Reuter, Katherine J., Ms May 2020 Geology Experimental Taphonomy of Penaeid Shrimp

REUTER, KATHERINE J., M.S. MAY 2020 GEOLOGY

EXPERIMENTAL TAPHONOMY OF PENAEID SHRIMP: ANALYSES OF

MORPHOLOGICAL DECAY IN DIFFERENT SEDIMENTARY CONDITIONS AND

OF METHODOLOGICAL PROTOCOLS ABSTRACT (79 pp.)

Thesis Advisor: Carrie Schweitzer, Ph.D.

Experiments using artificial sea water, sediment, and varying combinations thereof to represent different environmental settings yielded different decay rates of penaeid shrimp. Experiments were monitored by scoring decay-induced morphologies, including those commonly observed in compressed shrimp fossils. Changes in microstructural layering of cuticle and pH were also monitored throughout decay. Burial of the shrimp in kaolinite mud (powder saturated with artificial sea water) inhibited decay while the fastest decay occurred with shrimp lying unburied on the sediment-water interface. A small percentage of lime (10%) with kaolinite also resulted in slightly decreased decay rates in shrimp that experienced minor sedimentation within artificial sea water. Procedural and methodological concerns were identified from this series of sediment-based experiments. Experimental protocols and considerations for future decay experiments are suggested along with potential experimental conditions, the results of which could further taphonomic interpretations of taxonomically characteristic morphologies preserved in shrimp or other commonly compressed fossils.

EXPERIMENTAL TAPHONOMY OF PENAEID SHRIMP: ANALYSES OF

MORPHOLOGICAL DECAY IN DIFFERENT SEDIMENTARY CONDITIONS AND

OF METHODOLOGICAL PROTOCOLS

A thesis submitted

To Kent State University in partial

Fulfillment of the requirements for the

Degree of Master of Science

Katherine J. Reuter

May 2020

Except for previously published materials

Thesis written by

Katherine J. Reuter

B.S., Wittenberg University, 2016

M.S., Kent State University, 2020

Approved by

Carrie Schweitzer, Ph.D. , Advisor

Daniel Holm, Ph.D. , Chair, Department of Geology

James Blank, Ph.D. , Dean, College of Arts and Sciences

TABLE OF CONTENTS

TABLE OF CONTENTS ...... iv

LIST OF FIGURES ...... vii

LIST OF TABLES ...... viii

ACKNOWLEDGMENTS ...... ix

INTRODUCTION ...... 1

METHODS AND EXPERIMENTAL PROTOCOLS...... 3

Experiment Set 1: Artificial Sea Water without Sediment ...... 4

Experiment Set 2: Immediate Burial in Kaolinite with Compaction ...... 4

Experiment Set 3: Sediment – Water Interface...... 5

Experiment Set 4: Gradual Sedimentation of Kaolinite-Lime and

Pre-Punctured Carapace ...... 6

Morphological Decay...... 7

MORPHOLOGICAL RESULTS ...... 10

The Effects of Pre-Punctured Carapace and Variable Gradual

Sedimentation (Set 4) ...... 10

The Effects of Sediment ...... 10

Simulation of Rapid Burial ...... 12

Cuticle Decay ...... 13

Effects of Physical Agitation and Sieving Remains ...... 15

CHEMICAL RESULTS ...... 17

The Effects of Sediment and Other Factors of pH Variation...... 17

The Effects of Pre-Punctured Carapace and Variable Gradual Sedimentation

(Set 4) ...... 18

COMPARING RESULTS TO PREVIOUS EXPERIMENTS AND THE FOSSIL

RECORD ...... 20

The Effects of Sediment...... 20

Morphological Decay Trends ...... 23

Other Results ...... 25

TAPHONOMIC INTERPRETATIONS AND THE USE OF SEDIMENT IN

EXPERIMENTAL TAPHONOMY ...... 29

More Accurate Interpretations of Fossil Morphology and Taphonomy ...... 29

Complications with the Use of Sediment and the Importance of

Burial Scenarios ...... 31

Excavation Methods for Buried Specimens ...... 33

Monitoring Individual Specimens Versus Sequential Excavation of

Different Specimens ...... 35

Compression Simulation ...... 38

MEASUREMENTS OF DECAY AND THEIR IMPLICATIONS ...... 43

Methods for Tracking Morphological Decay and Total Amount of Decay ...... 43

Other Measurements of Decay ...... 50

MATERIALS AND OTHER CONSIDERATIONS FOR EXPERIMENTAL

PROTOCOL ...... 53

Raw Materials, Basic Observations, and Record of Information ...... 53

Experimental Containers ...... 55

CONCLUSIONS...... 57

The Use of Sediment and its Effects ...... 57

Measurements of Morphological and General Decay ...... 59

Other Protocol for Future Experiments and Universal Treatment

of Specimens ...... 61

FIGURES ...... 63

TABLES ...... 73

REFERENCES ...... 76

LIST OF FIGURES

Figure Page

1 Design for Experiment Set 2 ...... 63

2 Design for Experiment Set 3 ...... 64

3 Morphological scores for unaltered versus pre-punctured shrimp ...... 65

4 Morphological scores for 5g- versus 20g-sedimentation-shrimp ...... 66

5 Averaged morphological scores for all Experiment Sets ...... 67

6 Fresh and decayed cuticle samples (SEM) ...... 68

7 pH for Experiment Sets 1, 3, and 4 ...... 69

8 Stratigraphic changes in pH for Experiment Set 3 ...... 69

9 The effects of pre-punctured shrimp on pH ...... 70

10 Excavation of buried shrimp from trial experiments ...... 70

11 Experiment Set 2 shrimp excavated via mesh ...... 71

12 Sieved remains from Experiment Set 1 ...... 71

vii

LIST OF TABLES

Table Page

1 Character states and corresponding decay scores used to monitor decay rates

for all Experimental Sets. Modified from Klompmaker et al. (2017) ...... 72

2 Explanations for each character state, modifications during the experimental

Process, and some corresponding decay scores ...... 73

viii

ACKNOWLEDGMENTS

Thank you to the Amoco Alumni Scholarship for providing research support and to Dr. D.E.G. Briggs and Dr. D.A. Waugh for providing experimental and instrumentation advice. I am so grateful to my advisor, Dr. Carrie Schweitzer, for guiding me through my master’s program and for supporting the exploration of this taphonomic research. I am grateful to my committee members, Dr. Rodney Feldmann and Dr. Neil

Wells, for teaching me and providing guidance throughout my time at Kent State

University. I am also thankful for the comradery and mutual support from my fellow graduate students in the department. My utmost gratitude is to my parents and my loved ones for supporting me unconditionally.

INTRODUCTION

Solnhofen-type and Burgess Shale-type Lagerstätte have been a source of extremely abundant well-preserved fossils belonging to various taxa, most remarkably of soft-bodied organisms with low potential for preservation. However, well-preserved fossils of a given taxon still vary morphologically and taphonomically even when found associated within the same assemblage. Commonly but variedly observed decay features in fossil shrimp include the separation of the pleon from the carapace, separation of the anterior cephalothorax from the posterior cephalothorax, and displacement of the internal skeleton from the carapace, some or all of which hold implications for morphological interpretations that are significant in taxonomic and phylogenetic classifications. Because of this, morphological interpretations of the fossils must take into consideration all possible explanations such as taphonomic processes (the interrelated processes occurring between death and preservation), poor preservation potential of a weakly developed feature, or the possibility that presumed features are artifacts of taphonomy and they are not real. Fortunately, morphologic observations from decay experiments have aided in tapho-anatomic interpretations, such as distinguishing decay-related characters from real characters that might nonetheless be interpreted as the result of taphonomic processes.

These sorts of complications underscore the importance of taphonomic experiments such as this study. In particular, this study shows the importance of incorporating sediment in future experiments as its presence is fundamental to

fossilization because it provides protection and conditions for early diagenesis (Wilson and Butterfield, 2014).

The purpose of these experiments was to determine the effects of sediment type and the nature of its presence (e.g., burial versus non-burial) on the decay of specific morphologies of penaeid shrimp. Various methodologies were adapted and modified from previous decay experiments (Biggs and Kear, 1994; Wilson and Butterfield, 2014;

Klompmaker et al., 2017). Results from past experiments, as well as other methods and considerations we did not use, are compared in the Discussion. Moreover, preferential experimental protocols and considerations for future decay experiments are included, with particular emphasis on those that stem from the complications and possible experimental conditions following the introduction of sediment. These include excavation techniques, having a sufficient number of controls, compression simulation, measurements of morphological decay, and standardized treatment of specimens/samples.

It is important to note that even though these experiments focused primarily on the impact of taphonomy on morphology, the discussed protocols and considerations are applicable to any decay experiment involving sediment, no matter its purpose (i.e. testing other fundamentally important factors of fossilization like biomineralization, microbial activity, and chemically-promoting conditions).

METHODS AND EXPERIMENTAL PROTOCOLS

The shrimps were purchased from a local market, already dead, presumably brined (EDS revealed a Na and Cl coating on the surface of tissue), and stored in a freezer until used. For experiments involving sediment, varying combinations of Edgar

Minerals Inc.™ powdered kaolinite and Soil Doctorx ™ powdered Garden Lime were either mixed with Artificial Sea Water (ASW) to the designated consistency or added as suspended sediment in ASW to the experimental container. All experiment sets took place in 1000 milliliter glass beakers that were sealed with Glad PressN’Seal™ in order to replicate dysoxic conditions. The ASW had a standard marine salinity of 35 parts per thousand and Experiment Sets 1, 3, and 4 used ASW that was inoculated with

MicroBacter7™ Aquarium Bioculture. For Experiment Sets 1, 3, and 4, an Oakton pHTestr® was used to measure pH in both ASW and suspended sediment. Prior to use, the shrimp were thawed with warm water.

The methodology and purpose of each Experiment Set was modified based upon observations from the previous set, and the sets are not numbered in the chronological order they were performed. In other words, some methods and secondary purposes were unique to an Experimental Set in an attempt to eliminate procedural biases and to refine experimental protocols.

Experiment Set 1: Artificial Sea Water without Sediment

For this set of experiments, shrimp were placed into individual beakers with inoculated ASW for a total of 12 weeks. These data served to guide the morphological and pH controls for Experiment Sets 2, 3, and 4. The other purpose of this set was to determine the effects that invasive monitoring (observational methods using tools) might have on morphological decay trends. pH was measured once a week for 12 weeks with a probe at 700-900 ml and morphological observations were made once every 4 weeks.

Accurate morphological observations subjected the shrimp to minor probing when necessary. Morphological observations and pH measurements were not performed on half of the shrimp in order to determine if minorly invasive probing influenced morphological decay trends.

Experiment Set 2: Immediate Burial in Kaolinite with Compaction

The purpose of this experiment was to observe the effects of burial on decay rate as well as to observe the effects of burial and slight compaction of shrimp on their taxonomically defining traits. This set of experiments simulated complete burial within highly viscous mud – roughly 2-parts kaolinite and 1-part ASW – in order to simulate rapid burial and low energy conditions over geologic time. Shrimp were buried individually in the kaolinite mixture contained by glass beakers (Fig. 1). Upon burial, each layer of sediment was slightly compacted with a stainless-steel spoon to eliminate air pockets.

Shrimp were buried under 0, 400, and 800 g of additional pressure, created by loading the sediment surface with a petri dish with copper shot, which was used because

it was readily available. At 4, 8, 12, and 16 weeks, 3 shrimp were exhumed from their beakers, one from each burial regime.

Due to the difficulties experienced in trial experiments in exhuming decayed specimens, the beakers were lined with a strip of Adfors™ fiberglass mesh, and the mesh was also placed directly below and above the buried shrimp to ensure easy and less destructive exhumation. In order to further replicate dysoxic conditions, 2 ml of ASW was then added on top of the sediment and any weight.

Compression is discussed further in Compression Simulation. Exhumation techniques are discussed in more detail in Excavation Methods for Buried Specimens.

This is the only Experimental Set for which there was a different shrimp scored at each sampling time, the potential effects of which are discussed in Monitoring Individual

Specimens Versus Sequential Excavation of Different Specimens.

