The Knightia eocoena specimen was taken from Kutztown University (KU) fossil collection from Wards Scientific purchase ~ 1967 and was wrapped in the original packing. No additional preparation was carried out and no surficial preservative coatings were added (Fig. 2). The specimens were cut with a water saw. A supplemental specimen was sent to National Petrographics Inc. of Rosenberg TX where a thin section was cut parallel to bedding as opposed to the typical perpendicular to bedding orientation. The parallel section orientation was employed to mimic the surface that was examined under the FESEM analysis.
A second specimen was cut into manageable size pieces for the FESEM chamber size. The smaller pieces of the skeleton were cleaned of debris in an ultrasonic cleaner for 1 hour, dried, and placed on large SEM stubs with electrically-conductive carbon adhesives. Specimens were coated with 10 nm of 80% Au and 20% Pd to reduce charging and amplify the secondary electron signal. Samples were simultaneously imaged using a Everhart-Thornley secondary electron (SE) detector for surface textural detail and a backscatter electron (BSE) detector to identify compositional contrasts, with the signals from both detectors overlain to permit the recognition of the surface topography coupled with limited compositional contrast in the image. The backscatter (BSE) detector was used in conjunction with the Oxford EDS detector and processed with AZtec Software to determine elemental composition and generate spectra.
Geological setting
The early to middle Eocene Green River Formation Lake system (Bradley, 1963) developed in small, elongate intermontane basins linked to the eastern margin of the Sevier-Laramide fold and thrust belt (Fig. 1; Smith et al. 2008). Fossil Lake, the western extent of Lake Gosiute (Fig. 1) is the deepest with a maximum extent of 60 km by 30 km (Grande, 1984; Buchheim, 1994). The multiplex lake fill consists of three stages: over-filled (Road Hollow Member), balance-filled (Fossil Butte Member), and under-filled (Angelo Member) (Buchheim et al. 2011). The Fossil Butte Member contains numerous mass mortality horizons quarried commercially today, of which the KU Knightia eocaena specimen (Fig. 2) was most likely extracted. Typically, commercial fish fossils occur in a laminated carbonate facies (Buchheim and Eugster 1998). This is congruent with the lithology of the KU Knightia eocaena specimens in cross section and thin section. 95% of fish skeletons in the Green River deposits are fully articulated (Hellawell and Orr 2012).
Description
The Knightia eocaena specimen is approximately 9 cm in length and a dorsal fin to ventral length of 3 cm. The encasing carbonate matrix is yellowish gray (5 Y 8/1) whereas the fossil varies from light brownish gray (5 YR 6/1) to brownish gray (5 YR 4/1) (Fig. 2). Brownish gray is concentrated in the caudal fin, pelvic fin, ventral side, opercular area, and posterior to the orbitals.
The encasing rock is a lime mudstone, consisting of predominately micrite with grains long-axis lengths of 30 µm or less and are irregular-shaped (Fig. 3). FESEM analysis revealed the presence of two populations of carbonate grains in the micrite, ~ 20 µm and ~ 10 µm. EDS analysis of these two populations demonstrate the larger, more common elongate form population is composed of CaCO3, calcite, whereas the sparser euhedral crystal population of crystals is composed of dolomite CaMg(CO3)2 (Fig. 3).
Thin sections show the presence of organic substances and framboids associated with the Knightia eocaena fossil (Fig. 4). Thin section cuts through dorsal centrums display a sparry calcite cement infilling pores and apparent original bone fiber texture (Fig. 4A, B). Not all pores are infilled (Fig. 4B).
FESEM examination of the specimen revealed fractures, original bone, scales, and a multitude of microstructures including: 1) framboids, 2) filaments and 3) irregular spheres. Spectacular high concentrations of variable size framboids are present (Fig. 5). These features do not extend into the surrounding rock matrix without direct association with fossil materials. (Fig. 5C).
Closer examination of the bone surface reveals bone corrosion in the form of euhedral rhombohedral molds and crystal faces, most likely dolomite, scarring the outer rim of the bone (Figs. 6A, B). In addition, framboidal spheres composed of equidimensional microcrystal aggregate that together form the framboids are embedded in the bone and were plucked, probably during sample preparation revealing deep hemispherical pits in the bone surface with additional framboidal spheres deeply embedded inside the bone (Figs. 6C-F).
