr values (P = 0.197) (Table S10). When comparing the Sr content of inter-cloudinomorph cement to those measured from botryoids outside Cloudina tube 34 , no significant difference is found between these cements. This suggests that all cements found within the Cloudina tube irrespective of type retain a higher concentration of Sr compared to those cements situated outside tubes, where leaching was more extensive. This is supported by the higher mean Sr concentration of the intra-cloudinomorph cement compared to the inter-cloudinomorph cement. We find no statistical differences between the Cloudina-associated cements, the inorganic botryoids and dolomitised sediment, which suggests that they cannot be distinguished using this criterion. A similar conclusion was reached from study of Sr content of Cloudina from the Tamengo Formation of the Corumbá Group, Brazil 14 . On the basis of their timing of precipitation and the acicular, but non-botryoidal, texture, we conclude that all internal cements associated with Cloudina precipitated very early, but lack any distinctive Sr or Mg/Ca signature that might indicate either a diagenetic origin from a different pore fluid or biological fractionation.
Lamina thickness. The variation of paired lamina thicknesses noted could be due to deformation between the laminae, especially at Zebra River, as laminae are observed to be flexible at this site. However, these differences are more likely due to the different methods used to measure paired laminae thickness: laminae at Zebra River and Driedoornvlakte were measured using both CL and PPL images, but the CL images show thinner www.nature.com/scientificreports/ laminae compared to their PPL counterparts (Fig. 8B). When comparing data of laminae thickness collected from PPL images only, the data sets are not statistically different (T = 0.57) and so the null hypothesis that the paired laminae thickness at Zebra River and Driedoornvlakte is the same is supported, but due to the small sample size this is not significant (P-value = 0.58). This is not the case, however, when comparing the CL data, as T-Test values indicate the paired laminae thickness varies (T = 2.54, P-value = 0.029), especially when spalled laminae are not included with the calculations (T = 4.75, P-value = 0.002). When comparing the thickness of the moldic-paired laminae seen at Zwartmodder, the values fall in the range of paired laminae at other sites. This suggests that the assumed moldic laminae are paired laminae combined with inter-lamina cements, as observed at Driedoornvlakte and Zebra River.
Presumed Cloudina laminae at Zwartmodder also occupy a narrower range of paired laminae thicknesses than those from Driedoornvlakte and Zebra River (mean = 15.4 µm). These laminae are expressed as sparry-calcite infilled moulds formed by the dissolution of both the paired laminae and the inter-lamina cement, and so this dissolution may account for the increased range of laminae thickness at Zwartmodder.
Cloudina laminae from the Mooifontein Member have a thickness of 0.5-5 µm, and samples from Paraguay range between 0.5 and 8 µm 15 , so falling within the overall range found in this study (Fig. 8A). Although we note greater lamina thicknesses, we consider these to be likely artefactual due to thickening by dolomitisation at Driedoornvlakte.
Variability of wall thickness. The maximum thickness of the Cloudina wall and the thickness of the wall as a ratio of tube diameter is variable across the Zaris Subbasin (Fig. 8D,E). The weak positive correlation between the thickness of the tube and the maximum wall thickness at all localities suggests that the wall thickness was not a function of tube width. These data show that for a given tube width, the thickness of the wall is greatest at Driedoornvlakte, which is significantly and statistically greater than those at other sites.
This implies that wall thickness is environmentally-controlled, determining the distance between paired laminae sets and also potentially the volumetric extent of inter-lamina and inter-funnel cement formation. Driedoornvlakte was the most hydrodynamically energetic of those localities analysed, where rates of carbonate precipitation may have been higher, as shown by the abundant, volumetrically-significant syn-sedimentary botryoidal cements 27 . Such a regime may have promoted more rapid precipitation, and increased volumes, of internal cements. This is consistent with the observation that only brittle fracture is noted at Driedoornvlakte. Many other environmental parameters might have been important to produce a robust, more heavily calcified, and strong skeletal wall in this setting, however, such as enhanced food availability or as a response to currents.