Experiment Set 3: Sediment – Water Interface

This set of experiments simulated the sediment – water interface (SWI) – more specifically, a dysoxic, low energy SWI with an organism resting on the surface of the sediment (non-burial scenario). The purposes of this experiment were as follows: a) to observe any correlation between the presence of sediment and the decay rate or preservation of an in situ, deceased shrimp; b) to observe and compare sediment pH in varying proximity to the shrimp throughout decay; and c) to test the effects of invasive observation methods might have on morphological decay trends. Kaolinite was gradually added to 3 beakers already filled with ASW and allowed to settle naturally. After the sediment reached 700 ml, ASW was added to each beaker until there was approximately

200 ml of ASW above the sediment. One shrimp was then added to each beaker. The

shrimp did not disturb the sediment because each shrimp floated for a period of 7 to 14 days before naturally sinking (see Other Results). pH was measured once a week for 12 weeks with a probe at 800-900 ml, 400-500 ml, and 0-100 ml (defined in Fig. 2), and morphological observations were made every 4 weeks. One shrimp was left completely undisturbed in order to determine if minorly invasive probing influenced morphological decay trends like in Experiment Set 1.

Experiment Set 4: Gradual Sedimentation of Kaolinite-Lime and Pre-Punctured Carapace

This set of experiments simulated periodic sedimentation with powdered kaolinite-rich sediment – about 10% lime and 90% kaolinite to simulate the Solnhofen- type Lagerstätte. The purposes of this experiment set were as follows: a) to observe any correlation between the addition of a continuous source of sediment (and its composition) and morphological decay trends of the shrimp; b) to observe and compare chemical changes of the ASW in the presence of decaying shrimp as well as a continuous source of sediment; and c) to test if puncturing holes in the carapace would increase the sinking rate of the shrimp (see Other Results). If the holes helped the shrimp to sink faster, then they would promote a more realistic scenario – deceased shrimp would not experience sedimentation while floating on the surface, nor would they be exposed to same cycle of nutrients from the sediment. Shrimp were placed in individual beakers filled with about

700 ml of ASW. Half of the shrimp were punctured with 4 pin-sized holes on either side of the carapace while the other half remained unpunctured.

pH was measured once a week for 12 weeks with a probe at 700-900 ml and all specimens were subjected to invasive morphological observation once every 4 weeks.

Also every 4 weeks (after data was collected), sedimentation was simulated by evenly

sprinkling the kaolinite-rich sediment on top of the surface of the ASW; 0, 5, and 20 g of sediment was added to the respective unaltered and punctured specimen beakers. If the sprinkling of sediment caused disturbance or breakage of floating shrimp, it was noted but not scored until the next morphological observation time.

Morphological Decay

In order to monitor morphological changes throughout decay, important taphonomic features and observed decay character states were scored for all Experiment

Sets once every 4 weeks. This method and the character states were modified from

Klompmaker et al. (2017), and the character states were edited throughout a trial burial experiment as well as modified after Experiment Set 1 (ASW without sediment). The final list includes character states (Table 1) that were scorable across all of the

Experiment Sets; this was an extremely important quality when scoring under vastly different experimental conditions. As the numerical scores increase, the amount of decay increases – a score of 0 records no significant decay and scores with “incomplete” or

“absent” record complete decay of a trait. Character states that were scored as incomplete or absent were still closely monitored thereafter to make sure poor visibility did not account for that score. If a character state could not be accurately observed with minor invasive techniques alone, due to the position of the carcass, its current decay state, or clarity of the water, then it remained un-scored. For example, specimens submerged in

ASW (1, 3, & 4) were often difficult to reorient for thorough scoring as they were extremely fragile, and debris accumulated quickly: a hollow posterior cephalothorax could not be observed until the separation of the cephalothorax from the pleon. Excessive degrees of prodding and moving of carcasses was avoided for all but the buried

specimens. Reasons for including or combining certain characters and decay scores are explained in Table 2.

Morphological results between Experimental Sets were compared by averaging specimens’ numerical scores from each observation time. If a specimen was missing a score from one or more sampling times, the average was calculated without that score and it was removed from the total (for example, the scores 1, 0, X were averaged to 0.5).

Klompmaker et al. (2017) replaced missing scores with the conservative lower score or other various means: 0, X, 2 was reasonably interpreted or replaced with scores 0, 1, 2 or

0, 0, 2. This was not sufficient for the present character scores because, although they technically increase with degrees of decay, some character states and corresponding scores combine different parts of the shrimp or unrelated descriptions (1; fragmented cuticle, 2; hollowed carapace). Therefore, inserting the lowest or average score in the place of a missing score would not always be correct as “1” described the state of the cuticle while “2” described the state of soft tissue. The other reason missing scores were not replaced or compensated for was because Experiment Set 2 exhumed a different shrimp at each observation time, so assuming or calculating missing scores for different specimens would be inaccurate. However, it should be mentioned that Experiment Set 2

(compacted burial) specimens almost never had missing scores because the shrimp could be completely dissected as it did not have to be scored again at the next sampling time.

At the end of Experiment Sets 1, 3, and 4, all of the remnants from each beaker

(disturbed and undisturbed) were gently sieved through 850 µm mesh and gently rinsed

(for no more than 1 minute). The leftover remains from all Experimental Sets were stored in a drying oven at 110°F. Once dried, various identifiable remains (mostly carapace or

pleonal somite cuticle from the sieved remains) were chosen for SEM and EDS analysis.

In order to compare cross sectional microstructures of decayed cuticle between experimental conditions and decay times, pieces of dried cuticle were embedded in resin and polished.

MORPHOLOGICAL RESULTS

The Effects of Pre-Punctured Carapace and Variable Gradual Sedimentation (Set 4)

Pre-punctured holes in the carapaces of the shrimp (Experiment Set 4 specimens) had no effect on the decay rates (Fig. 3) nor did they have an effect on the sinking rate of the shrimp. Thus, in order to determine the effect of the amount of sedimentation on decay rates, the scores of the shrimp with holes and without holes were averaged together in order to have more morphological data for comparing 5 vs 20 g/month sedimentation.

For 10 out of the 15 character states, there was no notable difference between scores for specimens that experienced 5 g/month versus 20 g/month of sediment (Fig. 4).

Even for the 5 character states that showed a difference, there was no trend indicating that

5 g or 20 g of sediment consistently had the higher or lower scored traits. Because of this, the 5 g and 20 g scores were averaged together for the comparison of all averaged scores between all Experimental Sets (following section). The lack of significant difference in scores between 5 g/month versus 20 g/month of sedimentation could have been because the difference in the amount of sedimentation or perhaps the frequency of sedimentation was not enough to promote different scores.

The Effects of Sediment

Based on the scored character states, the specimens of Experiment Set 2

(compacted burial) experienced the least amount of overall decay, and Experiment Set 1

(ASW without sediment) specimens experienced the second least amount of decay. The

specimens of Experiment Sets 3 and 4 (SWI and gradual sedimentation) consistently had higher decay rates throughout the 12-week duration than Sets 1 and 2 (Fig. 5). As rapid burial is known to promote preservation, the buried specimens of Experiment 2 were expected to experience less decay. Moreover, the differences in experimental conditions, protocol, and results of Experiment Set 2 were significant and will therefore be discussed in more detail in the following section.

At every sampling time, Experiment Set 3 (SWI) shrimp either had the highest decay scores or shared the highest scores for all character states except state 12

(separation of pleonal somites). By week 12 though, the averaged scores for shrimp from all Experiment Sets indicated full separation between pleonal somites. Set 4 (gradual sedimentation) had nearly consistently the second highest decay scores. There were only

4 character states for which Set 4 scores were not the second highest, or tied for the second highest, by 12 weeks. The scores for the carapace cuticle (State 3), posterior cephalothorax (State 5), and the eyes (State 8) of Experiment Set 1 (ASW without sediment) specimens spiked above those scores for Set 4 by week 12. Experiment Set 4 also had significantly lower scores for the rostrum (State 9) for which Experiment Set 1 had the same scores throughout decay. Other than those four character states, Set 1 (ASW without sediment) specimens appear to have the lowest rate of decay as well as the overall lowest decay scores (out of Sets 1, 3, & 4). For the majority of the character states, the scores of Set 1 showed a more gradual rate of decay as they increased between sampling times while the scores of Sets 3 and 4 more often started with and continued to have the highest scores (Fig. 5). The character states for which Set 1 ended with higher scores than Set 4 suggests more taphonomic questions about the differences in

preservation potential of certain characters of shrimp over others (especially because the eyes were not hypothesized to be particularly decay-resistant in any experimental condition). Those four character states (3, 5, 8, and 9), as well as the overall comparison of scores between the experimental sets, also indicate differences in preservation between sediment type – when the sediment was 100% kaolinite (Set 3 – SWI), decay scores were the highest. When a small percentage of the sediment was lime (Set 4 – gradual sedimentation), decay scores were the second highest expect for those aforementioned character states for which Set 1 (ASW without sediment) scores were higher than Set 4.

So, kaolinite increased the decay rates for all character states and the gradual addition of a small percentage of lime helped to decrease the decay rate of most, but not all, of the character states.

Simulation of Rapid Burial

Experiment Set 2 results are reported with more character state details because the resulting specimens closely resembled compressed or other Lagerstätte fossils that preserve taxonomically defining characteristics. Complete burial in a thick mud delayed and even prevented decay; this was expected particularly in separation between large pieces of cuticle of the exoskeleton (States 1, 2, 12, 13 and 15). In fact, almost half of the averaged scores for Experiment Set 2 never increased from 0. Specimens were also consistent in not showing decay with respect to character states that didn’t record separation events (States 6, 10, 11). The soft tissues of the pleons (State 6), the internal skeletons (State 7), and the scaphocerites (State 10) remained nearly or complete up to week 12 (State 7 scored partial decay at week 12).

There was no consistent trend between separation events, cuticle fragmentation, or the decay of soft tissues influencing or even coinciding with one another as initially hypothesized. For example, the soft tissues of the anterior (State 4) and posterior cephalothoraxes (State 5) started to decay at week 4 even though the separation of the pleons from the carapaces (State 1) and the anterior cephalothoraxes from the posterior cephalothoraxes (State 2) never occurred. On the anterior half of the specimens, the internal skeletons (State 7) and the scaphocerites (State 10) remained complete (until week 12 for the internal skeletons) even though the carapace cuticle (State 3) started to decay by week 4. However, the posterior halves of the specimens did decay as hypothesized in that a lack of separation events and cuticle decay seemed to aid in the preservation of soft tissue. The soft tissues of the pleons (State 6) never experienced significant decay, nor did the cuticle of pleonal somites (State 13), and the pleonal somites never separated from one another (State 12). This is not indicative of the posterior halves of the specimens being more resistant to decay than the anterior halves; the pleopods (State 14) and the telsons (State 15) experienced significant decay as early as 4 and 8 weeks; thus, no consistent hypothesized trends occurred.

Cuticle Decay

Mircrostructural decay was observed between and within layers of the cuticle.

Fresh cuticle exhibited layering without any major separation or disruption while decayed cuticle from all Sets exhibited splitting between and/or within the exocuticle, the endocuticle, the epicuticle, and the membranous layer (Fig. 6). It is possible that slight disruption of layers in the fresh, non-decayed cuticle (Figs. 6.1 and 6.2) was caused by preparation of the tissue samples, but, it is minor compared to the degree of splitting in

the decayed cuticle (Figs. 6.3-6.6). Major separation of cuticle layers occurred by 8 weeks (Fig. 6.4) and could have occurred prior to that decay time, but that was the youngest decayed sample we had prepared for SEM examination. The oldest cuticle samples prepared for SEM were 20 weeks old (Figs. 6.5 and 6.6) from Experiment Set 1

(ASW without sediment) and Experiment Set 4 (gradual sedimentation) and between those and the 8-week-old cuticle (including few samples we prepared from in between those decay times), there was no correlation between the decay time of the cuticle and the degree of splitting between layers. There was also no correlation in the degree of splitting between differing experimental conditions, but, we did not have a well-sampled representation of cuticle from different Experiment Sets of the same age.

