DOI: 10.5061/dryad.k98sf7mcq
title: Data from: 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

description: 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. 
description: <em>Taxon
 selection </em><em> </em> Branchiopod
 crustaceans <em>Triops </em><em>longicaudatus</em>
 (Order Notostraca) were grown according to the instructions from the
 commercially available Toyops’ <em>Triops</em> 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.  
 <em>Sediment collection and
 preparation </em><em> </em> 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.   
 <em>Vial
 preparation</em><em> </em> 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.  
 <em>Triops micro-CT
 scanning </em><em> </em> To create a
 morphological comparison scan, one <em>Triops</em><em>
 </em>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).
 <em>Data analysis and
 visualization</em><em> </em> 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.  
description: ### \*\*Note to users:\*\* This README file includes combined instructions
 for two complementary Dryad datasets: 1. **The Dryad Archive of**
 **Dragonfly Sessions** works best when opened with the eponymous software
 ([https://dragonfly.comet.tech/;
 ](https://dragonfly.comet.tech/?uh=6b5ae8a230a2d222ba13a0f50f3f8cd0a88dea543e4694f4b763e4b6b372d5fc "https://dragonfly.comet.tech/")see details below), and includes all the information regarding the parameter used for visualizing the data used on the results and discussion of the published manuscript. **The Dryad Archive of Dragonfly Sessions can be accessed at DOI: 10.5061/dryad.k98sf7mcq** 2. **The Dryad Archive of TIFF Stacks** contains the raw data (after minimal processing / reconstruction from the micro-CT scanner) consisting of grayscale TIFF files that correspond to the tomographic sections of all the datasets analyzed. This option allows users to examine the data without the need to use Dragonfly, and can be accessed with other freeware options including SPIERS ([https://spiers-software.org/](https://urldefense.proofpoint.com/v2/url?u=https-3A__spiers-2Dsoftware.org_&d=DwMCAw&c=WO-RGvefibhHBZq3fL85hQ&r=RoBtBqLfBvA75aSCZXNUmOA0Zr30pao7udBKnzN6xJUFmq4F6NtAwHAvzg0J0Inr&m=oIs6PvI83yxVCgUqWr2ezfCDB0v30emCuEn7MMOituL8g9WrpLGfIPsRdoBF3qkM&s=1RQ0V1p2Yv5Ftf9DPkJLXaEdCpH3NOnhWi6T2mi2j1c&e= "https://urldefense.proofpoint.com/v2/url?u=https-3A__spiers-2Dsoftware.org_&d=DwMCAw&c=WO-RGvefibhHBZq3fL85hQ&r=RoBtBqLfBvA75aSCZXNUmOA0Zr30pao7udBKnzN6xJUFmq4F6NtAwHAvzg0J0Inr&m=oIs6PvI83yxVCgUqWr2ezfCDB0v30emCuEn7MMOituL8g9WrpLGfIPsRdoBF3qkM&s=1RQ0V1p2Yv5Ftf9DPkJLXaEdCpH3NOnhWi6T2mi2j1c&e=")), and Drishti ([https://github.com/nci/drishti](https://urldefense.proofpoint.com/v2/url?u=https-3A__github.com_nci_drishti&d=DwMCAw&c=WO-RGvefibhHBZq3fL85hQ&r=RoBtBqLfBvA75aSCZXNUmOA0Zr30pao7udBKnzN6xJUFmq4F6NtAwHAvzg0J0Inr&m=oIs6PvI83yxVCgUqWr2ezfCDB0v30emCuEn7MMOituL8g9WrpLGfIPsRdoBF3qkM&s=Fu-jwrajAOA3OGIX__PCs7UID_tL4wWrTgsNe247KcE&e= "https://urldefense.proofpoint.com/v2/url?u=https-3A__github.com_nci_drishti&d=DwMCAw&c=WO-RGvefibhHBZq3fL85hQ&r=RoBtBqLfBvA75aSCZXNUmOA0Zr30pao7udBKnzN6xJUFmq4F6NtAwHAvzg0J0Inr&m=oIs6PvI83yxVCgUqWr2ezfCDB0v30emCuEn7MMOituL8g9WrpLGfIPsRdoBF3qkM&s=Fu-jwrajAOA3OGIX__PCs7UID_tL4wWrTgsNe247KcE&e=")). **The Dryad Archive of Tiff Stacks can be accessed at DOI: 10.5061/dryad.nzs7h4525.** ## **Dryad Archive of Dragonfly Sessions** The week 2 scans were completed at the Digital Imaging Facility (DIF) at the Museum of Comparative Zoology, Harvard University, with a Bruker SkyScan 1173 micro-CT scanner. Scans were performed as 180° scans with Al 1 mm filter, 100 kV, 100 µA current, 32 µm voxel size, 12mm stage positioning with 0.4 angle step size and frame averaging of 3. Due to an update on the DIF facilities taking place during the duration of the experiment, week 20, week 42, and week 64 scans were produced on a new Bruker Skycan 1273 using 180° scans with Cu .5 mm filter, 120 kV, 125 µA current, 25.3 µm voxel size, 0.4 angle step size and frame averaging of 3. All scans were reconstructed as TIFF stacks in NRecon (Bruker Corporation) and visualized using the software Dragonfly 2019 4.0 (Object Research Systems, Montreal, Canada). The Dragonfly Session files are archived here. ## File types: The compressed ZIP files were compressed using Windows and will be unzipped to .ORSSession files. .ORSSession files