Paleobiology of the Early Cambrian Yanjiahe Formation in Hebei Province of South China

Home , Dengying Formation, Maceration (bone), Yanjiahe Formation

Jesse Broce

Thesis submitted to the faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Master of Science In Geosciences

Shuhai Xiao Benjamin C. Gill James D. Schiffbauer

April 30, 2013 Blacksburg, VA

Keywords: Acetic, Acid, Annulations, Brachiopod, Calcite, Cambrian, Cycloneuralia, Chancelloria, China, Diagenesis, Ecdysozoan, EDS, Embryo, Fossil, Grooves, Hyolith, Introverta, Limestone, Maceration, Markuelia, Micro-CT, Microfossil, Panarthropoda, Phosphatization, Priapulid, Pseudooides, Pyrite, Scalidophoran, SEM, Sponge, Taphonomy, Worm, Yanjiahe

Paleobiology of the Early Cambrian Yanjiahe Formation in Hebei Province of South China

Jesse Broce

ABSTRACT

Fossils recovered from limestones of the lower Cambrian (Stage 2-3) Yanjiahe Formation in Hubei Province, South China, recovered using acetic acid maceration, fracturing, and thin sectioning techniques were examined using a combination of analytical techniques, including energy dispersive spectroscopic (EDS) elemental mapping and micro-focus X-ray computed tomography (micro-CT). One important fossil recovered and analyzed with these techniques is a fossilized embryo. Fossilized animal embryos from lower Cambrian rocks provide a rare opportunity to study the ontogeny and developmental biology of early animals during the Cambrian explosion. The fossil embryos in this study exhibit a phosphatized outer envelope (interpreted as the chorion) that encloses a multicelled blastula-like embryo or a calcitized embryo marked by sets of grooves on its surface. The arrangement of these grooves resembles annulations found on the surface of the Cambrian-Ordovician fossil embryo Markuelia. Previously described late-stage Markuelia embryos exhibit annulations and an introvert ornamented by scalids, suggesting a scalidophoran affinity. In the Yanjiahe fossils illustrated herein, however, the phosphatized chorions and blastulas are not taxonomically or phylogenetically diagnostic, and the late-stage embryo is secondarily calcitized and thus poorly preserved, with only vague grooves indicative of Markuelia-type annulations. Consequently, their taxonomic assignment to the genus Markuelia is uncertain. If they indeed belong to the genus Markuelia, they are the oldest known Markuelia fossils from China, and represent both a new occurrence and possibly a new species.

ACKNOWLEDGMENTS

I am very thankful for the assistance and guidance provided by the Virginia Tech

Department of Geosciences, the faculty as well as the administrative staff. In particular, I would like to thank my advisor, Dr. Shuhai Xiao, whose incredible expertise and guidance have been invaluable to me in the completion of this project, as well as my personal development. I would like to thank the faculty and staff at the Virginia Tech Institute for Critical Technology and

Applied Science Nanoscale Characterization and Fabrication Laboratory for access to and training in the operation of the SEM. I would also like to thank Dr. Ge Wang and Kriti Sen

Sharma for assistance and training in the use of the Micro-CT. Financial support was provided by the Virginia Tech Geosciences Department, ICTAS and the National Science Foundation.

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TABLE OF CONTENTS

ABSTRACT …………………………………………………………………………………..… ii

ACKNOWLEDGEMENTS ………………………………………………………..…………… iii

TABLE OF CONTENTS ……………………………………………………………………….. iv

TABLE OF FIGURES …………………………………………………………………………... v

GEOLOGICAL BACKGROUND ………………………………………………………………. 1

FOSSIL ASSEMBLAGE ……………………………………………………………………….. 3

CAMBRIAN FOSSIL EMBRYOS ……………………………………………………………... 4

EMBRYO EXTRACTION AND EXAMINATION ……………………………………………. 7

FOSSIL DESCRIPTION ………………………………………………………………………... 9

Chorion ………………………………………………………………………………….. 9

Blastula-like embryos ………………………………………………………………….. 10

Fossils with grooves ……………………………………………………………………. 11

TAXONOMIC INTERPRETATION ………………………………………………………….. 12

TAPHONOMIC MODE ……………………………………………………………………….. 15

CONCLUSIONS ………………………………………………………………………………. 17

FIGURES AND FIGURE CAPTIONS ………………………………………………………... 19

REFERENCES ………………………………………………………………………………… 23

APPENDIX A: FIGURE REPOSITORY ……………………………………………………… 26

TABLE OF FIGURES

Figure 1. Geologic map and stratigraphic column …………………………………………… 19

Figure 2. Chorions and blastula-like fossils …………………………………………………… 20

Figure 3. Chorions and fossils with sets of parallel grooves ………………………………….. 21

Figure 4. Markuelia sizes through time ……………………………………………………….. 22

APPENDIX A: FIGURE REPOSITORY …………………………………………………… 26

Appendix A.1 Thin section images …………………………………………………….. 26

Appendix A.2 Macerated embryos …………………………………………………….. 52

Appendix A.3 In-situ …………………………………………………………………... 67

Appendix A.4 Counterpart ……………………………………………………………... 84

Appendix A.5 Interior ………………………………………………………………….. 85

GEOLOGICAL BACKGROUND

The fossils reported here were collected from the early Cambrian (Stage 1) Yanjiahe Formation at Wangzishi, near Changyang, Hubei Province, South China (Fig. 1.1). Geologically, the

Wangzishi area was situated on the Yangtze Platform, part of a southeast facing passive continental margin formed after the break-up of Rodinia supercontinent (Wang and Li, 2003).

During the early Cambrian, carbonates were deposited on a shallow platform and shales in a deep basin paleoenvironment, but intercalated carbonates and shales were deposited in the transitional facies (Jiang et al., 2012). The sampled section at Wangzishi is specifically located within this transitional facies, where the Yanjiahe Formation consists of both carbonates and shales.