Only approximate degrees of splitting between our sampled cuticle layers was used to compare between decay times and experimental conditions, which was subjective. Unlike Briggs and Kear (1994), who captured images of CaCO3 crystal bundles with a number of different crystal habits in samples of decayed Palaemon and

Crangon, we were unable to observe crystal habits or confident evidence of precipitation of minerals in our cuticle samples. This could have been for three reasons. First, cross- sectioned and polished cuticle was not an optimal orientation to observe potential crystal structures. Second, cuticle, and the specific morphologic regions from which we recovered samples, did not exhibit crystallization as well as other tissues would have –

Briggs and Kear (1994) found that certain morphologic regions exhibited mineralization and crystal bundles more than others. The third possibility is that instrumental limitations associated with tabletop SEMs and the 1200x magnification limit of the instrument we used made it difficult to observe the aforementioned features. Magnification, however,

was a less likely factor as Briggs and Kear (1994) observed crystal bundles as large as

300 µm. We did observe mud-crack-like (Fig. 6.1) and round (Fig. 6.6 – white in color) masses near and between layered cuticle, all of which were calcium phosphate just like the fresh and decayed cuticle layers. However, because those masses were observed in fresh and decayed samples, it could not be confirmed that they were newly precipitated minerals and not pieces of cuticle observed on different axes. Considerations for observing decayed tissue and the preparation of cuticle for SEM are discussed in Methods for Tracking Morphological Decay and Total Amount of Decay.

Effects of Physical Agitation and Sieving Remains

The effects of physical agitation was monitored for two Experiment Sets because

Briggs and Kear (1993) found significantly higher decay rates (measured by weight) of specimens in a rotary shaker; their agitation was much more extreme as the purpose was to separate sediment from remains. Nonetheless, the effects of more minor agitation were explored.

Experiment Sets 1 (ASW without sediment) and 3 (SWI) contained shrimp that remained undisturbed throughout the duration of the experiments. At the end of the experiments, shrimp that were undisturbed appeared almost entirely complete. One showed partial separation between the cephalothorax and pleon, and some showed disarticulation of pereiopods and chelae. However, the rest of the carcasses appeared complete and articulated (without using invasive methods for thorough confirmation).

The shrimp were also easier to see than those that were disturbed as there was less unidentified debris that formed at the bottom of the beakers. Briggs and Kear (1994) identified this as a bacterial-originated precipitate also appearing with their ASW

specimens. Our disturbed beakers had much cloudier water and more debris. Some of this was likely bits of shrimp tissue dissociated by observational prodding as well as the disturbance of the layers of biofilm that formed on the water surface between each sampling time. The films would always reform every week after pH sampling and sometimes exhibited small patches of white mold on top. When disturbed, the films would either break apart and sink or sometimes the entire film would sink as a unit – more common with the sedimentation in Experiment Set 4 – and settle on top of the shrimp.

If morphologically scored, it appeared as though the undisturbed shrimp would have had significantly lower scores than other Experiment Sets, many of which never would have increased from 0. However, as soon as the contents of the undisturbed beakers began to be poured out for sieving, most of the shrimp disarticulated and the soft tissue completely disintegrated. The results are inconclusive in determining if minor poking and prodding for morphological scoring affected the decay rate of the shrimp because the undisturbed shrimp were not observed or scored at all until the end of the experiment. This supports some advantages of the scoring method discussed in

Measurements of Decay and Their Implications.

CHEMICAL RESULTS

The Effects of Sediment and Other Factors of pH Variation

The presence of sediment and its composition had a significant effect on the initial pH of ASW for all of the Experiment Sets. The initial pH of Experiment Sets 1

(ASW without sediment) and 4 (gradual sedimentation) were the highest followed by the lowest pH at week 1 (Fig. 7). Experiment Set 3 (SWI) did not follow this trend as the sediment used for those experiments was kaolinite only making the initial pH more basic than the others. The pH did not remain basic for long though and by week 1, Set 3 mirrored the first-week decrease of Sets 1 and 4. By weeks 1 and 2, pH from all Sets

(except Set 2 – did not have pH data) appeared to stabilize at about pH 7 before steadily increasing throughout the rest of the experiment. The addition of 10% lime to kaolinite used in Set 4 (gradual sedimentation) caused slightly higher initial pH compared to the

ASW-only controls of Experiment Set 1. Experiment Set 4 also ended with the highest pH after that stabilization period of around pH 7. Also stabilizing at week 1-2 was a constant gradient in pH between proximities to the shrimp as well as within the settled kaolinite in Experiment Set 3 (SWI). A gradient in pH between the top, middle, and bottom occurred as early as the initial readings, grew in difference by the first week, and continued throughout the duration of the experiment (Fig. 8).

On average, the sedimentation rate of 20 g/month led to higher pH in Set 4 (Fig.

7). This was expected as more lime (however small its percentage of the sediment) was

being added continuously. Both sedimentation rates also showed a gradual increase towards a pH of above 8 like the other Sets (except for the ‘stabilization’ period that occurred in the first couple of weeks). It was expected however, that the addition of sediment, no matter the amount, would be more obviously represented by changes in pH throughout the experiment – in other words, every 4 weeks (when sediment was added) should have resulted in perhaps a more step-wise increase in pH rather than a gradual trend like the other Experiment Sets.

The degree to which different factors affected pH could only be hypothesized as the present Experiment Sets only suggested the effects of sediment composition. For example, the present pH results do not indicate (but conjure consideration for) how much of the rise in pH was caused by the decaying shrimp versus the presence of sediment. The pH of inoculated ASW by itself revealed that the shrimp were predictably significant, but perhaps more responsible for the majority of the change in pH over time throughout the experiment. A large container of ASW (inoculated with the aquarium microbes) was kept during the experiment. Its pH was measured occasionally and revealed a steady decline in pH, becoming more neutral (Fig. 7). Future experiments should incorporate appropriate controls in order to determine the degree to which the effects that decaying shrimp, inoculated ASW, and stagnant (no aquarium filter/not oxygenated) ASW has on pH, in addition to the presence and composition of sediment.

The Effects of Pre-Punctured Carapace and Variable Gradual Sedimentation (Set 4)

The pre-punctured carapaces had no significant effect on the pH of the ASW for

Experiment Set 4 (gradual sedimentation). The slight differences in pH, however, did reintroduce the question of the degree to which sediment affects pH versus the decaying

shrimp itself. The pre-punctured carapaces appeared to affect pH of ASW with sediment differently than ASW alone. In Set 4 beakers that received gradual sedimentation, pre- punctured shrimp caused a slightly higher pH than that of unaltered shrimp (Fig. 9). In

Set 4 beakers that did not receive gradual sedimentation (same conditions as Set 1 beakers), unaltered shrimp caused a slightly higher pH than that of the pre-punctured shrimp. The latter was not expected as it was initially hypothesized that pre-puncturing the cuticle of the shrimp would increase decay and therefore, increase pH. These results call for appropriate controls in the future that determine how much each factor is responsible for changes in pH throughout the experiments – as well, testing the possibility that each factor may change throughout different stages of decay. For example, decay of the shrimp could have had the dominant effect on pH for the 2-3 week-long stabilization period seen in all Sets (as the presence of shrimp was the common factor between all Sets), after which, perhaps sediment composition became the more dominant factor.

COMPARING RESULTS TO PREVIOUS EXPERIMENTS

AND THE FOSSIL RECORD

The Effects of Sediment

The effects of sediment vary slightly between previously published experiments, but it appears that when specimens decay in the presence of non-compacted, non-burying sediment (i.e. on the surface of sediment or under gradual sedimentation), decay rates of specimens generally increase. This is the same effect observed as Experiment Sets 3

(SWI) and 4 (gradual sedimentation) had the highest and second highest decay scores as early as 4 weeks. Plotnick (1986) also noted a slightly faster decay rate when sediment was present in a non-burial form. The same effect appears to occur with polychaetes;

Briggs and Kear (1993) found initial decay to be faster in a SWI scenario with both qualitative observations of morphology as well as microstructural evidence indicating faster degradation of the membrane covering the jaws. By 30 days, though, their experiments showed similar levels of decay (measured by weight – explained later) between the sediment and non-sediment associated specimens.

Previous results vary from ours slightly more with respect to buried specimens than non-burial scenarios. Some of this variation, however, could be caused by longer decay times (potentially reaching a plateau in morphological decay) and different measurements of decay. Substantial differences were observed in our averaged scores suggesting that our buried Set 2 specimens had a much higher potential for preservation.

In fact, Set 2 was the first non-trial experiment to take place and the carcasses remained so complete (compared to what we were expecting) that the original character state list and corresponding scores included much more detail (e.g., carapace spines and grooves, dorsal and ventral rostral spines, and 3rd maxilliped). It is important to note that the amount of morphological detail that remained was easily comparable to compressed fossils – this was of course aided by the very fact that the sediment helped to prevent disintegration upon excavation that occurred with the specimens submerged in ASW. The description by Schweitzer et al. (2014) for flattened penaeoid shrimp from Luoping

Yunnan, Province, China could effectively be used to describe the exhumed specimens from Experiment Set 2 at 8 weeks:

“The majority of the specimens observed tend to be complete but deformed. That

is, the cephalothorax and pleon are preserved together, although the pleon may be

somewhat displaced. Partial remains of pereiopods are often preserved as well

and, in some cases, pleopods are also visible. In several cases, the cephalothorax

and pleon are deformed to different degrees” (Schweitzer et al., 2014, p. 472).

Specific morphological comparisons to the fossil record are discussed in the following section.

Contrary to our Experiment Sets, Briggs and Kear (1994) saw no significance differences in decay rates when shrimp were buried in sediment at sampling times of 4 and 8 weeks. These results are extremely different from ours as our buried Set 2 shrimp rarely increased from a score of 0 even by week 12. Their morphological results though, were based on mostly qualitative observations as the complete burial of the shrimp

‘corrupted’ their quantitative measurements of overall decay using loss of mass with

weight measurements. Also varying from our results, Wilson and Butterfield (2014) noticed no significant difference in preservation between Crangon buried in sediment and the ASW-only control specimen. It is likely that, similar to the non-burial scenarios, there is a decay time after which the shrimp reach a similar level of decay under all experimental conditions. Their excavation time (4 months) was later than ours, and they even noted that the ASW control specimen had a significant amount of articulated cuticle by the end of the experiment. It is also possible that the scoring method gave us more quantitative data which required and demanded more strict observation of decayed morphology than the sole use of detailed observation could – even with our infrequent sampling times. The scoring method and other previously used decay measurements are discussed later in Measurements of Decay and Their Implications.

Sediment type also had an effect on our decay rates as it did for buried Crangon

(and Nereis) from Wilson and Butterfield (2014); they found their specimens were best preserved when buried in kaolinite and that calcite and quartz sediments seemed to promote decay. Sediment type had the opposite effect on our non-buried specimens.

When 100% kaolinite was present (Set 3 – SWI), decay appeared to be accelerated and it produced the highest decay scores between all of our Experiment Sets. A small percentage of lime with kaolinite (Set 4 – gradual sedimentation) generally produced slightly lower decay scores than Set 3. Of course, physically introducing fresh sediment every four weeks, no matter its composition, likely influenced the difference between Set

3 and 4 decay scores. However, because there was no significant difference between 5 and 20 g/month sedimentation decay scores, sediment composition appears to have a

more profound effect on preservation potential, and that effect differs between burial and non-burial decay conditions.

Morphological Decay Trends

There were similarities and differences between decay rates (and general decay stages) indicated by our averaged scores and observations of past decay experiments. For example, we observed similar ASW-only decay rates as those from Briggs and Kear

(1994) for the following morphologies: a) disarticulation and complete decay of the telson and uropods, pleonal somite separation, and disarticulation of the internal skeleton.