Framboids are “microscopic spheroidal to sub-spheroidal clusters of equidimensional and equimorphic microcrystals” (Ohfuji and Rickard, 2005) typically constructed of pyrite. and Diameters commonly range from 1 to 250 µm, with means between 10 and 20 µm (Wilken and Barnes, 1997). Framboids observed in Knightia eocaena thin section and under the FESEM are less than ~ 30 µm maximum in diameter (Figs. 5, 6, 7, and 8). Sheets of variable-sized framboids drape bone material (Fig. 5) and occur across ends of bone (Fig. 5B).
Framboids occur in a variety of preservation states (Figs. 6 and 7) and are ubiquitous throughout the sample. Equimorphic microcrystals characterize the outer surface (Figs. 6F, 7A and 7B), whereas others have a rind that partially obscures and smooths out the roughness of the underlying microcrystal structure (Figs. 7A, E, F, G and H). The rinds are ~ 200 nm or less in thickness (Figs. 7E, F, G, and H). The smooth rind, when breeched or when framboids are fractured when adhering to the bone, exposes external molds of pores ~ 500 nm in diameter (Figs. 7C-J). The shape of external molds varies from four-sided to near circular shaped. These molds form a sieve-like texture (Figs. 7C-J). The mold walls are composed of ~ 100 nm size spheres (Fig. 7J). Framboid surfaces commonly have ~ 1 µm crystal forms protruding above the spherical surface (Figs. 7I, 7K and 8). Rare framboids are hollow-shaped (Fig. 7K and L). On numerous surfaces of some framboids are 1 µm diameter pustular structures, some of which appear to have crystal faces, and others appear more spheroidal (Fig. 8A). On the exposed surfaces, ~ 2 µm irregular polygonal-shaped features with flat bases are defined by less than 100 nm-scale crystals forming ridges on the underlying feature (Fig. 8).
Numerous EDS analyses of the framboids were conducted. The EDS spectra of framboids reveal the presence of Fe and the absence of S (Fig. 9 spectra 1 and 3). The spectra indicate that the framboids are composed of siderite (FeCO3) with excess C. In addition, the host fish bone, note P and F peaks characteristic of bone, with embedded with a siderite framboids is not altered but may be coated with fine grained CaCO3 (Fig. 9 spectrum 2).
Spheres are ~ 0.5 to ~ 2 µm in diameter (Fig. 10). The spheres form individual clusters (Figs. 10A-C) or sheets (Figs. 10D and E). Spheres are hollow inside composed of walls of 100–200 nm equant subhedral to anhedral crystals and of mostly of uniform size (Fig. 10A, C, E and F). Some walls are crudely circular, whereas others are more complex ovals in cross section (Fig. 10B). Some surfaces are highly irregular, featuring overlapping, almost “vesicular”-like spherules covering the entire plane. (Fig. 10F). EDS analysis demonstrate the spheres contain CaCO3 and SiO2.
FESEM examination revealed the presence of numerous filament-like features comprised of 100–200 nm scale nanocrystals. These filaments range from single strands to complexly intertwined matrices (Fig. 11). Individual strands vary in form from straight, to curves, to sinuous calcite rods. Most filaments are are mostly in uniform size but display a bimodal distribution (Figs. 11A and D). Smaller filaments are more common and have widths that vary from ~ 300–400 nm to ~ 3–4 µm, and lengths of 2 to 4 µm (Fig. 11B). Smaller filaments coat surfaces and infill some cavities or fractures (Figs. 11B and F). Larger filaments have widths of 500 nm to ~ 5 µm (Figs. 11A and D). Longer filaments are 20–30 µm (Fig. 11G). The largest and rarest filaments are 3 µm in diameter with lengths of ~ 55 µm and associated with variable size framboids (Fig. 11I).
Nanocrystals compose all filaments and give the exterior a granular appearance (Fig. 12). Discrete individual 400–500 nanocrystals give the filaments a nodular looking surface fabric (Figs. 12B and C). Along the filament, the larger nodular structure gives way to the more typical small crystals 100–200 nm diameter down the filament (Figs. 12B).
Sheets of external molds of filaments are present and associated with framboids (Fig. 13). This type of preservation coats a limited number of flat surfaces. Filaments have widths of 0.5 µm and lengths of 6 to 8 µm. Framboids vary in size from maximum of 3 µm and appear on the surface of the sheets (Fig. 13B) as single or multiple-merged cluster forms (Figs. 13A and B). Filaments are preserved external molds with internal diameters of 400–500 nm with lengths of 4–5 µm. Within interstitial areas, the external molds are characterized by nm-scale spheres (Fig. 13C).