At Wangzishi, the Yanjiahe Formation overlies a thick-bedded dolostone of the

Ediacaran Dengying Formation and underlies black shales of the lower Cambrian (Stage 3)

Shuijingtuo Formation. The contact between the Dengying and Yanjiahe formations is an exposure surface likely representing a <5 myr unconformity based on regional correlation (Jiang et al., 2012). The Ediacaran–Cambrian boundary, however, is probably present within the lower

Yanjiahe Formation, based on carbon isotope chemostratigraphic and acritarch biostratigraphic correlations (Yao et al., 2005; Dong et al., 2009; Jiang et al., 2012). The contact between the

Yanjiahe and Shuijingtuo formations represents another sequence boundary, followed by rapid transgression as recorded by a thick succession of black shales with large carbonate concretions in the lower Shuijingtuo Formation. The Shuijingtuo Formation contains trilobite fossils and is regarded as Cambrian Stage 3 strata. From these bounding packages, the Yanjiahe Formation is constrained to be lower Cambrian (Stage 1–3), and the Wangzishi fossils described herein were

1 collected specifically from the uppermost Yanjiahe Formation, likely Cambrian Stage 2–3 (see below).

The lower Yanjiahe Formation at Wangzishi consists predominantly of shale, with thin- bedded phosphorite and chert. The middle Yanjiahe Formation contains interbedded limestones and shales. The upper sixteen meters of the Yanjiahe Formation consist of medium- to thick- bedded argillaceous limestones (Fig. 1.2), succeeded by lower Shuijingtuo black shales. The fossils described in this paper were collected from the upper argillaceous limestone unit of the

Yanjiahe Formation. Equivalent strata in the Yangtze Gorges area contain small shelly fossils characteristic of the Watsonella crosbyi Assemblage Zone (Chen et al., 1984; Steiner et al.,

2007), which has been correlated with the Tommotian Stage in Siberia and has been proposed as a biozone for the basal boundary of Cambrian Stage 2 (Li et al., 2011; Peng et al., 2012). To ensure accurate dating of the Wangzishi section, examination of the fossils was conducted via acetic acid maceration.

FOSSIL EXTRACTION

The Wangzishi section (30º33’01.9”N, 111º08’55.3”E) was measured downsection from the

Yanjiahe-Shuijingtuo boundary, with samples collected at one-meter intervals from the most fossiliferous horizons of the upper 16 meters of the Yanjiahe Formation. Limestone samples were digested using acetic acid maceration techniques (in a solution of 5% acetic acid, 45% water, and 50% of a calcium acetate buffer) in order to extract microfossils from rock matrix.

The calcium acetate solution was produced by previous acetic acid maceration of limestones and was used to prevent excessive etching of phosphatic fossils (Jeppsson et al., 1985). Macerate residues were extracted daily and acid was changed bi-weekly. The extracted residues were

2 washed in clean water, allowed to dry, and subsequently examined under a dissecting microscope to isolate and remove microfossils.

FOSSIL ASSEMBLAGE

Using this method, we recovered abundant sclerites of the coeloscleritophoran genus

Chancelloria. These sclerites acted as spiny armor for the Chancelloria organism. The sclerites of Chancelloria, like those in this study, are calcareous and hollow, which makes them vulnerable to acetic acid dissolution. As a result all Chancelloria sclerites were severely etched.

The interior of the sclerites was thought to be originally filled by soft tissue (Parkhaev, 2010)

The sclerites from this study, when completely preserved, most commonly exhibit six spines radially arranged on a single plane, and a seventh spine perpendicular to the other spines. Two of the recovered chancelloria sclerites exhibit a total of eight spines, with seven of those arranged radially and the eighth perpendicular. One way to ensure that these are Chancelloria sclerites, and not sponge spicules is to look for the basal pores, an opening in each spine located on the side of the sclerite opposite the perpendicular spine, but these sclerites are too damaged by the acid to see features like basal pores. (Parkhaev, 2010) Another common fossil type was calcareous, monaxon sponge spicules. These can be up to 2 mm in length and are below 100 μm in diameter. A single fragment of what might be a hyolith was recovered from the sample material. It is phosphatic, and exhibits transverse ridges.

We also recovered abundant linguliform brachiopods (mostly of the genera Eohadrotreta,

Palaeobolus, or Lingulotreta), which have much in common with brachiopods found in the lower Shuijingtuo Formation (Cambrian Stage 3) at other localities (Li and Holmer, 2004). The discrepancy between small shelly fossil-based and brachiopod-based biostratigraphic correlations

3 could be due to diachroniety of the Yanjiahe-Shuijingtuo lithostratigraphic boundary (i.e., the upper Yanjiahe Formation may be Cambrian Stage 2 in the Yangtze Gorges area but Cambrian

Stage 3 in the Wangzishi area); regardless, it is safe to conclude that the fossiliferous limestone unit of the upper Yanjiahe Formation is constrained to lower Cambrian Stage 2–3 deposits.

Some fossil brachiopod valves, in thin section, exhibit clay or pyrite association on one or both sides. Pyrite precipitation in marine sediments requires HS-, which can be produced by the decay of organic matter by sulfur reducing microbes. HS- requires anoxic, low pH, high Eh conditions to remain in solution, so pyrite precipitation favors restricted conditions around decaying organic matter (Faure, 1998). These conditions would be easier to produce on the underside of brachiopod valves, where the porewater was more isolated from oxygen in the water column, and the inside of brachiopod valves, where more organic matter was present.

Similarly, since clays settle out of the water column, clays might prefer the upper surface of brachiopod valves. Through EDS element mapping of brachiopod valves in thin section, association of pyrites and clays relative to brachiopod valves (above or below and inside or outside) was measured, but pyrite and clays did not show a preference to any particular side of brachiopod valves.

CAMBRIAN FOSSIL EMBRYOS

Some of the earliest metazoan fossils with clear affinities to extant animal clades appeared in the early Cambrian, several examples of which are known primarily from embryonic forms. With resolved phylogenetic affinities, these embryos provide crucial information about animal ontogeny and developmental biology near the root of the metazoan phylogenetic tree. Several

Cambrian fossil embryos, including Olivooides, Markuelia, and Pseudooides, offer

4 unprecedented insights into the evolutionary developmental biology of cnidarians, scalidophorans, and possibly arthropods, respectively (Dong et al., 2004; Steiner et al., 2004;

Dong et al., 2010; Dong et al., 2013). Of these, Markuelia and Pseudooides are relevant to the fossils described in this paper for comparison purpose.