They also observed similar timing of the separation event between the cephalothorax and pleon, occurring at 3 to 7 weeks under ASW-only conditions. Plotnick (1986) noted that the separation event would occur very early on in decay as the articular membrane securing the two segments was extremely thin. Conversely, Klompmaker et al. (2017) saw this event occur between 50-100 days for shrimp in ASW only, twice the amount of time that we observed in Set 1 specimens. Upon first consideration, their delayed timing of that event could have been because they used fresh shrimp (killed right before the experiment via lowering water temperature) and they did not poke or prod the shrimp for observation. Our shrimp were already dead, believed to be brined, and were subjected to minor disturbances for observation. Like Klompmaker et al. (2017), Briggs and Kear

(1994) used fresh shrimp and did not report any intrusive methods on the specimens but they still saw results closer to ours. So, major variations in rate of decay may not be methodological (in terms of observation methods) but could demonstrate a level of variation that can naturally occur in morphological decay rates even under similar conditions.

Not only was there variation in decay rates between past decay experiments with similar conditions, but we had scores of individual shrimp varying within the same experimental sets. Some of those instances could very well be explained by the long intervals between morphological scorings. In other words, specimens could have reached the same score within a delay of days or weeks while remaining unrecorded until the next observation period four weeks later. However, in some cases, the variation in scores was as large as a character state of one shrimp never experiencing a score greater than 0 whereas that character state of another shrimp from the same Set showed complete decay by the end of the experiment.

Similar degrees of variation in preservation occur in the shrimp fossil record, even in single assemblages, although the causes of that variation may be numerous localized taphonomic processes associated with each carcass. Carapace, pleonal somite, and endophragmal boundaries are obscured as often as they are preserved between and even within specimens; measurements and positions of somites are used for taxonomic classification. When the preservation of pleonal somites (and other segments) vary, their various ornamentation becomes more likely to be obscured or not preserved. Grooves and spines of the carapace, tergum, pleuron, and telson often vary between fossil biota.

Even though preservation variation of the same fossil traits raises questions about properties of preservation potential and localized taphonomic pathways, it has many times resulted in more accurate interpretations and classifications. For example,

Anisaeger brevirostrus Schweitzer et al., 2014, from the Luoping biota (Middle Triassic of China) was classified with pereiopods 1-3 having chelae even though no one specimen had all chelae preserved. The same described new genus and species had postorbital

spines, hepatic spines, and development of the cervical groove preserved in some specimens but not others due to distorted cuticle (Schweitzer et al., 2014). Similarly, all but one specimen of Macropenaeus sidiaichensis Boukhalfa et al., 2017, from the Early

Cretaceous Sidi Aïch Formation in Algeria lacked a rostrum. From the same described new species, the following morphologies were preserved on some specimens but not others: scaphocerite keel, antennular peduncles, biflagellate antennules, and most of an antennae (Boukhalfa et al., 2017). These and other cases of varied morphologic preservation from the same assemblages allow for more accurate classification.

Moreover, the same variations occurred throughout our experimental sets and added valuable additions to our averaged scores.

Other Results

Floatation occurred regularly for our non-buried specimens, and this was also referenced in several previous decay experiments with shrimp. Briggs and Kear (1994) noted immediate sinking of freshly killed Crangon and Palaemon after which floating was rare (other than one experimental condition altered with additional bicarbonate) and the carcasses only had a tendency to become slightly buoyant by 8 weeks. Conversely,

Allison (1986) reported that Palaemon carcasses (decaying on top of sediment) floated to the surface of the water within three days, a response attributed to decay gasses. Perhaps differences in experimental temperatures increased or decreased gas retention in tissue and caused different results for floating specimens (Briggs and Kear, 1994). Temperature would have also affected osmotic and diffusion rates. Klompmaker et al. (2017) was unable to include a picture of the shrimp carcass (and the horseshoe crab carcass) at the

100-day observation because it was ‘lifted by black algae’. These inconsistencies with

floating shrimp carcasses and the proposed reasons from previous experiments may not be exactly urgent but the range of time by which floating occurred as well as the lack of its mentioning is interesting to note.

Every single one of our non-buried specimens floated on top of the ASW as soon as they were placed in the beakers and they continued to float for 1 to 2 weeks. Segments of the shrimp usually sank at the same rates except for some of Experiment Set 4 specimens (gradual sedimentation); sometimes the cephalothorax and pleon separated and one segment would sink before the other. The addition of sediment on top of the

ASW while the shrimp were still floating certainly promoted the separation event. This did not skew our score data though, because we already know from our other experiments and previous decay experiments that the separation of cephalothorax from pleon occurs very early on and was usually scored as separated at the first morphological sampling at 4 weeks. The floatation of specimens would have skewed our morphological scores if our observation times were more frequent (we could have compared exact sinking times to the timing of decay within days). However, Briggs and Kear (1994) documented no noticeable difference in morphological decay between their only floating shrimp (with additional bicarbonate) and the rest of their shrimp specimens. The immediate floating of our specimens could have been caused by an additional number of factors not applicable to the previous experiments. For example, the commercially purchased shrimp were frozen and likely had some air pockets trapped in the carcass. Also, brined or treated specimens would have different diffusion rates than those with altered tissue and cuticle.

Once the shrimp finally sank, they never floated to the surface again or showed any signs of a more buoyant state. Regardless, the timing of flotation or its lack should be recorded

in future experiments, particularly in consideration of any effect floating or sinking would have on morphological decay trends.

Another decay-induced occurrence was observed in every single one of our specimens across all experimental conditions. A hardened, red-brown mass formed inside the posterior cephalothorax by 4 weeks in every single specimen. This formation of a red mass was originally included as a character state but was removed because there was no variation in its formation. However, keeping it as a character state would have helped track more exact timing of its formation between experimental conditions and should be included in future character state lists with the addition of more frequent sampling times.

In some cases, the red mass formed as early as 2-3 weeks. After being left in the drying oven, the hardened mass would change color (to white or a darker red-brown) and always became very brittle and delicate. It was also delicate as soon as it formed in experimental conditions, but it still resisted disintegration (unlike the majority of the rest of the shrimp carcass) after sieving and could, therefore, have implications on the interpretation of fossils. This hard mass has been noted in past decay experiments with shrimp; Hof and

Briggs (1997) believed it to be the hepatopancreas which mineralized in the form of

CaPO4 microspheres in mantis shrimp. Briggs and Kear (1994) found the hepatopancreas of Crangon and Palaemon were frequently mineralized but their granular texture revealed no structural detail. We encountered difficulty preparing and mounting the hardened hepatopancreas for SEM as most samples were extremely fragile after they were fully dried, and they crumbled upon mounting them to the adhesive tape. When the hardened hepatopancreas could be observed, no structural detail was observed, and it was compositionally the same (calcium phosphatic) as our cuticle samples. Future

experiments should follow traditional methods and embed the hardened hepatopancreas in resin (as we did with specimens of cuticle) because once dried, they become extremely delicate and difficult to mount for SEM. That was how Briggs and Kear (1994) were able to observe the hepatopancreas as well as delicate muscle fibers of decayed Palaemon and

Crangon. Or, perhaps if an eSEM is used to observe future samples, samples could be mounted before they have fully dried and have become difficult to prepare.

TAPHONOMIC INTERPRETATIONS AND THE USE OF SEDIMENT IN

EXPERIMENTAL TAPHONOMY

More Accurate Interpretations of Fossil Morphology and Taphonomy

The preservation of shrimp as relatively complete and intact carcasses despite having thin cuticle and delicate articulation strongly suggests rapid burial of the carcass, as major decay and disarticulation events occur as early as days after death (Plotnick,

1986). This is supported by our buried specimens and the resulted low decay scores. The need for rapid burial to counter rapid disarticulation may argue that complete fossils represent live burial of organisms because even a week-old shrimp carcass would not stay articulated against any slight deposition event or mild currents. Whether an organism is living or dead upon burial may not be particularly crucial in fossil taxonomy, but because decay experiments should be striving for realistic conditions, the manner in which the experimental specimens are killed should be considered (oxygen deprivation, temperature, etc.).

Variable experimental decay rates also mirrors the variable preservation potential of the same traits within and between fossils of the same biota. Decay experiments can determine if this variation was caused by natural decay rates, other taphonomic processes, or a combination of the two. This distinction must be made for accurate classification of fossil shrimp. Many soft-bodied fossils are preserved as flattened specimens, meaning there will always be some kind of distortion from the original morphology. Shrimp fossils

are no exception and they, and their traits, are often reported as distorted, obscured, cracked, or displaced. Decay experiments have revealed that some damage is actually naturally occurring during decay events, so morphologies that might have been disregarded due to the fear that they were created via other taphonomic processes can more confidently be interpreted. In other words, preserved morphologies can more confidently be used to classify fossils instead of their presence or absence being attributed to other taphonomic processes such as transportation, predation, or compression. Otherwise, interpretations must remain unbiased or rather, all possibilities

(taphonomic processes, poor preservation potential of a weakly developed feature, or the very fact that a feature never existed) must be considered and documented. Schweitzer et al. (2014) used the lack of a diaeresis to classify the new genus and type species

Anisaeger brevirostrus Schweitzer et al., 2014. However, it was also noted that the diaeresis of extant shrimp is weakly developed compared to lobsters, so it was concluded that the diaeresis was simply not preserved in those specimens.

These ambiguities are the very reason Allison and Briggs (1991) first assigned

‘taphonomic thresholds’ defined by key stages of decay in experimental specimens. A stage could be loosely identified in a given fossil, making the absence of some morphologies more likely to be ‘not preserved’ or ‘was never there’. (For more on

‘taphonomic thresholds’, see Measurements of Decay and Their Implications). For example, the separation of the carapace from the internal skeleton as well as the displacement of the pleon is frequently reported in compressed shrimp fossils and because of decay experiments, they can now be attributed to the natural decomposition of the shrimp and not shear stress. If those decay events had instead been attributed to shear

stress or other taphonomic processes, then other morphologies would more likely be interpreted as obscured or distorted traits, leading to less confident taxonomic classifications.

In conclusion, experimental rotting of specimens in ASW has led to and validated fundamental taphonomic interpretations of fossils, like the importance of rapid burial of marine organisms leading to potential preservation. ASW decay experiments also act as a sort of control to interpret fossils more accurately in terms of identifying naturally occurring decay events and stages. The natural step forward for commonly compressed, soft-bodied fossils would be to incorporate experimental burial and compression of specimens to further add to taphonomic interpretations of fossils. Just like ASW-only experiments have helped to interpret ‘tapho-anatomic ordering’ of fossils (Wilson and

Butterfield, 2014), the use of burial experiments will further those interpretations because they recreate more realistic conditions under which fossils form; sediment promotes diagenetic processes which induce shifts in the potential for preservation (Wilson and

Butterfield, 2014).

Complications with the Use of Sediment and the Importance of Burial Scenarios

Just like ASW-only decay experiments, those that involve sediment have a similar fundamental complication in that it is impossible to recreate the exact paleoenvironmental conditions under which the comparative fossils formed (e.g., extinct subject specimens and microbiomes). In addition, analyses of experiments involving sediment rely heavily on analogies with soil science and organic chemistry, the models for which do not readily address implications on specific morphology and therefore cannot immediately be applied to paleontological studies (Wilson and Butterfield, 2014).

The involvement of sediment also immediately adds to the number of variables that can contribute to decay which makes it harder to find appropriate control groups as well as pinpoint the factors responsible for changes in morphological results. For example,

Wilson and Butterfield (2014) tested the effects of sediment type on preservation potential of buried Nereis and Crangon with ASW-only specimens used as the controls.

In addition to the consideration of differences between the controls and buried specimens

(e.g., pH and oxygen levels), they also had to consider grain size, permeability, and other sediment-specific factors that could affect the numerous influences of preservation.

The use of sediment in experiments in any form – compacted, suspended, burial scenario, etc. – also introduces major methodological complications. In fact, Briggs and

Kear (1994) omitted their weight-loss data from their sediment experiments because it was impossible to fully separate organic tissue from the sediment without damaging the tissue itself. This happened despite their inserting a piece of nylon cloth between the bottom layer of sediment and the carcass in an attempt to aid in the recovery of the remains from conditions comparable to our SWI experiments. The inability to effectively separate sediment from tissue would have skewed their decay results measured via weight loss and would have obscured their mineralogical analyses via SEM. No matter the purpose of the decay experiment (morphological, fossilization, diagenetic, etc.), the involvement of sediment adds logistical complications that hinder the recovery of quantitative effects of the sediment (Martin et al., 2004; Wilson and Butterfield, 2014).