Three additional types of rare structures are present: capsule form (Fig. 12B blue arrow), multiple-branching form (Fig. 14) and robust filaments (Fig. 15).
The capsule form is ~ 2 µm long and 0.5 µm wide giving it a 4:1 length to width ratio (Fig. 12B). The multiple-branching filaments are ~ 40 µm in length and is composed of a thick central filament with a maximum width of 2 µm and constructed of multiple twisted filaments (Fig. 14B). These twisted filaments are 100–200 nm in width (Fig. 14D). The main structure branches off into successively smaller filaments (~ 600 nm to ~ 100–200 width) and attaches to the surrounding grains (Fig. 14B).Filaments branch, then merge together (Fig. 14A). A spheroidal structure is present and attached to the filaments (Fig. 14D). Rhombohedral grains are attached to the main filament cluster (Fig. 14B)
Interpretation
The preserved features are best interpreted as the preservation of microbial organisms, mainly bacteriomorphs, and their associated metabolic byproducts (Southam and Donald 1999; Westall 1999; Westall et al. 2001; Schopf 2012; Kremer and Kaźmierczak 2017). The microorganism’s cell walls and extracellular polysaccharides substances (EPS) act as nucleation sites for variety of mineral precipitates aiding in early preservation (Westall et al 1995; Newman et al 2017). Intercellular carbonate precipitation can also take place, within cyanobacteria (Benzerara et al., 2014). Generally, this type of mineral precipitation, biomineralization, commonly produces external molds of the microorganisms and does not necessarily preserve the cell walls (Westall et al 1995).
Clusters of uniform diameter sphere-shaped structures with wrinkled exteriors in the Green River Formation fish are best interpreted as external molds of coccoid-type bacteria (Alterman 2001; Westfall et al., 2001; Toporski et al 2002; Kazmierczak et al 2009; Cosmidis et al 2013; Kremer and Kaźmierczak 2017). They are nearly 10 times smaller than spheres attributed to cyanobacteria, but of overall similar gross morphology (Kasmierczak et al. 2009; Chacón et al., 2018; Guo et al. 2018). Cell walls and sheaths are preferentially preserved over the interiors of the cells (Guo et al., 2018). Based on the clustered morphological arrangement, coupled with sphere diameter, these forms are best assigned the generic descriptive form staphylococci. Staphylococci are important as they are components of many types of bacterial biofilms (Otto 2008).
The bimodal filamentous forms are interpreted as preserved filamentous bacteria (smaller forms) and cyanobacteria (larger forms) (Samanta et al 2011). These filamentous bacteria and cyanobacteria molds are composed of nanocrystals of calcite that were most likely precipitated in the encapsulating extracellular polysaccharides substances (EPS) (Wright and Altermann, 2000).
Sausage-shaped structures with a ~ 3 to 1 ratio and smooth exterior are also present in the Precambrian and interpreted as bacilliform bacteria (Westall et al., 2001). This morphology is similar to the Fe (II)-oxidization bacteria used in experimental studies (see Posth et al., 2014).
The multiple-branching filaments are composed of a thick central strand consisting of numerous twisted filaments, progressively smaller offshoots and merging of filaments are characteristic of fungal hyphae (Riquelme et al., 1998; Cavagnaro et al 2001; Grim et al., 2005; Vídal-Diez de Ulzurrun et al., 2017; Lagree et al., 2018; Gutiérrez-Medina and Vázquez-Villa 2021). A spheroidal structure attached to the filaments may represent the development of a spore (Riquelme et al., 1998; Cavagnaro et al 2001; Grim et al., 2005). Filamentous fungus are common components in freshwater lacustrine ecosystems (Wurzbacher et al., 2010; Lepère et al., 2019) and are important components in microbial mats (Gerdes et al., 1993; Krings and Harper, 2019; Carreira et al. 2020), so a parasitic or communalistic fungus on the deceased fish cannot be ruled out (Gomez and Primm, 2021; Harper et al., 2021).