Markuelia comes in a wide range of sizes, from 275 μm (Haug et al., 2009) to 500 μm

(Bengtson and Yue, 1997). While no adult Markuelia fossils are known, some exceptionally preserved embryos appear to be at a very late stage of development, providing hints about adult characteristics. These late-stage Markuelia fossils appear wormlike, and are coiled into a spherical or subspherical embryo. The mouth, revealed by synchrotron X-ray tomography, is located inside the introvert that is covered by scalids (Donoghue et al., 2006a). Annulations extend through length of the entire trunk, as do spines in some species (M. secunda, M. lauriei,

M. qianensis, and M. waloszeki), and the tail ends with three pairs of spines (Dong et al., 2010).

This annulated wormlike morphology with several rings of scalids around an introverted mouth suggests a scalidophoran affinity, closely resembling modern priapulid worms. This taxonomic placement is supported by cladistic analysis (Dong et al., 2010), which positions Markuelia within the scalidophoran total group. As such, Markuelia establishes phylogenetic importance as one of the earliest fossil ecdysozoans.

A potential sister group representative to the cycloneuralians, Pseudooides is a possible arthropod embryo exhibiting germ band formation (Steiner et al., 2004). Pseudooides specimens from a wide range of developmental stages have been described, providing a nearly complete embryonic developmental sequence. The cleavage stage embryos display irregular radial cleavage. A median furrow and a central pore then develop, either of which could represent the blastopore. Four curved lateral furrows and 12 body divisions develop in more advanced

5 ontogenetic stages. The early germ bands appear pinched, and Pseudooides specimens with germ bands range in size from 250 to 425 μm in diameter (Steiner et al., 2004).

The stratigraphic and geographic distribution of Markuelia and Pseudooides has been documented in a number of studies. Markuelia has been found in Cambrian–Ordovician rocks in

Siberia, China, Australia, and the United States (Bengtson and Yue, 1997; Dong et al., 2004;

Dong et al., 2005; Donoghue et al., 2006a; Haug et al., 2009; Dong et al., 2010; Zhang et al.,

2011). Its stratigraphic occurrences range from the lower Cambrian Stage 2 (Bengtson and Yue,

1997) to the lowest Ordovician (Donoghue et al., 2006a; Dong et al., 2010), and there are currently six named species and one unnamed species of Markuelia. The earliest and first- described species, M. secunda, is found in the lower Tommotian (Stage 2) Pestrotsvet Formation,

Siberia. Other later Markuelia representatives include: M. hunanensis, M. spinulifera, and M. qianensis from the middle-upper Cambrian in South China; M. lauriei and M. waloszeki from the middle Cambrian in northern Australia; and a single specimen of Markuelia sp. from the Lower

Ordovician Vinini Formation, Nevada, United States (Dong et al., 2010). In comparison,

Pseudooides has a relatively limited species diversity and stratigraphic distribution; this genus include only one described species, P. prima, which has been so far only known from the

Fortunian Kuanchuanpu Formation in southern Shaanxi of South China (Qian, 1977; Steiner et al., 2004).

The taphonomy of previously reported Markuelia and Pseudooides fossils is related to phosphatization. Of these two genera, Markuelia is better understood in terms of its taphonomy and can serve as a taphonomic model for Pseudooides. Markuelia is typically preserved entirely in calcium phosphate, including both the enveloping chorion and the embryo proper. In some cases, the embryo has shrunk away from the more rigidly preserved chorion, an expected result

6 of decay prior to mineralization. If preserved within an intact chorionic envelope, the shrunken embryo is commonly attached to the chorion by phosphatic filaments, which may represent mucous strands, filamentous bacteria, or fungal hyphae (Xiao and Knoll, 1999; Xiao and

Schiffbauer, 2009; Dong et al., 2010). Likely similar in composition to the fertilization envelope of modern panarthropods, the chorion is more resistant to decay, and therefore exhibits better preservational potential (Dong et al., 2010). The superior decay resistance of cuticular chorions versus the lability of embryos has been empirically observed in taphonomic experiments of cycloneuralian and panarthropod embryos, including lobster (Martin et al., 2003, 2005), horseshoe crab (Martin et al., 2005), brine shrimp (Gostling et al., 2009), and priapulid eggs

(Dong et al., 2010). Fossil chorions are typically preserved with a smooth outer surface, but an inner surface commonly coated with calcium phosphate microspherules. In some specimens, only the chorion is preserved in calcium phosphate (Dong et al., 2010).

EMBRYO EXTRACTION AND EXAMINATION

Although abundant well-preserved brachiopod fossils were recovered, most embryo fossils extracted using this method were severely damaged because of their mostly calcitic composition.

The same was true for other calcitic fossils such as Chancelloria. The chorion surrounding the embryos (Figs. 2.1–2.2, 3.1–3.2), however, is composed of calcium phosphate and was largely resistant to damage from the buffered acetic acid solution. Unfortunately, as confirmed by closely-monitored acetic acid etching experiments, the chorion did not serve as an adequate barrier between the embryo proper and the acetic acid; that is, the acid infiltrated the chorion and began to dissolve the surface of the embryo before the fossil itself was released from the host limestone. As a result, the structural integrity of the micrometers-thick chorion was

7 compromised, only a few extracted embryo fossils retained an intact chorion, and the calcitic interior was mostly damaged. In samples that were especially argillaceous, the samples disaggregated very slowly in the acetic acid solution, and the macerate residue was comprised of small flakes instead of slurry. No embryo body fossils were recovered from such samples, but hemispherical depressions of matching size (200 μm) were visible in the rock flakes, corresponding to external molds of the spherical embryo fossils. Other calcitic fossils, such as

Chancelloria, were only seen as molds in the residue of these samples. Because embryo fossils and their counterparts are visible on rock fragments, the absence of embryo fossils in maceration residues indicates that the fossils dissolved faster than the disintegration of host argillaceous rock.

Fossils were also physically exposed by chiseling samples into mm- and cm-sized fragments. This method was unable to remove the fossils from the rock matrix completely, but allowed in-situ observation of embryo fossils and their counterparts. Some physically exposed specimens were further revealed or extracted by acetic acid etching. While a relatively small number of rock samples was processed using in this fashion, the vast majority of the embryo fossils in this study were discovered using this method. This indicates that the acetic acid maceration technique described above was too aggressive and can be destructive.

Standard petrographic thin-sections were also made from all samples and examined under plane and cross-polarized light microscopy. In thin-section, embryo fossils were easily distinguishable with their circular shape and a thin, opaque, phosphatized chorion (Fig. 2.1–2.2).

This method had the advantage of providing an interior view of embryo fossils, and allowing for mineralogical characterization and understanding petrographic context, but necessarily could not provide 3D information.