Experimental complications notwithstanding, rapid burial is clearly crucial in the preservation of both Burgess Shale-type fossils and carbonaceous compression fossils, so the use of sediment with respect to burial experiments is absolutely necessary.

Fossilization requires burial, after which the immediately adjacent grains may be altered via diagenetic processes (Martin et al., 2004). The degree to which levels of decay vary in and between fossils depends on whether or not diagenetic, authigenic, or other decay- slowing processes (like lower temperatures) occur at a faster rate than decay itself

(Briggs, 2003). Simulating a more realistic environment by adding sediment and/or burying specimens will lead to better taxonomic and taphonomic interpretations just like

ASW-only decay experiments vindicated the importance of rapid burial and helped identify naturally occurring decay stages in the fossil record.

Excavation Methods for Buried Specimens

The careful excavation of Experiment Set 2 shrimp was particularly important since these experiments focused primarily on in situ morphological decay trends.

Excavation methods were not needed for shrimp from Sets 1, 3, and 4 as they could be scored from within the beakers. However, the potential advantages of their in situ excavation are discussed in the following section. Future excavation methods will depend entirely on the purpose of the experiments and how overall decay is measured, which is discussed in Measurements of Decay and Their Implications. The following recommendations and discussions use scored morphological traits to measure decay.

For the first burial trial – the results of which are not recorded in this study – the shrimp were exhumed in the following manner: any excess water on top was siphoned off or decanted (Wilson and Butterfield, 2014). Then, the contents of the container were immediately removed as a single block by scraping the perimeter of the container and gently squeezing the outside of the plastic container (see Experimental Containers).

Finally, the shrimp were exhumed by carefully scraping away the sediment from above

and around the sediment block. This method, for the most part, did result in impression- like, in situ profiles of the shrimp (Fig. 10). The moisture content of the block of sediment – in other words, how long the block of sediment sat before removal – greatly influenced the excavation methods. In another trial, excess water was decanted and the sediment was left to dry in the container for a day (following Wilson and Butterfield,

2014). This sediment was harder to scrape away, and some shrimp were exhumed by physically breaking open the dried-out block of sediment. This made it harder to keep track of any now-brittle pieces of shrimp as the sediment block was broken open. In addition to this, the pressure needed in order to scrape away sediment meant less accuracy and more damage to the shrimp, which degraded morphological analysis.

Different excavation methods were tested. Letting the contents of the containers dry out completely led to using traditional excavation and preparation methods for fossils. Specifically, a microjack was tested on an already partially exhumed shrimp that had been in a drying oven for over 72 hours. This allowed for more careful and intentional removal of the sediment. However, the cuticle became extremely brittle and cracked often, with more chances for ‘fly-away’ bits. Furthermore, drying the contents neither prevented nor reduced adherence of sediment to the shrimp, so the microjack disturbed the cuticle as well as the surrounding sediment.

For Experiment Set 2, the sediment was not allotted any time to dry out and mesh was used for excavation. The higher moisture content of the mud with the added mesh kept the shrimp from becoming too brittle. In fact, the shrimp were possibly even more delicate, but fragmented tissue was more malleable and was less likely to get blown away as it adhered to the mesh and could be recovered and permitted data collection. The mesh

did not have any obvious effects on the morphology of the shrimp even with the added weight for compression. It aided in efficient excavation of the shrimp and in most cases, the mesh aided in reducing the amount of sediment that adhered to the shrimp. There were a couple of cases in which the mesh adhered to the shrimp, so peeling the mesh back actually removed some cuticle and tissue (Fig. 11). In contrast, preparation by scraping off sediment typically disrupted pieces of cuticle and tissue and made those pieces unrecognizable for scoring. It should be noted that the instances in which the mesh did adhere to the shrimp did not correlate with the amount of weight added on top of the sediment or the duration of decay. Some considerations should also be taken into account both for the results discussed in this paper and for future experiments. The composition of the mesh – or whatever material is used for the same purpose as the mesh – may affect chemical or mineralogical analyses of the cuticle, ASW, or the surrounding sediment.

Also, even though the amount of weight we used did not cause the mesh to affect morphology, its use should certainly still be considered as a complication when morphological observations are made. Finally, the use of the mesh, including its placement and dimensions, may also affect microbial activity around the shrimp by changing the surface area of substrate available to microbes.

Monitoring Individual Specimens Versus Sequential Excavation of Different Specimens

Discussion of excavation methods must also consider the advantages and disadvantages of observing the same specimen versus a different specimen each sampling time. The same advantages and disadvantages apply to and should be considered for analyses and samplings of organic tissue, ASW, and sediment. Regardless of which protocol is used – observing the same or different specimens each time – future

experiments should use the same protocol for each series, including controls. This is because there is a chance that monitoring the same, individual specimens throughout some experiments versus different, sequentially excavated specimens for others will skew data. For example, our buried specimens from Experiment Set 2 were the only ones studied by sequential excavation, and those shrimp were more thoroughly observed and therefore, more accurately scored, because there was no need to avoid excess prodding that would affect the next evaluation. This was one of the main advantages of using different, sequentially excavated specimens. The first character state list was vastly more detailed than the final list partially because the buried specimens were so well preserved, but also because the carcasses could be thoroughly dissected because they wouldn’t be scored again later. Sequentially removing different specimens was necessary for

Experiment Set 2 as it would not be realistic to excavate and re-bury the same specimen throughout an experiment. Excavation and re-burial techniques aside, that would have had a number of affects like introducing oxygen and unavoidable damage to the specimen with excess handling.

Since burial scenarios required removal of different specimens and since in situ excavation of the buried specimens allowed for better scoring, specimens in all future experimental conditions should undergo the same protocol. In situ excavation of non- buried specimens would be difficult as shrimp that were not buried in compacted mud were always saturated in ASW (even if sediment was present in a non-burial form) making them extremely fragile and difficult to observe even early on in the experiment.

Beyond disarticulation, the remains started to disintegrate and created debris. So, when specimens are left unburied (to contrast with buried specimens), the difference in

extraction techniques alone may cause artefacts in the experiment’s results. But, if the control shrimp could be physically exhumed from the ASW in an in situ state, it could undergo the same level of observation as the buried shrimp. For instance, decanting water from experiments that did not involve burial resulted in many detached and unrecognizable pieces of tissue that were impossible to score (Fig. 12). Perhaps an extremely fine mesh, equivalent to fine filter paper, could remain at the bottom of the experimental container until the observation time. Alternatively, the majority of the excess ASW could be siphoned off and the rest left to evaporate until the remains become properly observable, although not in a drying oven because the remains would then become too brittle for detailed scoring. Using only different, sequentially excavated specimens and the ability to remove ASW-saturated specimens would also eliminate missing morphological scores as they were almost always caused by the combination of poor visibility in ASW and trying to avoid excess probing or re-orientation of continuously monitored specimens.

Continuous observation of individual specimens did not provide any obvious advantages in terms of morphological scoring. It was hypothesized that the decay of some characters would lead to the decay of others and that there would be a trend between separation events and further cuticle fragmentation, with the decay of soft tissues influencing other decay paths. It was further hypothesized that continuous monitoring of individual specimens might allow observation of more subtle changes in morphology.

However, no such trends were observed in the averaged scores of our continuously monitored specimens of Experiment Sets 1, 3, and 4. This suggests several possible conclusions. First, using scoring as the measurement of decay inhibited the record of such

subtle changes that may have indicated the hypothesized trends. Second, our other methodologies (combining unrelated character states and scores, too few and insufficiently frequent scoring times, etc.) combined with using continuously monitored specimens obscured or reduced the ability to record the hypothesized trends. Three, the natural variability in morphological decay might never produce the hypothesized trends, no matter the measurement of morphological decay.

Continuously monitored specimens and sampling, however, may be beneficial for long term, chemical analyses. Chemical properties of fossilization, particularly when being monitored with differing proximities to organic decay, should be analyzed in realistic conditions which would require undisturbed decaying specimens, which would only be possible with installed instrumentation such as pH and O2 probes. If sampling

(either of the ASW, the sediment, or the specimens themselves) for numerous instrumental analyses is required, then continuously monitored specimens could not be used, as any disturbance would affect subsequent analyses, unlike the use of different, sequentially excavated specimens.

In conclusion, excavation methods and sampling protocol depend on the purpose of the experiment (as well as the measurements of decay) but are crucial for accurate observations of future burial experiments. Future experiments should explore different burial scenarios using sequential removals of different specimens and samples, including controls.

Compression Simulation

Extra weight was added on top of the sediment for Experiment Set 2 in order to simulate compression of the shrimp by varying degrees of burial. The objective was to

determine if compression either created, destroyed, or obscured morphological traits that are used to systematically identify fossil shrimp preserved as flattened specimens.

Boukhalfa et al. (2017) suggested that laterally oriented carapaces of fossil shrimp provide little taxonomic value because they are subject to various taphonomic modifications – most commonly, alteration of observed carapace ornamentation caused by the morphology of the underlying endoskeleton. The addition of simulated compression to burial experiments will aid in tapho-anatomic interpretations of compressed fossils and considerations for future experiments are discussed.

Methods for simulating experimental compression should be explored. Additional weight was also added for the first burial trial experiments but, instead of using petri dishes filled with copper shot, two sandstone cores weighing a total of 400 to 840 grams were placed on wooden platforms atop the sediment. These sandstone cores were used because they were a readily available source of weight for compression. In retrospect, using two sandstone cores resulted in uneven weight distribution. Therefore, Experiment

Set 2 used copper shot, filling a petri dish, because that was thought to provide an even distribution of weight over the buried shrimp. This method is not recommended for two reasons. First, the 400 and 800 g of weight had notably less effect than the sandstone cores in distorting the shrimp in Experiment Set 2. Second, the copper may have contaminated chemical analysis of the sediment and may have affected the decay rates of the shrimp. The chosen method for compression simulation must also be applicable to control specimens, both non-buried and non-compressed which may be difficult depending on experimental conditions.

Different methods for simulating compression might also affect the experimental container. For example, reasons for the lack of an effect of weight on Experiment Set 2 versus the trial experiments are unknown, but it is important to note that the dimensions of the plastic food storage containers used in the trial were different than that of the 1000 ml glass beakers. The food storage containers were shorter in height (held less sediment) and had a wider opening than the glass beakers, and thus had more surface area and likely allowed for more displacement of sediment around the shrimp. Rex and Chaloner (1983) used a slender, rectangular apparatus with plexiglass sides within which synthetic material was placed (saw dust instead of sediment; circular piece of foam instead of organic tissue). This was then compressed in order to recreate and understand forces leading to compression plant fossils. They introduced a number of considerations and complications similar to what would be expected in shrimp compression experiments.

They considered ranges (as percentages) of potential vertical and horizontal growth of different parts of woody plants caused by their compression. They also omitted any kind of water from the experiments despite crediting compression to the decrease of pore space caused by the movement of water through sediment. This was because they did not have the mechanical means to provide a continuous flow of water up and out of their compression apparatus.

Another variable to consider for compression experiments is that of the orientation of the compressed shrimp. A lateral attitude is most common for shrimp from

Solnhofen-type limestone localities, but it is not the only reported orientation. Schweitzer et al. (2014) described Triassic shrimp from the Luoping biota and suggested the unique orientation of some shrimp could have been caused by a shear force acting on the still

soft remains; the shrimp were described as not being completely ventrally or laterally preserved, but instead had the lateral aspect of one side exposed with a substantial portion of the other lateral side exposed as well – this gave the shrimp a ‘twisted’ appearance.

Future experiments should consider different orientations of the shrimp as well as the type/ direction of the compression acting on the shrimp.