The close association of bacteriomorphs with the iron-rich framboids argues for a biological origin, a hypothesis that has been around for over a century (e.g. Schneiderhöhn, 1923; Love, 1957; Love and Arnstutz, 1966; Folk, 2005). Framboids are commonly composed of iron sulfides (pyrite (FeS2)), and less common as iron oxyhydroxides, or magnetic iron minerals. They are found in a range of lithologies that developed in diverse geochemical conditions (Ohfuji and Rickard, 2005). Experimentally, well-developed framboids have been formed at elevated temperatures above those commonplace in sedimentary environments (e.g. Berner, 1969; Sweeney and Kaplan, 1973; Graham and Ohmoto, 1994). Framboids are associated commonly with redox interfaces and the presence of organic carbon-rich surfaces in sediments in diverse sedimentary environments (Kohn et al., 1998; Sawlowicz 2000; Grimes et al., 2001, 2002; Large et al., 2001; Propa et al., 2004; Rickard, 2012; Pesquereo et al. 2015). Polysaccharide-dominated biofilm surfaces provide ideal nucleation sites for iron sulfides, in particular Fe2+ ions, and may also play a role in stabilizing the framboids in sedimentary environments (Large et al., 2001; Chatellier and Fortin, 2004; Chan et al., 2009; Hao et al., 2016).
Non-pyritic Fe-rich framboids are commonly interpreted to have formed by replacement of primary pyrite framboids originally produced by bacterial sulfate reduction and the anaerobic oxidation of methane. (Cavalazzi et al. 2012). Pyrite forms rapidly by microbial processes in the matter of hours to weeks following burial (Liu, 2015). Longer timeframes from hours to five years have been proposed for pyrite framboid formation, with larger forms requiring longer growth periods (Wilkin et al., 1996; Ohfuji and Rickard, 2005; Schieber and Schimmelmann, 2007; Richard, 2019).
Large et al. (2001) recognized two types of framboids: 1) protoframboids with a smooth or cauliflower surface texture, and 2) well-developed framboids. Either type of framboid may be composed of greigite to pyrite. In addition, well-formed framboids developed flattened contacts during compaction indicating that internal crystals in the framboid were loosely bound with the enclosing biofilm maintaining structural integrity (Large et al. 2001).
Biofilms are an organic template for the growth and aggregation of pyrite microcrystals with anhedral crystal faces (MacLean et al., 2008). Biofilm coatings are documented from the outer surface of complete pyrite framboids and the surfaces of individual microcrystals within framboids (Large et al., 2001; MacLean et al., 2008). Additionally, partial proto-framboids were discovered embedded within biofilms (MacLean et al., 2008).
Fe-oxide and carbonaceous microspheres have been identified in Miocene bone with similar sieve-like structure as observed in the Green River framboids (Pesquereo et al. 2015). Additionally, a recognizable colloidal exterior that was an oxidized product and sieve-like internal texture portion is consistent with an iron sulfate (melanterite) oxidation product in this case (Soliman and El Goresy, 2012). Maclean et al., (2008) described a bacterial biofilm from a drill core with a sieve-like matrix between the individual pyrite crystals within the framboids similar to textures in the samples in this study. The matrix scaffolding was enriched in carbon relative to pyrite microcrystals suggesting “bacterial capsules”. The sieve-like structure developed as segregated compartments, before infilling by pyrite (Maclean et al 2008). In contrast, in other studies the voids in sieve-like structure of framboids are interpreted as the product of oxidation of iron-sulfide into iron-oxides (Sawlawicz and Kaye, 2006).
Iron-oxide spheres, formerly pyrite, with similar morphology have been described from pores in trabecular dinosaur bone and attributed to endocasts of bacterial biofilms composed of fibers and spheres that mimicked blood vessels (Kaye et al., 2008). Preserved capsule-shaped cell structure has been reported as precipitated iron-sulfide of probable bacterial origin, occurring as 1 x 3 mm oblong and as < 1 mm spherical forms (Fig. 2 of Large et al., 2001). Nanometer-scale iron sulfides are contained in elongated cellular structures within these structures.
The preservation of bacteriomorphs and the absence of preserved soft tissue under FESEM analysis indicates that soft tissue was consumed in its entirety. All taphonomic pathways to soft-tissue preservation require some degree of microbial decay (Sagemann et al., 1999). Betts et al. (2014) demonstrated adipocere formation in fish carcasses in massive fish kills in a freshwater lake. Such adipocere forms by the anaerobic bacterial decay of triglycerides in fat to form free fatty acids (Mant 1957; Smith and Wuttke, 2012). Cyanobacteria biofilms have been documented to cause structural damage to bone by endolithic activity when left exposed in dig sites and museum settings (Marano et al., 2016).