Chemically extracted and physically exposed specimens were examined using two scanning electron microscopes (SEM), a Hitachi TM3000 desktop thermionic (tungsten filament source) SEM and a FEI Quanta 600 field emission variable-pressure SEM. Backscattered electron (z-contrast) and secondary electron images were acquired, and energy dispersive X-ray spectroscopy (EDS) elemental maps were collected. EDS point analyses were used when more precise stoichiometric compositional data were required, whereas elemental mapping provided spatial, but qualitative, compositional information. Selected embryo fossils were examined using an Xradia micro-CT, including a fossil chemically extracted through bulk maceration, a fossil extracted through etching, and a fossil physically exposed on a rock fragment. The fossil in the rock fragment had not been exposed to acetic acid, but the quality of the scan was adversely impacted by the similar composition and density of the embryo and surrounding rock material as both are calcitic. Micro-CT scan parameters varied depending on the attenuation of individual samples, but all samples were scanned under 80 kV source voltage and a 40× objective lens.

Micro-CT data visualization and volume reconstructions were conducted using SkyScan

DataViewer, Xradia XMReconstructor, and Seg3D2.

FOSSIL DESCRIPTION

Chorion.—The embryo fossils are sometimes enclosed in a phosphatic envelope (Figs.

2.1–2.2), which is interpreted as the chorion. The chorion is 0.6–5.1 µm in thickness (mean = 2.3

µm; n = 90), and intact chorion range are 109–240 µm in diameter (mean = 191.8 µm; n = 27).

The chorion may be partially or completely lost, however, during fossil preparation through acetic acid maceration (Fig. 3.1, 3.4–3.6). Thin-section observation and EDS analyses indicate that the chorion is secondarily phosphatized (Fig. 2.1–2.2, 3.7–3.9) and that the interior space

9 surrounded by the chorion is typically filled with blocky calcite (Fig. 2.1–2.3). The outer surface of the chorion is smooth, black, and commonly reflective under reflected light, but the inner surface has a rough texture, covered by micrometer-scale (mean = 1.0 μm; n = 50) calcium phosphate spherules, indicating that phosphatic minerals nucleated and grew on the inner surface of the original chorion. In one of the specimens, part of the phosphatized chorion is fragmented, with pieces captured within the calcite interior (Fig. 2.2). This preservation indicates an earlier phosphatization of the chorion than the mineralization of the interior embryo; in other words, the chorion is preferentially phosphatized versus the interior soft tissues.

Blastula-like embryos.—Five spherical fossils have a polygonal pattern on the surface

(Fig. 2.4–2.9). The polygons are mostly pentagons and hexagons, 20.5–53.1 µm in size (mean =

30.6 µm; n = 29). They are tightly and seamlessly packed, without much gap in between. The edge of the polygons manifests as raised ridges (Fig. 2.4–2.5) or as shallow furrows (Fig. 2.7–

2.9) on the fossil surface; both preservation styles have been reported from Cambrian blastula fossils (e.g., compare fig. 1a–1b of Dong et al., 2004, and fig. 1C of Zhang and Pratt, 1994) and are probably related to how the cell boundary vs. cell lumen are phosphatized (Schiffbauer et al.,

2012). The polygons can be taphonomically transparent through multiple layers of mineral coating (i.e., the topographic ridges or furrows are inherited over multiple layers of possibly isopachous mineral overgrowth; Fig. 2.7–2.9). The polygonal surface is remarkably similar to that of Parapandorina-stage blastula embryos from the Doushantuo Formation (Xiao and Knoll,

2000), blastulas associated with Cambrian Markuelia fossils (Dong et al., 2010; Zhang et al.,

2011), blastulas associated with Cambrian trilobites (Zhang and Pratt, 1994), and fossils interpreted as blastulas of Olivooides (Yue and Bengtson, 1999) or as yolk-pyramid stage embryos of arthropods from the basal Cambrian Kuanchuanpu Formation (Chen et al., 2004).

The blastula fossils cited above are all pervasively phosphatized, and their polygonal patterns can be shown to correspond to polyhedral blastomeres, which sometimes become spherical likely due to degradation (Raff et al., 2006; Xiao et al., 2012). The Yanjiahe fossils, however, are internally calcitized and only a vague polygonal pattern is visible on the surface. Thus, it is impossible to extract them using acid maceration techniques or to reveal interior structures using micro-CT because often the fossils are internally replicated by recrystallized calcite (Fig. 2.1–

2.3). We have found no fossils with spherical blastomeres, either because our sample size is small (only five specimens) or because such fossils would be unrecognizable when pervasively calcitized. Nonetheless, the similarity to other Ediacaran and Cambrian blastula fossils prompt us to interpret the Yanjiahe fossils as blastulas, which are estimated to have 25–28 polyhedral blastomeres.

Fossils with grooves.—The calcitic interiors of the embryo fossils are the main focus of this study. The surface of the calcitic interior is best seen in “naked” specimens (without chorions) extracted via acetic acid maceration. The observed specimens range from 170–220 µm in diameter (mean = 195.6 µm; n = 8), and their surface is covered by sets of parallel grooves

(Fig. 3.4–3.6–2.5). The grooves are slightly curved, averaging 42.6 μm in length and 10.2 μm in spacing between adjacent grooves (n = 24). Apart from the grooves, the surface is covered with euhedral calcite rhombs and secondary cracks.

One acid extracted specimen was scanned using micro-CT, and volume rendering confirms the presence of sets of parallel grooves across much of the surface (Fig. 3.4–3.5).

However, no interior structures are preserved, consistent with thin section observation that shows the interior space surrounded by the chorion is typically filled with secondary blocky calcite

(Fig. 2.1–2.3). Micro-CT surface rendering also indicates that the calcitic interior was partly

11 dissolved during acid digestion. To minimize the destruction of acid digestion, we also scanned a mechanically exposed and subsequently etched specimen (Fig. 3.2), as well as a partially exposed specimen (Fig. 3.3), using micro-CT; but attempts to digitally extract any anatomical features were unsuccessful because the interior is completely calcitized. This is not an apparent resolution issue, as the micro-CT system used has submicron-scale resolvability, but rather a preservation issue resulting from the secondary calcitization.