Rex and Chaloner (1983) also emphasized that fractures in the rock that expose plant fossils can affect this compressed form; the fracture is influenced by the matrix, the fossil-matrix interface, and the original shape and orientation of the plant. Ideally, a fracture that exposed a compressed fossil splits to create a compression fossil on one side and an impression (or a mold of surface features) on the other side (Rex and Chaloner,

1983). However, any given compressed fossil usually exhibits more than one form of preservation. Conway Morris (1979) stated that the locations of fracture planes led to varying exposures of different parts of Canadia spinosa in the Burgess Shale Formation – specifically, many of the invertebrates there exist across more than one layer of microbedding and some arthropod appendages are even interrupted by thin layers of sediment. Whittington (1975) also attributed the likelihood of a fracture plane to the orientation, or angle to bedding, of the animal in addition to the surface area and width of the appendages of the animal. Jones et al. (2014) classified Devonostenopus pennsylvanienses Jones et al., 2014, from a fractured specimen that preserved the pleon as a cast on one part and the carapace as a mold on the counterpart, both of which had partial carbonized cuticle. Compression experiments will have to consider preferential fracture planes, and the variously preserved characters they expose could also influence potential experimental excavation methods.

In conclusion, the variables, as well as their variations for controls, involved in successfully recreating compressed fossils is extensive. In addition to the aforementioned complications associated with the addition of sediment as well as the excavation methods, compression experiments must consider the following:

• appropriate controls to any dependent variables being tested (sediment type,

amount of weight, carcass orientation, etc.)

• the amount of added weight as well as the means of applying or simulating

compression

• the amount of time for which a specimen is compressed and at what stage of

decay (compression could continue/increase/decrease throughout decay)

• the experimental container and its dimensions compared to the specimen

• the physical properties of the sediment itself (grain size, porosity, etc.)

• the orientation of the compressed specimen as well as the direction(s) of the

force(s) acting on the carcass

• the use of synthetic versus more realistic material as well as the use of water

MEASUREMENTS OF DECAY AND THEIR IMPLICATIONS

Methods for Tracking Morphological Decay and Total Amount of Decay

Scoring morphological features in order to monitor both morphologic-specific and general decay has potential but for the present Experiment Sets, but there was likely a large bias caused by the extreme difference between experimental conditions. In other words, as much as the compact sediment in Experiment Set 2 aided in preservation, it also allowed for closer and more careful morphological observation than Sets 1, 3, and 4.

Nevertheless, the scoring method did effectively determine that certain characteristics and decayed more or less in different decay conditions. The scoring method also mirrored the amount of variation that occurs in both experimental and fossil specimen preservation under the same experimental conditions and biota. Also, as previously mentioned, scoring produced quantitative data and ensured consistency and thoroughness that could otherwise have been lost. Because of this, it is possible that in addition to a naturally occurring variation in decay rates, differences in decay rates between our and past experiments was caused by our different measurements of decay. Wilson and Butterfield

(2014) photographed exhumed Crangon which were buried for 17 weeks in montmorillonite, which looked extremely similar to our trial shrimp that were buried for

16 weeks (Fig. 9). Their decayed Crangon ‘occurred as thin cuticular films adhering to un-collapsed sedimentary matrix’. However, unlike our observations, they ‘observed no significant difference’ between their buried shrimp and their ASW control shrimp. Other

methods for monitoring morphological decay should be further researched, but scoring

(or a similar method) should be considered for future experiments for the following advantages: it produces quantitative data; it detects naturally occurring variation between morphological decay rates when multiple specimens are used; and it requires strict thoroughness at each observation time.

Aside from those advantages of scoring, there are some criticisms that should be mentioned about the present character states scored for these Experiment Sets. Some character states and their corresponding scores combined different parts of the shrimp or unrelated characteristics (like combining hollow and cracked carapace when one indicates the decay of soft tissue while the other indicates cuticle decay). This made it difficult to look at the averaged scores of each character state and correlate timings of specific decay events to others. Another limitation of the present Experimental Sets, not directly related to the character states or the scoring method, is that our morphological observation times were too infrequent: once every 4 weeks did not provide an ideal amount of data and should be as frequent as once a day for the first 2 weeks and once a week thereafter like the pH readings.

In addition to separating unrelated characters and more frequent sampling times, future experiments should consider adding the following decay characteristics: denatured cuticle characterized by cuticle coloration and carapace translucency (Klompmaker et al., 2017) as well as the discoloration of the ASW (Briggs and Kear, 1994) or sediment (Martin et al., 2004; Naimark et al., 2016) throughout the experiment. The difficulty of those characters though, is their qualitative nature which, if a scoring method is used, will have to be translated into a semi-quantitative observation. For example, the Munsell Soil Color

Chart could be used to monitor sediment color throughout decay and changes in the numbered colors would provide quantifiable data that could be compared to future decay experiments. The present character state list also did not extensively include decay of soft-tissue which can also be preserved in the fossil record. However, thoroughly observing those features was very difficult for non-buried specimens and will require different methods (see Monitoring Individual Specimens Versus Sequential Excavation of

Different Specimens). A more extensive inclusion of commonly preserved soft-tissue in future experiments will help to distinguish decay-related features from morphologies used in systematic studies.

Some other previously used methods for monitoring morphological decay should be considered or used in addition to the scoring method used here. Briggs and Kear

(1994) and Hof and Briggs (1997) defined numbered stages for decay of shrimp that included key morphological changes that began with (a) a ‘freshly killed’ stage, followed by a (b) ‘swollen’, (c) ‘ruptured’, (d) ‘hollow’, (e) ‘disarticulated’, and finally ending with (f) ‘fragmented’ stage of decay. They designated similar stages (Briggs and Kear,

1993) which were specific to polychaete worms. In fact, the decay stages were similar between many soft-bodied organisms. A number that represented each decay stage was then assigned or identified in experimental specimens at given observation or sampling times. This method was fundamentally similar to the scoring method, but it had important implications and applications for fossil specimens. The definitions of each decay stage acted as a ‘taphonomic threshold’ (Allison and Briggs, 1991) and even though the definitions of each decay stage weren’t as specific or in depth as our character states, they included commonly seen stages and distortions preserved in fossils (e.g., separation of

the pleon from the carapace, separation of the anterior cephalothorax from the posterior cephalothorax, displacement of the internal skeleton from the carapace, etc.). Those numbered decay stages can also be identified in fossil specimens, leading to more accurate tapho-anatomic interpretations. For example, the absence of a morphological character could be interpreted as more likely never having existed in a ‘freshly killed’ fossil versus more likely not to have been preserved in a ‘disarticulated’ or ‘fragmented’ fossil. Sansom et al. (2010), Sansom and Wills (2013), Wilson and Butterfield (2014 and refs. therein), and Klompmaker et al. (2017) also considered the implications their morphological decay results would have on already classified fossil taxa and the importance of those comparisons on phylogenetic classifications. Defining and applying

‘taphonomic thresholds’ to experimental and fossil specimens is advantageous and extremely important. However, it is important to consider our conflicting decay times with those of Briggs and Kear (1994) – as well as the other previously mentioned publications – and the possibility that those variations could have stemmed from the different methodologies for measuring morphological decay. Identifying decay by morphological stages is insufficiently quantitative (Briggs and Kear, 1994). Even though our averaged scores indicated very similar decay trends to those of their defined decay stages, the variation within our own scores (under the same experimental conditions) could be significant to track, which our scoring method was able to do. This is why it is likely beneficial to include more than one measurement of morphological decay when comparing between different experimental conditions – even if the most major difference is an ASW-only control for buried specimens. Of course, more than one measurement of

decay can create further inconsistencies if the methods for excavation and treatment of specimens differ.

Microstructural morphologies should also be used to monitor decay in specimens.

Briggs and Kear (1994) and Hof and Briggs (1997) observed tissue of decayed shrimp via examination of carcasses and molts (and bacterial films) by scanning electron microscope. They also used SEM to observe crystal structures and their mineralization, which we were not able to observe. Even so, we were able to observe disruption of cuticle layers due to decay. For example, where the cuticle split (between and/or within the epicuticle, endocuticle, and/or exocuticle) and the degree of separation might correlate to decay time or correspond to differing experimental conditions, but we were unable to quantify that, probably due to lacking tissue samples from key decay times.

Moreover, the deterioration of structure and layers as decay goes on could have implications for the surface area of cuticle and for the potential for biomineralization of each layer of cuticle. The identification of specific layers of cuticle (and their condition or degree of damage) in a given fossil have an effect on morphological interpretations and preparation methods of said specimen. For example, the thin epicuticle of shrimp may commonly be interpreted to have not likely been preserved in fossils. Or, it is assumed that fossilized cuticle of Crustacea commonly splits naturally on the contacts between distinguished epicuticle, endocuticle, and exocuticle while preparing specimens with a microjack. These affect the interpretation of carapace ornamentation – preserved spines and grooves could be epicuticle or could be a less pronounced internal mold lying on top of the endocuticle or exocuticle – leading to assumptions about potential

dimorphism and general taxonomic classifications of fossilized shrimp, crabs, and lobsters.

Furthermore, continued and refined research of decayed epi, endo, and exocuticle

(and preparation thereof) could add microstructural details to strengthen future

‘taphonomic thresholds’ identifiable in fossils and could further interpretations of cuticle ornamentations. Because of this, future experiments should utilize microstructural imaging of decayed cuticle, including identification of crystal structures and EDS analysis when possible, and should consider the following notes on preparations of tissue samples prompted from the present experiments. First, consider using small pieces of recovered cuticle when embedding samples in resin; the cuticle samples were laid flat and glued to a primary layer of hardened resin, and using larger pieces of carapace and pleonal somite made it more difficult for air bubbles to escape from underneath the tissue. Second, the hardened hepatopancreas (and other delicate, less rigid tissues) should be prepared with traditional embedding or coating methods, which was not required for our samples observed with eSEM but would have been beneficial regardless. An additional point regarding the first two suggestions is that polishing samples may not create optimal surfaces for observing the precipitation of minerals, and imperfect surfaces may reveal additional (or clearer) characteristics, including those potentially preserved in/between decayed cuticle layers. Finally, prepare cuticle samples via critical point drying as it is commonly used to preserve microstructures for SEM analysis. At the time, there were no obvious differences between oven-dried and critical-dried fresh cuticle samples taken from the same specimen. However, later review suggested slightly more disturbance of layers in the oven-dried cuticle that was likely caused by rapid shrinking

(Figs. 6.1 and 6.2). Samples of the hardened hepatopancreas did not undergo critical point drying out of concern that they would disintegrate once submerged (or re- submerged for Experiment Sets 1, 3, and 4) in liquid and that any structural characteristics would be lost. Thus, preferential drying methods of the hardened hepatopancreas could not be confirmed for future decay experiments.

In addition to monitoring decay of specific morphologies and microstructures, potential measurements of total decay such as loss of organic material should be researched further, and it could be beneficial to compare that data to the more specific morphological and chemical observations. For example, Hof and Briggs (1997) recorded initial weights of mantis shrimp before decay but were able to exclude any statistical correlations between the initial weight of the shrimp and final percentages of weight loss.

With a measurement of total decay, similar statistical analyses could be tested in association with sexual dimorphism, molting cycles, and between differing experimental conditions. Briggs and Kear (1993, 1994) and Hof and Briggs (1997) measured total weight loss during decay by using the percent difference between initial and final wet weights and dry weights. They reported that dry weights provided a more reliable indication of differences in decay rate because wet weights reflected/were influenced by any factors that also affect osmotic factors – salinity, permeability of the cuticle, rupture events of the gut and cuticle (which, like our decay rates could potentially vary even within the same experimental conditions). Again, total weight loss should be paired with morphological measurements of decay because differences in final weights do not record trait-specific decay events. Perhaps additional scoring of the dried, sieved remains could represent total amount of decay (when compared to decay scores from sampling times)

and results could be compared to weight loss results from the methods of Briggs and

Kear (1993 and 1994) and Hof and Briggs (1997).

It is reasonable to assume that some portion of morphological loss could be attributed to some level of methodological error. However, as long as the measurement of morphological decay (as well as accompanying excavation and treatment of carcasses) remains consistent and is universally applied to all varying experimental conditions, then at least that percentage of methodological error is consistent across the varying conditions. This is why ASW-controls should be carefully excavated in the same manner as any future buried specimens. In conclusion, quantitative measurements of morphological decay should be further researched and refined, and more than one method should be used for comparison.

