Stuck in the mud: experimental taphonomy and computed tomography demonstrate the critical role of sediment in stabilizing the three-dimensional external morphology of arthropod carcasses during early fossil diagenesis - DRAGONFLY sessions

Exceptionally preserved fossils provide critical information on the morphology and ecology of extinct organisms, but their formation requires complex pathways that affect the process of decay in any given depositional environment. The field of experimental taphonomy has produced critical insights that allow us to better understand the physical and chemical mechanisms responsible for fossil preservation. However, taphonomic experiments designed to assess the rate of morphological information loss typically require that they are performed in artificial sea water without sediment to clearly quantify any observable changes over time. Here, we utilize micro-computed tomography to non-invasively investigate changes in carcasses of the branchiopod crustacean Triops longicaudatus for over a year of post-burial decay. After 64 weeks, specimens are still detectable as three-dimensional low-density volumes that capture various external morphological features in life position, including the body outline, carapace, and limbs. Our results show that sediment plays a critical role in carcass stabilization in arthropods, maintaining the external integrity of the body over much longer periods of time than previously demonstrated experimentally. We hypothesize that the low-density volumes produced during decay most likely produce sites for subsequent mineral precipitation needed for exceptional three-dimensional fossilization of non-biomineralized arthropod carcasses and structures as observed in the fossil record. 

Methods
Taxon selection  

Branchiopod crustaceans Triops longicaudatus (Order Notostraca) were grown according to the instructions from the commercially available Toyops’ Triops kit until they reached maturity and desired size of 3-5cm at three weeks after hatching. All specimens were euthanized by refrigeration, being frozen for 3 hours, and then thawed in a refrigerator overnight.  

Sediment collection and preparation  

Fresh estuarine sediment was collected from Belle Isle Marsh Reservation in Boston, Massachusetts. At low tide, one 5-gallon bucket of sediment was collected from each of the two 0.5-0.75 meters deep pits, one located one meter toward the stream from the peak of high tide (42.392208, -70.991179) and another 5 meters closer to the center of the tidal stream (42.392157, -70.991088). The sediment was collected from dark gray fine grain mud with a strong sulfidic smell, indicating the presence of sulfate-reducing bacteria in an anoxic environment. Large plant fragments and live organisms were manually discarded to remove excess organic matter. Sediment was mixed with artificial seawater (Instant Ocean®, Aquarium Systems), passed through a 2 mm sieve to remove remaining pieces of plant matter, shells, and organisms. Sieved sediment was placed into sealed containers with 100 mL of additional artificial seawater to create a thick slurry. A small sample of the sieved sediment was analyzed for bulk organic carbon content by weight. The organic carbon content of the sediment was 3.4%, measured by decarbonation with HCl and comparing dried sample mass before and after combustion of the organic matter.  

A loosely lithified smectite (clay mineral) was provided by Robert Gaines (Pomona College, California), collected in 2008 from Fish Creek Wash in Anza Borrego Desert State Park, Imperial County, California. X-ray diffraction analysis of oriented clay separates indicates that the clay is comprised primarily of illite with subordinate smectite and a negligible organic carbon content (Robert Gaines, personal communication). 

The sediment enriched in sulfate-reducing bacteria and the clay minerals were mixed at a ratio of 1:3 by weight to produce a fine grain sediment with less 1% organic carbon by weight but with the natural bacteria population from the marsh sediment. The resulting mixture was 0.65% organic carbon by weight. Organic carbon was measured using a basic geochemistry/carbon isotope technique, taking a small sample of sediment, decarbonating it with HCl, drying the sample, identifying the ratio of carbon to nitrogen in the sediment and directly measuring the amount of nitrogen in the sample and calculating the carbon percentage based on the ratio.   

Vial preparation 

Anoxic seawater was produced by boiling spring water in a flask with a septum and two needles while inputting nitrogen gas in one septum needle which was then used to make artificial seawater and set aside before adding the 0.5mM iron(ii) chloride solution in a dysoxic glove bag. The experimental vials were also prepared for the experiment inside a glove bag filled with dinitrogen gas to create a dysoxic, low oxygen, environment.  

Triops micro-CT scanning  

To create a morphological comparison scan, one Triops specimen was euthanized by submersion in 95% ethanol, stained in a solution of 1% iodine metal and 100% ethanol, left overnight, and rinsed with 100% ethanol. The iodine-stained specimen was wrapped in synthetic cotton batting in a plastic vial with 100% ethanol in Bruker SkyScan 1173 micro-CT scanner at the Digital Imaging Facilities (DIF) in the Museum of Comparative Zoology (MCZ) with voltage of 100kv, wattage of 80uA, 1mm Al filter, exposure of 750 ms, 0.3 angle degree step, and voxel size of 10.088 µm. The scans were reconstructed as TIFF stacks in NRecon (Bruker Corporation) and visualized using the software Dragonfly 2019 4.0 (Object Research Systems, Montreal, Canada).

Data analysis and visualization 

Scans were reconstructed as TIFF stacks in NRecon (Bruker Corporation). Reconstructions used a variety of post-scan settings, unique to each scan comparing with fine tuning within the program to maximize reconstruction quality, generally with ring reduction of 1, smoothing of 1, and misalignment compensation between -7 and 7.