TAXONOMIC INTERPRETATION

In synchrotron microCT-based studies of Markuelia embryos (Donoghue et al., 2006a; Dong et al., 2010), anatomical structures of the embryo have been useful for taxonomic and morphological identification. For the Yanjiahe fossils, however, because only the chorion is phosphatized, very little morphology is preserved in the calcitic interior. The polygonal pattern on the blastula-like fossils is not taxonomically or phylogenetically diagnostic, and the grooves are so poorly preserved to allow any definitive taxonomic identification. Below, we compare the

Yanjiahe fossils with Markuelia and Pseudooides based on the specimens with grooves.

A possible comparative analogue for the Yanjiahe fossils is Markuelia. As elucidated in studies of Markuelia morphology (Bengtson and Yue, 1997; Dong et al., 2004; Haug et al., 2009;

Zhang et al., 2011), the developing Markuelia embryo is curled up within the chorion such that the head and tail lie adjacent to one another, pointing in opposite directions. The part of the body behind the head arcs around the tail as the part in front of the tail arcs around the head. On the reverse side of the embryo, the body is sharply folded twice. Parallel annulations originate from just behind the introvert and extend through the entire length of the body. In sections of the body that lie parallel to one another, the annulations on both sections are oriented in the same

12 direction. The geometry of the annulations is different where the body arcs around the head or tail, and where the body undergoes a tight fold. In both cases, the annulations cannot be parallel; they instead fan out and form sharp angles with annulations on juxtaposing sections of the body.

By using these geometric rules, it is possible to interpret the annulations on incompletely preserved specimens or those partially damaged during acid extraction, like the Yanjiahe fossils.

Ignoring the differences between the head and the tail, and the orientation of spines along the body of Markuelia, neither of which are visibly preserved in the Yanjiahe specimens, the folding geometry of Markuelia is rotationally symmetric. The Yanjiahe fossils show grooves that are arranged in parallel sets, and sets of grooves can be juxtaposed at an angle, similar to previous observations of annulations on Markuelia embryos (Bengtson and Yue, 1997; Dong et al., 2004;

Haug et al., 2009; Zhang et al., 2011). Thus, it is possible that the grooves observed in the

Yanjiahe specimens may represent boundaries between annuli. However, there are noticeable differences between the Yanjiahe fossils and Markuelia. First, the spacing of the grooves in the

Yanjiahe fossils is ~10.2 μm on average, small for Markuelia annuli. For comparison, annulus length is 20–25 μm in M. secunda, the largest known species (Dong et al., 2004), 10–25 µm in

M. hunanensis (Dong et al., 2005), and 15 µm in M. lauriei (Haug et al., 2009). The Yanjiahe fossils have much shorter annuli by comparison, but they are also generally smaller in overall size than all described Markuelia species (Fig. 4). Markuelia fossils from other studies range from 275 µm to 550 μm in maximum diameter (Bengtson and Yue, 1997; Dong et al., 2005).

Even the minimum diameter of the smaller discoid-shaped Markuelia fossils, such as M. lauriei and M. waloszeki (Haug et al., 2009; Dong et al., 2010), are around 200 µm (Fig. 4). For comparison, the Yanjiahe fossils are all close to spherical in shape and average only 194.7 μm in diameter (n = 35). The smaller size of the Yanjiahe fossils are unlikely due to taphonomic

13 shrinkage, which usually results in collapsed embryos and deflated chorions (Xiao and Knoll,

1999; Dong, 2009b; Dong et al., 2010). In the Yanjiahe fossils, however, the chorion is always spherical or brittly broken (Figs. 2.1–2.2, 3.1–3.2), indicating its phosphatization before taphonomic deflation. Also, the calcitic interior has a tight fit within the chorion, so that even if the chorion is not present the calcitic interior is a good proxy for the chorion diameter. Thus, the average diameter of ~200 μm must be the original size of the Yanjiahe fossils, and it is volumetrically smaller than all known Markuelia species (Fig. 4). For this reason, if the Yanjiahe fossils belong to the genus Markuelia, they may represent a new species and the oldest

Markuelia fossil in South China, but these fossils are too poorly preserved to describe in adequate detail. More importantly, the Yanjiahe fossils lack key features characteristic of

Markuelia; no introvert, tail, or scalids are preserved in the Yanjiahe fossils. Without these diagnostic features, the Yanjiahe fossils cannot be confidently identified as Markuelia, and even if they do represent poorly preserved Markuelia, they may not be identified with any existing species given the differences in size and annulation length.

An alternate comparative analog for the Yanjiahe fossils is the Cambrian animal embryo

Pseudooides. As the Pseudooides embryo develops, it forms wrinkled germ bands (Steiner et al.,

2004; Donoghue et al., 2006b). The germ band can be pinched, causing the wrinkles to deform accordingly. The germ band is divided into 12 series divisions, each potentially representing a body segment, and the wrinkles are discontinuous at segment boundaries. A major difference between the wrinkles on Pseudooides germ bands and annulations in Markuelia is that the wrinkles are mostly longitudinal structures following the length of the germ band, whereas the annulations are transverse structures. When the Markuelia body folds, the orientation of transverse annulations can change, and sets of annulations with different orientations can be

14 juxtaposed with each other. In contrast, wrinkles in Pseudooides follow the length of the germ band and deformation occurs only at segment boundaries and where the germ band is pinched laterally. The grooves in the Yanjiahe fossils are relatively short and are arranged in distinct sets illustrating different orientations (Fig. 3.4–3.6). Thus, Markuelia annulations appear to be a better morphological equivalent than Pseudooides wrinkles.

TAPHONOMIC MODE

While Markuelia embryos are commonly preserved in calcium phosphate, the Yanjiahe fossils only exhibit a phosphatized chorion, with the interior filled with calcite. Phosphatization is a means of exceptional preservation of soft tissues, whereas calcite rarely preserves soft tissues with a similar level of biological fidelity. Modern seawater is supersaturated with respect to apatite, but conditions instead typically favor calcium carbonate precipitation. The chemical conditions required for phosphatization of soft tissues as opposed to calcite precipitation are very particular. This delicate chemical balance is likened to a “switch” between calcium carbonate and calcium phosphate precipitation regime. These conditions may toggle during fossil mineralization in the microenvironment surrounding a degrading organism, resulting in partial preservation of a fossil in calcium phosphate and partial preservation in calcium carbonate

(Briggs and Wilby, 1996). One requirement for calcium phosphate preservation is low pH. The degradation of organic matter by microbes produces dissolved carbon dioxide and hydrogen sulfide, which can create localized microenvironment with lower pH and facilitate localized precipitation of calcium phosphate (Taylor and Macquaker, 2011). As the process of organic decay slows, local pH can again rise, flipping the switch back to calcium carbonate precipitation, potentially resulting in calcite overgrowth on phosphate (Briggs and Wilby, 1996). Furthermore,

15 the switch to calcite precipitation can also be driven by increasing bacterial sulfate reduction relative to aerobic degradation as organic decay continues, as this process generates bicarbonate rather than carbon dioxide, providing the alkalinity needed for calcite precipitation.