Other Measurements of Decay

Chemical analysis should accompany the physical and morphological analyses.

Like any experimental design, more quantitative data allows for adequate statistical analyses to determine the major components affecting decay. This is why quantitative measurements of morphological decay and overall decay (like weight loss) should be used for future experiments.

In addition to pH, the following analyses should be considered for the experimental ASW, sediment, and specimen remains: O2, sulfur content, phosphate content, TOC, CHN ratios, microbial growth and activity, and EDS analysis (Briggs and

Kear, 1993 and 1994; Hof and Briggs, 1997; Martin et al., 2004; Wilson and Butterfield,

2014; Naimark et al., 2016). Depending on experimental designs and purposes, chemical analyses will vary. For example, if the same specimen container is going to be sampled

continuously throughout the experiment, instrumental probes are ideal. If the specimens or containers are different each sampling time (if the specimen has to be exhumed), then other instrumentation is required which might require different treatment and storage of samples. Differences in treatments and instrumental procedures further increases the difficulty of keeping methods consistent across different experimental conditions. For example, adding sediment to experiments increases differences immediately, as it introduces more particulate solid matter. Making procedures universal across differing experimental conditions may be as simple as rotary tumbling ASW-control remains so that they undergo to same procedure as buried specimens even though the only purpose of tumbling is to separate sediment from organic tissue. However, if microstructures are being compared between the two conditions, rotary tumbling of just the buried specimens may create microstructural damage that the other condition(s) would not exhibit. The same precautions towards methodological bias should be made for chemical analyses as many instruments are only capable of compositional analysis of certain physical materials; moreover, the ASW, sediment, and respective organic contents should all be chemically analyzed and the sampling, storage, treatment, and possibly the instrumentation itself differ greatly between analysis of liquids versus solids.

Briggs and Kear (1993, 1994) performed analyses of structural protein, chitin, water-soluble protein, carbohydrate, and phospholipids of specimens by homogenizing the remains (powdered via mortar and pestle). When sediment was involved, the remains underwent rinsing and rotary tumbling in an attempt to remove the sediment but, they reported that sediment was never fully removed from the carcasses. This methodology should be considered for chemical analyses of organic remains and sediment in future

experimental specimens because sieving and oven-drying specimens is already an efficient storage method of specimen remains for morphological and SEM analysis.

Future experiments with both extensive morphological and chemical analysis (or if treatment and preparation of samples varies greatly enough between different chemical analyses or instrumentation) may need multiple parallel experiments per sampling/decay time for use in different types of analysis.

MATERIALS AND OTHER CONSIDERATIONS FOR EXPERIMENTAL

PROTOCOL

Raw Materials, Basic Observations, and Record of Information

For the purpose of this paper, raw materials refer to the constituents within the experimental containers and those that physically surrounded the shrimp that have yet to be discussed. This includes the composition and origins of the sediment, details of the individual shrimp and their origins (or other specimens of interest), and the composition and origins of the artificial or collected sea water as well as the methods of inoculation.

The experiments described here used commercial grade sediment, market-bought (dead) shrimp, and aquarium microbes for efficiency and availability. Past studies have used sampled and fresh materials like local ocean water and marine sediment; commercially bought or locally collected experimental specimens and inoculatory specimens (like mussels or marine rocks) have also been used in past decay experiments (Briggs and

Kear, 1993 and 1994; Hof and Briggs, 1997; Martin et al., 2004; Wilson and Butterfield,

2014; Klompmaker et al., 2017; Naimark et al., 2016). It is ideal to follow the latter and use the freshest and most natural materials in order to simulate the most realistic conditions. Most of the prior publications ensured incubation periods in aquariums for the living specimens, sediment, and ASW prior to the start of the experiments.

Some observations that were not made for the present Experimental Sets are recommended for future experiments. The use of commercial-bought shrimp caused

floating, but did not appear to influence results, as specimen-floating did not affect decay scores and overall decay trends matched those of past decay experiments.

However, obtaining and caring for live shrimp until experimental use allows tracking of molting cycles, as the time since molting might affect decayed morphological or ornamentation results. Variables likely to be affected include ‘wrinkled’ cuticle and plastic deformation versus fractured cuticle and brittle deformation. Schweitzer et al.

(2014) suggested differences in molting cycles could explain why fossilized shrimp from the same biota displayed both brittle and plastic cuticle deformation: wrinkled cuticle is a form of deformation rarely observed in lobsters and crabs as they exhibit a more strongly calcified cuticle which tends to exhibit brittle deformation in similar flattened fossils.

However, this does not preclude other factors – varied cuticle deformation could well be yet another example of vastly different results of decay under nearly identical conditions, thereby strengthening the importance of having multiples of experimental conditions per sampling time. In addition to time since molting, the sex of the shrimp should also be noted because the variability of pristinely preserved traits between specimens from the same biota are often speculated to be sexual or sexual size dimorphism. Schweitzer et al.

(2019) suggested that size variation in the second pleonal somites between fossilized specimens distinguished mature females from immature females and male specimens.

No matter where the raw materials came from, record of their origins and any other known details of the materials are crucial and the implications they may have on the results and analyses of the experiments should be considered. In addition to including origin, brand, or locality of raw materials, raw data (chemical and quantitative-

morphological) should be included in publications or a statement of archived data should be included so that it is accessible to future decay experiments of the same taxon.

Experimental Containers

Plastic food storage containers were used for the first burial trial – the results of which are not included in this study. For the official Experiment Sets, the switch to glass was made to ensure the containers could be disinfected and sterilized for the next round of experiments. Chemical contamination from low-grade plastic was also a concern. For the first burial trial, the plastic containers lacked lids and were simply covered with a cut- to-fit piece of plywood firmly pressed on top of the mud to reduce air pockets. The origin and type of the plywood was unknown and small arthropods were found in the sediment upon excavation. After 16 weeks, the shrimp were significantly more decayed than those from Experiment Set 2. The infestation (and possibly the higher levels of decay) was likely due to the un-lidded containers.

Glass containers were preferred for our experiments for the purpose of sterilization and re-use of the beakers for the following set of experiments. However, the experimental container will depend entirely on the purpose of the decay experiment and could even be modified or changed throughout an experiment. For example, all of the aforementioned considerations for compression experiments will greatly influence the experimental vessel, some of which may compromise other desired experimental conditions. This was the very reason Rex and Chaloner (1983) abandoned the use of water and sediment in their experiments in order to focus on the properties of force that create laterally flattened plant fossils; they designed their experimental container around that objective and sacrificed some of the more realistic conditions under which

compression would occur. Decay experiments that are more oriented to chemistry than morphology cannot forgo the use of oxygenated water and probably requires the use of aquariums with filters. Experiments should continue to research which decay containers or apparatuses are best for appropriate purposes, which could be extremely beneficial for experimental taphonomy. This is especially true for morphology-based burial experiments since burial adds numerous complications and compromises in realistic materials may have to be made.

CONCLUSIONS

The Use of Sediment and its Effects

Sediment affected our morphological results in several ways that mirror past experiments as well as the fossil record. Results from this study and other researchers’ published studies suggest that the presence of sediment as a substrate but without actual burial generally increased decay rates of shrimp. This study suggests that complete burial in a thick mud significantly delayed decay, resulting in shrimp that closely resembled compressed or other Lagerstätte fossil shrimp that preserve taxonomically defining characteristics. Previous burial experiments varied from ours slightly more than previous experiments on exposure without burial as some observed no difference in decay levels between buried shrimp and controls in artificial sea water without burial. Those differences in results could have been caused by longer decay times (potentially reaching a plateau in morphological decay in all conditions) or could result in part from different measurements of decay. Composition of sediment also had an effect on decay rates: shrimp in the presence of 100% kaolinite exhibited slightly greater decay than those in the presence of kaolinite with 10% lime. The effect of sediment type was only seen in non-burial conditions (our burial experiments used only kaolinite). However, composition likely affects buried shrimp differently than non-buried shrimp. Wilson and

Butterfield (2014) observed that kaolinite preserved buried shrimp better than either calcite or quartz sediments.

The use of sediment complicates logistical properties of decay experiments. One of the most prominent complications for morphologically-focused experiments is the recovery of remains and determining quantitative effects of all sedimentary properties

(composition, permeability, porosity, etc.). The addition of sediment also increases the number of controls needed for morphological and chemical analyses – this includes but is not limited to the ASW, ASW with specimen, dried sediment only (or sediment with distilled water), sediment with ASW, specimen only, and all of the prior with and without inoculated ASW. The inclusion of these and additional appropriate controls should be used in future experiments particularly because the degree of effect each variable has on morphological and chemical results likely shifts throughout the experiment. The extensive inclusion of controls is emphasized because the present experiments lacked a control beaker with sediment and ASW (without a shrimp) and therefore, we could not determine changing degrees of influence of each factor on pH throughout the experiments.

Nonetheless, despite those complications, sediment makes the experimental conditions more realistic to those in which fossils form with respect to actual fossilization. The results from sediment-decay experiments will improve tapho-anatomic interpretations of fossils just as ASW-only experiments have done. Future decay experiments should incorporate sediment into experimental conditions in order to observe the implications of sediment composition, the nature of sediment-presence (burial versus non-burial), and other taphonomic processes including compression.

Measurements of Morphological and General Decay

Scoring states of morphological decay likely produced more accurate records of decay than the sole use of detailed observation because it required stricter observation of specific decayed traits and produced quantitative data. It also recorded a naturally occurring variation between scores of the multiples from the same experimental conditions – a variation that also occurs in shrimp fossils from the same biota. Moreover, despite the discussed criticisms of the present character states and corresponding scores, our specimens were scored across all of the Experimental Sets despite the vastly different experimental conditions. It is for these reasons that the scoring method, or a similarly quantitative method, should be used for monitoring morphological decay in future experiments. Even for purposes that are not solely morphologically (and phylogenetically) oriented towards given taxa, scoring the decay of compositionally and structurally varied tissues under different conditions will aid in fossilization research and the bio-mineralogical, sedimentary, and bacterial factors involved.

In addition to the scoring method, ‘taphonomic thresholds’ – defined decay stages commonly observed in a fossil taxon that can be identified in experimental specimens

(Allison and Briggs, 1991) – should be used. This method promotes application of experimental results to the fossil record which improves taphonomic and phylogenetic interpretations of given taxa. For example, the absence of a morphological character could be interpreted (more confidently) as more likely never having existed on a fossil that is recognized to have barely decayed prior to preservation. Conversely, the absence of a morphological character on a fossil that exhibits severe decay-related features (like separation of the pleon from the carapace, separation of the anterior cephalothorax from

the posterior cephalothorax, and displacement of the internal skeleton from the carapace in shrimp fossils) increases the likelihood that that character has not been preserved. This method should not be used as the only observation of decay as the number of decay stages and corresponding definitions are limited to those observed in fossils and will not be as detailed and specific as decay stages observable in experimental specimens. For this reason (and the fact that our scoring method produced both similar and different decay rates from past decay experiments), more than one measurement of morphological decay will likely be needed and should be used in order to thoroughly track decay in future experiments. Observation times in future experiments should be as frequent as once a day for the first two weeks because a considerable amount of decay, as well as changes in pH, occurred quite early. After two weeks, sampling may decrease to once a week. Once every four weeks did not provide an ideal amount of data and could have contributed to the lack of hypothesized trends between separation events, cuticle fragmentation, or the decay of soft tissues influencing one another.

In addition to morphology-specific decay measurements, methods for determining a total amount of decay – like the weight loss method used in Briggs and Kear (1993 and

1994) and Hof and Briggs (1997) – should be further researched and applied to future experiments. With an accurate representation of total organic loss of a given specimen, statistical correlations to sexual dimorphism, molting cycles, and between differing experimental conditions could be beneficial to future fossil interpretations as well as fossilization-promoting conditions.