maybe be opened in Dragonfly using a free noncommercial license. Dragonfly 2024.1 [Computer software]. Comet Technologies Canada Inc., Montreal, Canada; software available at [https://dragonfly.comet.tech/](https://dragonfly.comet.tech/?uh=6b5ae8a230a2d222ba13a0f50f3f8cd0a88dea543e4694f4b763e4b6b372d5fc "https://dragonfly.comet.tech/") ## Uploaded files and descriptions Triops_refscan_Fig2D.ORSSession This file corresponds to the stained *Triops longicaudatus* anatomical scan in Figure 2D. The Dragonfly Session may be opened in the Dragonfly program. week2_viala.ORSSession This zipped file corresponds to the scan of vial a after 2 weeks of decay, in Figure 3A. The Dragonfly Session may be opened in the Dragonfly program. week2_vialb.ORSSession This zipped file corresponds to the scan of vial b after 2 weeks of decay, in Figure 3B. The Dragonfly Session may be opened in the Dragonfly program. week2_vialc.ORSSession This zipped file corresponds to the scan of vial c after 2 weeks of decay, in Figure 3C. The Dragonfly Session may be opened in the Dragonfly program. week2_viald.ORSSession This zipped file corresponds to the scan of vial d after 2 weeks of decay, in Figure 3D. The Dragonfly Session may be opened in the Dragonfly program. week2_viale.ORSSession This zipped file corresponds to the scan of vial e after 2 weeks of decay, in Figure 3E &F. The Dragonfly Session may be opened in the Dragonfly program. week20_viala.ORSSession This zipped file corresponds to the scan of vial a after 20 weeks of decay, in Figure 4A. The Dragonfly Session may be opened in the Dragonfly program. week20_vialb.ORSSession This zipped file corresponds to the scan of vial b after 20 weeks of decay, in Figure 4B. The Dragonfly Session may be opened in the Dragonfly program. week20_vialc.ORSSession This zipped file corresponds to the scan of vial c after 20 weeks of decay, in Figure 4C. The Dragonfly Session may be opened in the Dragonfly program. week20_viald.ORSSession This zipped file corresponds to the scan of vial d after 20 weeks of decay, in Figure 4D. The Dragonfly Session may be opened in the Dragonfly program. week20_viale.ORSSession This zipped file corresponds to the scan of vial e after 20 weeks of decay, in Figure 4E &F. The Dragonfly Session may be opened in the Dragonfly program. week42_viala.ORSSession This zipped file corresponds to the scan of vial a after 42 weeks of decay, in Figure 5A. The Dragonfly Session may be opened in the Dragonfly program. week42_vialb.ORSSession This zipped file corresponds to the scan of vial b after 42 weeks of decay, in Figure 5B. The Dragonfly Session may be opened in the Dragonfly program. week42_vialc.ORSSession This zipped file corresponds to the scan of vial c after 42 weeks of decay, in Figure 5C. The Dragonfly Session may be opened in the Dragonfly program. week42_viald.ORSSession This zipped file corresponds to the scan of vial d after 42 weeks of decay, in Figure 5D. The Dragonfly Session may be opened in the Dragonfly program. week42_viale.ORSSession This zipped file corresponds to the scan of vial e after 42 weeks of decay, in Figure 5E &F. The Dragonfly Session may be opened in the Dragonfly program. week64_viala.ORSSession This zipped file corresponds to the scan of vial a after 64 weeks of decay, in Figure 6A. The Dragonfly Session may be opened in the Dragonfly program. week64_vialb.ORSSession This zipped file corresponds to the scan of vial b after 64 weeks of decay, in Figure 6B. The Dragonfly Session may be opened in the Dragonfly program. week64_vialc.ORSSession This zipped file corresponds to the scan of vial c after 64 weeks of decay, in Figure 6C & D. The Dragonfly Session may be opened in the Dragonfly program. week64_viale.ORSSession This zipped file corresponds to the scan of vial e after 64 weeks of decay, in Figure 6E &F. The Dragonfly Session may be opened in the Dragonfly program.   ## **Dryad Archive of Tiff Stacks** The week 2 scans were completed at the Digital Imaging Facility (DIF) at the Museum of Comparative Zoology, Harvard University, with a Bruker SkyScan 1173 micro-CT scanner. Scans were performed as 180° scans with Al 1 mm filter, 100 kV, 100 µA current, 32 µm voxel size, 12mm stage positioning with 0.4 angle step size and frame averaging of 3. Due to an update on the DIF facilities taking place during the duration of the experiment, week 20, week 42, and week 64 scans were produced on a new Bruker Skycan 1273 using 180° scans with Cu .5 mm filter, 120 kV, 125 µA current, 25.3 µm voxel size, 0.4 angle step size and frame averaging of 3. All scans were reconstructed as TIFF stacks in NRecon (Bruker Corporation). These reconstructed TIFF stacks are archived here. ## File types: The compressed ZIP files were compressed using Windows and will be unzipped to folders of .tif image files. Folders of reconstructed .tif stacks may be visualized in many programs. Authors used Dragonfly 2019 4.0 (Object Research Systems, Montreal, Canada), please see the associated Dryad repository for this archive. Other visualization tools can import the images here including the free software Drishti, cited below. Ajay Limaye; Drishti: a volume exploration and presentation tool. Proc. SPIE 8506, Developments in X-Ray Tomography VIII, 85060X (October 17, 2012). [https://github.com/nci/drishti/wiki](https://github.com/nci/drishti/wiki) Triops_recon *[NOTE that for size constraints, this file is uploaded to this DRAGONFLY dataset, not the TIFF dataset]* This file corresponds to the stained *Triops longicaudatus *anatomical scan in Figure 2D. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_2_viala_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial a after 2 weeks of decay, in Figure 3A. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_2_vialb_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial b after 2 weeks of decay, in Figure 3B. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_2_vialc_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial c after 2 weeks of decay, in Figure 3C. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_2_viald_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial d after 2 weeks of decay, in Figure 3D. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_2_viale_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial e after 2 weeks of decay, in Figure 3E & F. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_20_viala_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial a after 20 weeks of decay, in Figure 4A. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_20_vialb_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial b after 20 weeks of decay, in Figure 4B. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_20_vialc_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial c after 20 weeks of decay, in Figure 4C. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_20_viald_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial d after 20 weeks of decay, in Figure 4D. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_20_viale_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial e after 2 weeks of decay, in Figure 4E & F. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_42_viala_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial a after 42 weeks of decay, in Figure 5A. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_42_vialb_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial b after 42 weeks of decay, in Figure 5B. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_42_vialc_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial c after 42 weeks of decay, in Figure 5C. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_42_viald_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial d after 42 weeks of decay, in Figure 5D. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_42_viale_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial e after 42 weeks of decay, in Figure 5E & F. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_64_viala_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial a after 64 weeks of decay, in Figure 6A. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_64_vialb_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial b after 64 weeks of decay, in Figure 6B. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_64_vialc_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial c after 64 weeks of decay, in Figure 6C & D. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software. week_64_viale_recon This zipped folder of reconstructed .tif images corresponds to the scan of vial e after 64 weeks of decay, in Figure 6E & F. The .tif files may be opened in Dragonfly, Drishti, or other 3D image processing software.