In addition to local pH conditions, other factors critical for calcium phosphate precipitation includes a source of phosphate to maintain localized supersaturation and the availability of nucleation sites or substrates. Certain microbes are known to concentrate polyphosphate in their cells (Schulz and Schulz, 2005) and can provide a phosphorus source for phosphatization; in addition, microbes can also form biofilms on degrading tissues, thus forming a pseudomorph of, and therefore promoting the preservation of, soft tissues (Raff et al., 2008).

Other sources of phosphorus include the degrading carcass itself (Briggs and Wilby, 1996) and the abundant linguliform brachiopods coexisting with the Yanjiahe embryo fossils. When the localized supersaturation level with respect to calcium phosphate is reached and a suitable nucleation substrate is available, phosphate precipitation may occur and result in phosphatization of soft tissues.

In the Yanjiahe fossils, the only part that was preserved in calcium phosphate is the chorion. It is possible that the chorion may have been cuticular and more resistant to decay

(Dong et al., 2010). As a result, the chorion was more likely to serve as a substrate for calcium phosphate precipitation; indeed, taphonomic experiments have shown that the chorion is preferentially preserved through calcium phosphate encrustation (Martin et al., 2005; Hippler et al., 2011). In the Yanjiahe fossils, the chorion is replicated with a smooth outer surface and no trace of microbes are visible on the chorion surface, so they are preserved with the “substrate” microfabric (Wilby and Briggs, 1997); this style of preservation is different from the “microbial” microfabric characteristic of more labile and degraded tissues such as shrunken cells of

Parapandorina-stage embryos that are typically covered with filaments and coccoids (Xiao and

Knoll, 1999, 2000). In the Yanjiahe fossils, however, small spherules of calcium phosphate can be seen on the inner surface of chorions, suggesting that calcium phosphate nucleated and encrusted on the inner surface of the original organic chorion; similar encrustation patterns have been documented in the Doushantuo embryo fossils (Xiao and Knoll, 1999; Xiao and

Schiffbauer, 2009) and likely require rapid phosphatization (Schiffbauer et al., 2012). None of the Yanjiahe fossils we examined have a completely phosphatized embryo within the chorion, and much of the interior space is filled with blocky calcite with only vague grooves interpreted as possible annulations. These observations, together with evidence showing that the chorion was phosphatized before the interior was calcitized (Fig. 2.1–2.2), suggest that: (1) the embryo within the chorion degraded rapidly after death; (2) the chorion has greater preservation potential than the embryo proper likely due to its recalcitrance to decay; (3) localized supersaturation with respect to calcium phosphate promoted encrustation on the inner surface of the chorion; and (4) mineralization of the interior space was later than the chorion mineralization, after the localized microenvironment switched back to calcite precipitation, causing calcitization with poorly preserved (if any at all) anatomical structures. The low preservation quality of the embryo proper is exacerbated by recrystallization, which resulted in polycrystal aggregates of calcitic rhombs that further compromise taphonomic fidelity.

CONCLUSIONS

The samples from the Wangzishi locality are from the Cambrian Stage 2-3. They contain a variety of fossils from the small shelly assemblage. While the brachiopods sometimes show clay or pyrite association, there is no preferential orientation of clay or pyrite coatings. The embryo

17 fossils described here are poorly preserved, with a phosphatized chorion surrounding a phylogenetically uninformative blastula or a calcitic interior with sets of grooves that tantalizingly resemble the transverse annulations of the Cambrian-Ordovician scalidophoran embryo Markuelia. The differential preservation of the chorion and embryo proper suggests that the chorion had a greater preservational potential due to its recalcitrance to decay and that phosphatization in the Yanjiahe sediments was short-lived and quickly taken over by calcite precipitation.

If the Yanjiahe fossils are indeed Markuelia, they represent the oldest occurrence of this genus in South China. Additionally, they are smaller than any previously described species of

Markuelia and therefore may represent a new taxon. The preservational bias of the chorion relative to the embryo proper indicates that many empty phosphatic vesicles recovered by acid digestion from Cambrian carbonates may be fossilized chorions. Indeed, many of the Cambrian embryo fossils were first described as empty chorions (Qian, 1977; Qian et al., 1979), which continue to be some of the most abundant yet least studied fossils in many Cambrian assemblages recovered using acetic acid maceration techniques.

FIGURES AND FIGURE CAPTIONS

FIGURE 1.—Geological map and stratigraphic column. 1, Geological map of the Yangtze Gorges and surrounding region, showing the fossil locality (arrowhead) at Wangzishi in Hubei Province.

Modified from Jiang et al. (2012). 2, Stratigraphic column of the Yanjiahe Formation at

Wangzishi. Fossil occurrences marked by dots to the right of the stratocolumn. Dashes mark horizons where samples were collected, but no embryo fossils were found. Abbreviations: m, micritic limestone; w, wackestone; p, packstone; g, grainstone; DY: Dengying Formation; SJT,

Shuijingtuo Formation.

FIGURE 2.—Chorions (1–3) and blastula-like fossils (4–9) observed in thin section (1–3) and mechanically exposed specimens (4–9), using transmitted light microscopy (1, plane polarized light; 2, cross-polarized light) and BSE SEM (3–9). 1, Spherical phosphatic chorion and calcitic interior, sample 11-WSZ-YJH-3.0-ts-1; 2, Brittly broken phosphatic chorion, 11-WSZ-YJH-0.6;

3, Chorion barely visible, 11-WZS-YJH-0.8; 4, Polygons defined by ridges, 11-WSZ-YJH-0.6;

5, Magnified view of 4; 6, Faintly preserved polygons on a spherical fossil, 11-WZS-YJH-0.8; 7,

Polygons are visible on multiple layers of mineral overgrowth, 11-WSZ-YJH-0.6-1; 8–9,

Magnified views of 7. Arrowheads mark corners of polygons. Scale bars represent 50 µm.