Other Protocol for Future Experiments and Universal Treatment of Specimens

Due to the extremely different conditions between this study’s experiments, universally applicable methods and treatments between future experimental conditions and controls – excavation, sampling, measurements of decay, storage, etc. – is emphasized in order to avoid methodological artefacts. This becomes increasingly problematic once sediment is introduced. Specific methodologies and protocols should be further researched and refined. A frequently encountered method used in past morphological experiments was rotary tumbling remains in an attempt to separate sediment from organic tissue, which was never reported to be fully successful. It is crucial though, that if tumbling (or an alternative method) is utilized, that future experiments apply the same methods to their control specimens (even when sediment is not involved) as tumbling could induce microstructural damage that could be mistaken for decay.

Another notable consideration for the universal treatment of specimens is the observation of different, sequentially excavated specimens for all experimental conditions including ASW-controls. Our buried experiments were the only ones for which a different shrimp was observed at each excavation time and the shrimp were dissected because they did not have to be scored again. Therefore, our buried specimens likely received more accurate scoring than the same, continuously observed specimens for which excess prodding was avoided. In situ excavation of the extremely delicate ASW- control specimens will prove difficult and methods for this should be refined. But, if specimens from all experimental conditions could be physically exhumed in their in situ state, then they could all undergo the same level of observation as specimens set in mud.

In addition to this, the sole use different, sequentially excavated specimens would reduce obscured or hindered morphological observations as they were almost always caused by the combination of poor visibility within ASW or efforts to avoid excess probing or re- orientation of continuous specimens. Precautions against methodological biases are also emphasized for future chemical analyses as the ASW, sediment, and respective organic contents should all be considered for analysis and all will require different instrumentation with specific sampling, treatment, and other protocols.

FIGURES

Figure 1—Design for Experiment Set 2 (compacted burial) showing the placement of the mesh. Shrimp were placed flat on their sides at about 400 ml and had mesh below and above them for efficient excavation. This diagram represents beakers without additional weight; for weighted experiments, petri dishes filled with copper shot rested on top of the sediment and were submerged in ASW.

TOP

MIDDLE

BOTTOM

Figure 2—Design for Experiment Set 3 (SWI) with approximate positions for pH readings. Three beakers were filled with over 700 ml of ASW. Powdered kaolinite was added gradually, allowing it to settle until sediment reached about 700 ml. Each beaker was then topped with about 200 ml of ASW (if needed) and one thawed shrimp was placed in each beaker. pH was measured for Beakers 3A and 3B every week at three different levels. Measurements were taken between 800-900 ml, 400-500 ml, and 0-100 ml. This is Beaker 3A after 5 weeks.

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DECAY TIME (DAYS) CS. 9 CS. 10 CS. 11 CS. 12 2.5 2.5 2.5 2.5

2 2 2 2

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DECAY TIME (DAYS)

Figure 3—Morphological scores for Experiment Set 4 (gradual sedimentation) unaltered shrimp (NH) versus shrimp with pre-punctured carapaces (HO) for Character States 1-8 and 9-15. Scores from unaltered and pre-punctured shrimp for Experiment Set 1 (ASW without burial) included. None of the scores have been averaged – i.e. each score represents a single specimen.

66 CS. 15 2.5

1.5 1A,B,C,D AVE

4A NH-HO AVE 1 4B NH-HO AVE 0.5

0 0 2 4 6 8 10 12 14

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CS. 10 CS. 11 CS. 12 2.5 2.5 2.5 CS. 15 2 2 2 2.5 1.5 1.5 1.5

1 1 1 Experiment Set 1 (ASW) 2 0.5 0.5 0.5

0 0 0 0 2 4 6 8 10 12 14 1.50 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 Experiment Set 4: 5g- sedimentation (NH+HO) CS. 13 CS. 14 CS. 15 2.5 2.5 2.5

2 2 1 2 Experiment Set 4: 20g- sedimentation (NH+HO) 1.5 1.5 1.5

1 01 .5 1

0.5 0.5 0.5

0 0 0 0 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

Figure 4—Morphological scores for Experiment Set 4 (gradual sedimentation) 5 g/month versus 20 g/month sedimentation for all character states. Scores of unaltered shrimp (NH) and shrimp with pre-punctured carapaces (HO) were averaged because the holes had no significant effect on decay. Averaged Experiment Set 1 (ASW without sediment) scores included.

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Character State 15 2.5 Experiment Set 1

2 Experiment Set 2 1.5

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0.5 0.5 0.5

0 0 0 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

Figure 5—Averaged morphological scores for all Experiment Sets and character states. Experiment Set 1 – ASW without sediment; Experiment Set 2 – compacted burial; Experiment Set 3 – SWI; and Experiment Set 4 – gradual sedimentation. Set 4 includes scores from both unaltered and punctured shrimp, as well as 5 and 20 g/month sedimentation shrimp. Negative decay trends of Set 2 scores were caused by the excavation of a different shrimp at each sampling time.

1 2

3 4

5 6

Figure 6—Fresh and decayed cuticle samples of penaeid shrimp. All figures captured all remaining layers of cuticle (separated and/or intact) with the most external layers (epicuticle and exocuticle) oriented upward except for Fig. 6.4 that is focused on a separated piece of internal cuticle. 1, fresh carapace prepared via drying oven, unidentified mass located below horizontal cuticle layers is compositionally the same as cuticle (calcium phosphatic); 2, fresh carapace prepared via critical point drying; 3, Experiment Set 1 (ASW without sediment) pleonal somite, decayed 12 weeks; 4, ASW- only (inoculated) trial-experiment carapace, decayed 8 weeks; 5, Set 1 carapace, decayed 20 weeks; 6, Set 4 (20 g/month sedimentation) carapace, decayed 20 weeks.

Experiment Set 3 (TOP)

H 6 Experiment Set 1 p Experiment Set 4 (5g) Experiment Set 4 (20g) 4 Inoculated ASW

0 0 2 4 6 8 10 12 14 Week

Figure 7—pH for Experiment Sets 1, 3, and 4 (Set 2 lacks pH data). Experiment Set 3 (SWI) included averaged pH from only the TOP 800-900 ml measurements were used as that was the condition most similar to the other Sets – i.e., not in suspended sediment. Set 4 (gradual sedimentation) included the pH from beakers with unaltered shrimp only. Inoculated ASW had no sediment or specimen.

9 8 7 6 5

H TOP AVE p 4 MID AVE 3 BOT AVE 2 1 0 0 2 4 6 8 10 12 14 Week

Figure 8—Stratigraphic changes in pH for Experiment Set 3 (SWI). TOP (800-900mL) measurements were taken in ASW. MIDDLE (400-500mL) and BOTTOM (0-100mL) measurements were taken in suspended kaolinite.

Exp. Set 4 (5g-sedimentation) Exp. Set 4 (20g-sedimentation) 10.5 10.5 10 10 9.5 9.5 9 9

8.5 8.5

H H p p 8 8 7.5 7.5 7 7 6.5 6.5 6 6 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 Day Beakers 4B-NH,HO (20g sedimentation) Day 10.5 Exp. Set 1 (ASW ) 10 10.5 10 9.5 9.5 9

9 8.5 H

8.5 p No Holes (NH)

H 8 p 8 7.5 7.5 Holes (HO) 7 7 6.5 6.5 6 6 0 10 20 30 40 050 6020 70 4080 60 80 Day Day

Figure 9—The effects of pre-punctured carapaces on pH for Experiment Set 4 (gradual sedimentation) versus Set 1 (ASW without sediment). None of the results have been averaged – i.e. each pH represents a single specimen.

Figure 10—Excavation of buried shrimp from trial experiments via breaking open the block of sediment. Specimen was buried for 12 weeks.

Figure 11—Experiment Set 2 (compacted burial) shrimp excavated via mesh. Specimen was buried for 8 weeks.

Figure 12—Sieved remains from Experiment Set 1 (ASW without sediment – left) after 12 weeks and Experiment Set 3 (SWI – right) after 12 weeks. Both are the remains of 2 shrimp.

TABLES

Table 1—Character states and corresponding decay scores used to monitor decay rates for all Experimental Sets. Modified from Klompmaker et al. (2017).

Character State Score 1. Separation of (0) no, (1) yes pleon and cephalothorax 2. Separation of anterior cephalothorax (0) no, (1) partial, (2) yes from posterior cephalothorax 3. State of cephalothorax cuticle (0) no fragmentation, (1) partial fragmentation, (2) incomplete or absent 4. State of anterior cephalothorax (0) complete, (1) partial, (2) hollow, (3) absent 5. State of posterior cephalothorax (0) complete, (1) partial, (2) hollow 6. State of pleon (0) complete, (1) partial, (2) hollow 7. State of internal skeleton (0) no fragmentation, (1) partial fragmentation or detached pereiopods, (2) incomplete or absent 8. State of eyes (0) no fragmentation or disarticulation, (1) partial fragmentation or disarticulation, (2) incomplete or absent 9. Rostrum completeness (0) complete, (1) fragmented, (2) absent 10. State of scaphocerite (0) no fragmentation, (1) partial fragmentation or disarticulation, (2) incomplete or absent 11. State of pereiopods (0) complete, (1) partial fragmentation, (2) absent 12. Separation between 2 or more (0) no, (1) yes pleonal somites 13. State of pleonal somite cuticle (0) no fragmentation, (1) partial fragmentation 14. State of pleopods (0) no fragmentation or disarticulated, (1) partial fragmentation but articulated, (2) disarticulated/absent 15. State of telson (0) no fragmentation or disarticulation, (1) partial fragmentation, (2) disarticulation/absent

Table 2—Explanations for each character state, modifications during the experimental process, and some corresponding decay scores.

Character Description and Explanations State 1 Complete separation of pleon from cephalothorax; no score for partial separation was needed. 2 Score for partial separation was added when attachment via minimal amounts of tissue was observed. 3 Distinguished from Character State 5 in order to describe the cuticle of the cephalothorax. Score for partial fragmentation was added due to brittle fractures in cuticle during Exp. Set 2 (buried) and singular tears in the cuticle during Exp. Sets 1 (ASW), 3 (SWI), and 4 (sedimentation). 4 Scores 0, 1, and 2 refer to the contents of the anterior cephalothorax. Score 3 was added when total decay of the anterior cephalothorax was observed and because there is no Character State that distinguishes the state of the cuticle of the head from the contents of the anterior cephalothorax. 5 Distinguished from Character State 3 in order to describe the contents of the posterior cephalothorax. 6 Distinguished from Character State 14 in order to describe the contents of the pleon. 7 Scores 1 and 2 are strongly associated with a slight to total separation of the internal skeleton from the carapace. The disarticulation/ detachment of the pereiopods from the internal skeleton is included with this Character State because Character State 12 was mostly scored via the identification and state of the claws. 8 Fragmentation and disarticulation were combined into a single score (1) because neither state had an isolated occurrence. 9 Score 1 is associated with Exp. Set 2 (buried) (specimens were observed closely enough to identify missing dorsal and rostral spines). Score 2 represents the rostrum separating from the cephalothorax and its total absence for all Experiment Sets. 10 Same as Character State 8. 11 These scores relied heavily on the identification of the claws. However, the Character State was not changed to identify only claws due to possible excavation flaws of Exp. Set 2 (buried). A general statement on the state of pereiopods was more universal between experimental conditions and results. 12 Character State was added when pleonal somites started to separate during Experiment Sets 1 (ASW), 3 (SWI), and 4 (sedimentation). 13 Distinguished from Character State 6 in order to describe the cuticle of the pleonal somites. Score for partial fragmentation was added due to brittle-like cracks in cuticle during Exp. Set 2 (buried) and singular tears in the cuticle during Exp. Sets 1 (ASW), 3 (SWI), and 4 (sedimentation). 14 Disarticulation and absence were combined into a single score (2) because in Exp. Sets 3 (SWI) and 4 (sedimentation), neither state had an isolated occurrence. 15 Score 1 was added for Exp. Set 2 (buried) when the tissue was observed closely enough to notice minor fragmentation. Disarticulation and absence were combined into score 2 because whenever the telson was observed to have detached, it decayed completely (i.e. was never located after disarticulation from the pleon for Exp. Sets 1, 3, and 4).

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