Conclusions
The consistent lamina thickness of Cloudina along the Zaris Subbasin shelf suggest that lamina formation was under biological control (Fig. 8E). The moldic or replacive dolomitised preservation of lamina indicate calcification of an organic-rich structure, potentially during life, from which early, acicular pseudomorphed aragonitic cements could nucleate. The precipitation of these cements pre-dates breakage prior to sediment infill, transport, and pseudomorphed aragonitic botryoid precipitation. The presence of such internal cements is a widespread feature of Cloudina, although diagenetic expression varies. Geochemical analysis (Mg/Ca; Sr concentrations), however, shows no statistically significant differences between these cements and the surrounding sedimentary matrix, and so no signature of biological fractionation is detected. We conclude that these cements associated with Cloudina formed rapidly, but it is not clear if they formed during life, post-mortem, or in parts of the Cloudina skeleton that were abandoned by soft-tissue as the animal grew to occupy younger parts of the skeleton. But the formation of these cements, particularly the inter-lamina and inter-funnel cements, would impart rigidity to the Cloudina tube, and the inter-cloudinomorph cements would create attachment between adjacent tubes.
The variation of sinuosity in Cloudina in different populations across the ramp of the Zaris Subbasin (Fig. 8E) implies that the curvature of the tube is environmentally-controlled, perhaps to maximise feeding efficiency in any given setting. This complements the findings that Cloudina tube diameter is also environmentally variable within the Nama Basin 10 . Variability in the cloudinomorph wall thickness is not a function of tube width and also differs between localities (Fig. 8E), further suggesting the influence of environmental factors in determining the distance between paired laminae sets and the volumetric extent of inter-lamina and inter-funnel cement formation. This may have been controlled by factors such as carbonate supersaturation or hydrodynamic energy, as thicker walls and only brittle fracture are noted in high-energy reef settings.

Material and methods
ImageJ (Fiji) software (https://imagej.net) was used to quantify the size of features from photographs, hand specimens, and thin sections. Sinuosity, the degree of curvature, of the cloudinomorph tubes and wall thickness from bedding surface images was determined using ImageJ. Sinuosity, a term mostly associated with river morphology, is defined by dividing the length of an object by the length of the straight-line distance from bedding plane surfaces (see Fig. 2A). Values of < 1.1 indicate straight linear objects with higher values indicating increasing sinuosity. Percentage shortening is the amount of shortening of the tube in comparison to the original and assumed straight Cloudina tube and is calculated as a percentage ((straight line distance/ midline distance)*100/ midline distance). A large number of measurements were obtained to overcome any systematic bias due to use of 2D measurements.
Highly-polished thin sections were used for plane polarised light (PPL) and cathodoluminescent (CL) petrography on a cathodoluminescence Cold Cathode CITL 8200 MK3A attached to a Nikon optiphot microscope at the University of Edinburgh. Samples from Zwartmodder were imaged using a Carl Zeiss SIGMA HD VP Field Emission scanning electron microscope (SEM) at the University of Edinburgh. Sections from Driedoornvlakte www.nature.com/scientificreports/ and Zebra River were used to quantify major element concentrations (Ca, Mg, Sr) of Cloudina and associated diagenetic components via Electron Microprobe analysis (EMPA) following CL images to test for differences in original mineralogy, diagenetic phase, or evidence of vital fractionation. EMPA was undertaken on a Cameca SX100 Electron Microprobe at the University of Edinburgh using a 80 s count time, a beam diameter of 3 µm, an accelerating voltage of 15 kV, and a beam current of 35 nA. All data were statistically analysed using the Kruskal-Wallis Test, after data normalisation, using MS Excel 2016. Z-Tests were used on sample sizes where n > 50, such as sinuosity, and T-Tests were undertaken where sample sizes were n < 50 to provide a statistical comparison of each site using MS Excel 2016, variance was tested to determine which T-Test function to use, i.e. whether data sets had equal or unequal variance.