FIGURE 3.—Chorions (1–3, 7–9) and fossils with sets of parallel grooves (4–6). 1 and 4–6 were extracted by bulk acid maceration, 2 was first exposed mechanically and then extracted by etching, and 3 and 7–9 were mechanically exposed. 1–3 and 6–7 are BSE SEM images, 4 is SE

SEM image, 5 is micro-CT volume rendition, and 8–9 are EDS elemental maps. Specimens

21 illustrated in 2–4 were scanned using micro-CT. 1, Partial chorion preservation, 11-WSZ-YJH-

0.8-4; 2, Partial chorion preservation, 11-WSZ-YJH-0.6; 3, 11-WSZ-YJH-0.6; 4, Specimen with three sets of parallel grooves, and a prominent crack, 11-WSZ-YJH-6.3; 5, Same specimen as 4, showing the opposite side with two sets of parallel grooves; 6, Poorly preserved specimen with two visible sets of parallel grooves and several cracks, 11-WSZ-YJH-0.8-5, arrowheads in 3–5 denote orientation of grooves; 7, Phosphatic chorion with dark patches of organic carbon, 11-

WSZ-YJH-0.8; 8–9, Phosphorus and carbon maps, respectively, of same specimen as in 7, showing chorion enriched in phosphorus and organic carbon enrichment areas matching dark patches in 7. Lighter color means relatively greater elemental content. Shadow on the right side of element maps is an artifact of topography. Scale bars represent 50 μm.

FIGURE 4.—Size and age ranges of Markuelia species in comparison with the Yanjiahe fossils.

For discoidal fossils (M. waloszeki and M. lauriei), measurements of maximum and minimum

22 dimensions are presented in black and grey bars, respectively. The diameter of spherical fossils, such as M. secunda, is marked only in black bars. Brackets show ranges and box shows one standard deviation from the mean. Data from (Dong et al., 2004; Dong, 2009a; Haug et al., 2009;

Dong et al., 2010; Zhang et al., 2011).

REFERENCES

BENGTSON, S. AND Z. YUE. 1997. Fossilized metazoan embryos from the earliest Cambrian. Science, 277:1645-1648. BRIGGS, D. E. G. AND P. R. WILBY. 1996. The role of calcium carbonate-calcium phosphate switch in the mineralization of soft-bodied fossils. Journal of the Geological Society, London, 153:665-668. CHEN, J., A. BRAUN, D. WALOSZEK, Q. PENG, AND A. MAAS. 2004. Lower Cambrian yolk- pyramid embryos from southern Shaanxi, China. Progress in Natural Science, 14:167- 172. CHEN, Y., S. ZHANG, G. LIU, X. XIONG, AND P. CHEN. 1984. The Sinian-Cambrian boundary in the eastern part of the Yangtze Gorges, Hubei, p. 14-35. In Y. Xing, Q. Ding, H. Luo, T. He, and Y. Wang (eds.), The Sinian-Cambrian Boundary of China; Bulletin of the Institute of Geology, Chinese Academy of Geological Sciences, No. 10. Geological Publishing House, Beijing. DONG, L., S. XIAO, B. SHEN, C. ZHOU, G. LI, AND J. YAO. 2009. Basal Cambrian microfossils from the Yangtze Gorges area (South China) and the Aksu area (Tarim Block, northwestern China). Journal of Paleontology, 83:30-44. DONG, X.-P. 2009a. The anatomy, affinity, and developmental sequences of Cambrian fossil embryos. Acta Palaeontologica Sinica, 48:390-401. DONG, X.-P. 2009b. Cambrian fossil embryos from western Hunan, South China. Acta Geologica Sinica, 83:429-439. DONG, X.-P., S. BENGTSON, N. J. GOSTLING, J. A. CUNNINGHAM, T. H. P. HARVEY, A. KOUCHINSKY, A. K. VAL'KOV, J. E. REPETSKI, M. STAMPANONI, F. MARONE, AND P. C. J. DONOGHUE. 2010. The anatomy, taphonomy, taxonomy and systematic affinity of Markuelia: early Cambrian to early Ordovician. Palaeontology, 53:1291-1314. DONG, X.-P., P. C. J. DONOGHUE, H. CHENG, AND J.-B. LIU. 2004. Fossil embryos from the Middle and Late Cambrian period of Hunan, south China. Nature, 427:237-240. DONG, X., J. A. , S. CUNNINGHAM, C. BENGTSON, J. THOMAS, M. LIU, STAMPANONI, AND P. C. J. DONOGHUE. 2013. Embryos, polyps and medusae of theEarly Cambrian scyphozoan Olivooides. Proceedings of the Royal Society, 280:1-8. DONG, X., P. C. J. DONOGHUE, J. A. CUNNINGHAM, J. L. AND, AND H. CHENG. 2005. The anatomy, affinity, and phylogenetic significance of Markuela. Evolution & Development, 7:468- 482.

DONOGHUE, P. C. J., S. BENGTSON, X.-P. DONG, N. J. GOSTLING, T. HULDTGREN, J. A. CUNNINGHAM, C. YIN, Z. YUE, F. PENG, AND M. STAMPANONI. 2006a. Synchrotron X-ray tomographic microscopy of fossil embryos. Nature, 442:680-683. DONOGHUE, P. C. J., A. KOUCHINSKY, D. WALOSZEK, S. BENGTSON, X.-P. DONG, A. K. VAL’KOV, J. A. CUNNINGHAM, AND J. E. REPETSKI. 2006b. Fossilized embryos are widespread but the record is temporally and taxonomically biased. Evolution & Development, 8:232-238. GOSTLING, N. J., X. DONG, AND P. C. J. DONOGHUE. 2009. Ontogeny and taphonomy: An experimental taphonomy study of the development of the brine shrimp Artemia salina. Palaeontology, 52:169-186. FAURE, G. 1998. Principles and Applications of Geochemistry. Prentice Hall. Upper Saddle River, New Jersey. HAUG, J. T., A. MAAS, D. WALOSZEK, P. C. J. D. AND, AND S. BENGTSON. 2009. A new species of Markuelia from the Middle Cambrian of Australia. Association of Australasian Paleontologists Memoir, 37:303-313. HIPPLER, D., N. HU, M. STEINER, G. SCHOLTZ, AND G. FRANZ. 2011. Experimental mineralization of crustacean eggs leads to surprising tissue conservation: new implications for the fossilization of Precambrian-Cambrian embryos. Biogeosciences Discussions, 8:12051- 12077. JEPPSSON, L., D. FREDHOLM, AND B. MATTIASSON. 1985. Acetic acid and phosphatic fossils: A warning. Journal of Paleontology, 59:952-956. JIANG, G., X. WANG, X. SHI, AND S. XIAO. 2012. The origin of decoupled carbonate and organic carbon isotope signatures in the early Cambrian (ca. 542-520 Ma) Yangtze platform. Earth and Planetary Science Letters, 317-318:96-110. LI, G. AND L. E. HOLMER. 2004. Early Cambrian lingulate brachiopods from the Shaanxi Province, China. GFF, 126:193-211. LI, G., X. ZHAO, A. GUBANOV, M. ZHU, AND L. NA. 2011. Early Cambrian mollusc Watsonella crosbyi: a potential GSSP index fossil for the base of the Cambrian Stage 2. Acta Geologica Sinica, 85:309-319. MARTIN, D., D. E. G. BRIGGS, AND R. J. PARKES. 2003. Experimental mineralization of invertebrate eggs and the preservation of Neoproterozoic embryos. Geology, 31:39-42. MARTIN, D., D. E. G. BRIGGS, AND R. J. PARKES. 2005. Decay and mineralization of invertebrate eggs. Palaios, 20. PENG, S., L. E. BABCOCK, AND R. A. COOPER. 2012. The Cambrian Period, p. 437-488. In F. M. Gradstein, J. G. Ogg, M. Schmitz, and G. Ogg (eds.), Geological Time Scale 2012. Elsevier, Oxford. QIAN, Y. 1977. Hyolitha and some problematica from the Lower Cambrian Meishucun Stage in central and southwestern China. Acta Palaeontologica Sinica, 16:255-275. QIAN, Y., M. CHEN, AND Y. CHEN. 1979. Hyolithids and other small shelly fossils from the Lower Cambrian Huangshandong Formation in the eastern part of the Yangtze Gorge. Acta Palaeontologica Sinica, 18(3):207-232. PARKHAEV, P. Y., Y. E. DEMIDENKO. 2010. Zooproblematica and Mollusca from the Lower Cambrian Meishucun Section (Yunnan, China) and Taxonomy and Systematics of the Cambrian Small Shelly Fossils of China. Paleontological Journal, 44(8):883-1161 RAFF, E. C., K. L. SCHOLLAERT, D. E. NELSON, P. C. J. DONOGHUE, C.-W. THOMAS, F. R. TURNER, B. D. STEIN, X. DONG, S. BENGTSON, T. HULDTGREN, M. STAMPANONI, Y. CHONGYU, AND R. A. RAFF. 2008. Embryo fossilization is a biological process mediated

by microbial biofilms. Proceedings of the National Academy of Sciences of the United States of America, 105:19360–19365. RAFF, E. C., J. T. VILINSKI, F. R. TURNER, P. C. J. DONOGHUE, AND R. A. RAFF. 2006. Experimental taphonomy shows the feasibility of fossil embryos. Proceedings of the National Academy of Sciences, USA, 103:5846-5851. SCHIFFBAUER, J. D., S. XIAO, K. SEN SHARMA, AND G. WANG. 2012. The origin of intracellular structures in Ediacaran metazoan embryos. Geology, 40:223-226. SCHULZ, H. N. AND H. D. SCHULZ. 2005. Large sulfur bacteria and the formation of phosphorite. Science, 307:416-418. STEINER, M., G. LI, Y. QIAN, M. ZHU, AND B.-D. ERDTMANN. 2007. Neoproterozoic to early Cambrian small shelly fossil assemblages and a revised biostratigraphic correlation of the Yangtze Platform (China). Palaeogeography, Palaeoclimatology, Palaeoecology, 254:67- 99. STEINER, M., M. ZHU, G. LI, Y. QIAN, AND B.-D. ERDTMANN. 2004. New Early Cambrian bilaterian embryos and larvae from China. Geology, 32:833–836. TAYLOR, K. G. AND J. H. S. MACQUAKER. 2011. Iron minerals in marine sediments record chemical environments. Elements, 7:113-118. WANG, J. AND Z.-X. LI. 2003. History of Neoproterozoic rift basins in South China: Implications for Rodinia break-up. Precambrian Research, 122:141-158. WILBY, P. R. AND D. E. G. BRIGGS. 1997. Taxonomic trends in the resolution of detail preserved in fossil phosphatized soft tissues. Geobios, Memoire special, No. 20:493-502. XIAO, S. AND A. H. KNOLL. 1999. Fossil preservation in the Neoproterozoic Doushantuo phosphorite Lagerstätte, South China. Lethaia, 32:219-240. XIAO, S. AND A. H. KNOLL. 2000. Phosphatized animal embryos from the Neoproterozoic Doushantuo Formation at Weng'an, Guizhou, South China. Journal of Paleontology, 74:767-788. XIAO, S., A. H. KNOLL, J. D. SCHIFFBAUER, C. ZHOU, AND X. YUAN. 2012. Comment on “Fossilized nuclei and germination structures identify Ediacaran ‘animal embryos’ as encysting protists”. Science, 335:1169c. XIAO, S. AND J. D. SCHIFFBAUER. 2009. Microfossil phosphatization and its astrobiological implications, p. 89-117. In J. Seckbach and M. Walsh (eds.), From Fossils to Astrobiology. Springer-Verlag, New York. YAO, J., S. XIAO, L. YIN, G. LI, AND X. YUAN. 2005. Basal Cambrian microfossils from the Yurtus and Xishanblaq formations (Tarim, north-west China): Systematic revision and biostratigraphic correlation of Micrhystridium-like acritarchs from China. Palaeontology, 48:687-708. YUE, Z. AND S. BENGTSON. 1999. Embryonic and post-embryonic development of the Early Cambrian cnidarian Olivooides. Lethaia, 32:181-195. ZHANG, X. AND B. R. PRATT. 1994. Middle Cambrian arthropod embryos with blastomeres. Science, 266:637-639. ZHANG, X., B. R. PRATT, AND C. SHEN. 2011. Embryonic development of a middle Cambrian (500 myr old) scalidophoran worm. Journal of Paleontology, 85:898-903.

Appendix A: Figure Repository A.1 Thin Section Images-

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