Understanding the geobiology of the terminal Ediacaran Khatyspyt Lagerstätte (Arctic Siberia, Russia)

The Khatyspyt Lagerstätte (~544 Ma, Russia) provides a valuable window into late Ediacaran Avalon‐type ecosystems with rangeomorphs, arboreomorphs, and mega‐algae. Here, we tackle the geobiology of this Lagerstätte by the combined analysis of paleontological features, sedimentary facies, and lipid biomarkers. The Khatyspyt Formation was deposited in carbonate ramp environments. Organic matter (0.12–2.22 wt.% TOC) displays characteristic Ediacaran biomarker features (e.g., eukaryotic steranes dominated by the C29 stigmastane). Some samples contain a putative 2‐methylgammacerane that was likely sourced by ciliates and/or bacteria. 24‐isopropylcholestane and 26‐methylstigmastane are consistently scarce (≤0.4% and ≤0.2% of ∑C27‐30 regular steranes, respectively). Thus, Avalon‐type organisms occupied different niches than organisms capable of directly synthesizing C30 sterane precursors among their major lipids. Relative abundances of eukaryotic steranes and bacterial hopanes (sterane/hopane ratios = 0.07–0.30) demonstrate oligotrophic and bacterially dominated marine environments, similar to findings from other successions with Ediacara‐type fossils. Ediacara‐type fossils occur in facies characterized by microbial mats and biomarkers indicative for a stratified marine environment with normal–moderate salinities (moderate–high gammacerane index of 2.3–5.7; low C35 homohopane index of 0.1–0.2). Mega‐algae, in contrast, are abundant in facies that almost entirely consist of allochthonous event layers. Biomarkers in these samples indicate a non‐stratified marine environment and normal salinities (low gammacerane index of 0.6–0.8; low C35 homohopane index of 0.1). Vertical burrowers occur in similar facies but with biomarker evidence for stratification in the water column or around the seafloor (high gammacerane index of 5.6). Thus, the distribution of macro‐organisms and burrowers was controlled by various, dynamically changing environmental factors. It appears likely that dynamic settings like the Khatyspyt Lagerstätte provided metabolic challenges for sustenance and growth which primed eukaryotic organisms to cope with changing environmental habitats, allowing for a later diversification and expansion of complex macroscopic life in the marine realm.

2.22 wt.% TOC) displays characteristic Ediacaran biomarker features (e.g., eukaryotic steranes dominated by the C 29 stigmastane). Some samples contain a putative 2-methylgammacerane that was likely sourced by ciliates and/or bacteria. 24-isopropylcholestane and 26-methylstigmastane are consistently scarce (≤0.4% and ≤0.2% of ∑C 27-30 regular steranes, respectively). Thus, Avalon-type organisms occupied different niches than organisms capable of directly synthesizing C 30 sterane precursors among their major lipids. Relative abundances of eukaryotic steranes and bacterial hopanes (sterane/hopane ratios = 0.07-0.30) demonstrate oligotrophic and bacterially dominated marine environments, similar to findings from other successions with Ediacara-type fossils. Ediacara-type fossils occur in facies characterized by microbial mats and biomarkers indicative for a stratified marine environment with normalmoderate salinities (moderate-high gammacerane index of 2.3-5.7; low C 35 homohopane index of 0.1-0.2). Mega-algae, in contrast, are abundant in facies that almost entirely consist of allochthonous event layers. Biomarkers in these samples indicate a non-stratified marine environment and normal salinities (low gammacerane index of 0.6-0.8; low C 35 homohopane index of 0.1). Vertical burrowers occur in similar facies but with biomarker evidence for stratification in the water column or around the seafloor (high gammacerane index of 5.6). Thus, the distribution of macro-organisms and burrowers was controlled by various, dynamically changing environmental factors. It appears likely that dynamic settings like the Khatyspyt Lagerstätte provided metabolic challenges for sustenance and growth which primed eukaryotic organisms to cope with changing environmental habitats, allowing for a later diversification and expansion of complex macroscopic life in the marine realm.
This study tackles the geobiology of the Khatyspyt Lagerstätte by combining detailed paleontological and sedimentological observations with systematic organic biomarker analyses of different facies. This integrative approach taps the full geobiological potential of the Khatyspyt record, providing valuable insights into ecosystem functioning at this critical transition of Earth's history. We will reconstruct biological communities and environmental conditions in the Khatyspyt Lagerstätte and test potential controls on the distribution of Avalon-type macro-organisms and mega-algae. Furthermore, we will discuss our results in the light of findings from other Ediacaran marine ecosystems (e.g., Oman, Baltica, South China).
The Khatyspyt Formation is laterally not extensive and typically less than 190 m thick Pelechaty, Kaufman, et al., 1996). It can be subdivided into four members mainly comprising limestones (partly dolomitized) and shales   (Figure 1). In the type area, sedimentary facies indicates deposition in low-energy carbonate ramp environments below storm wave base (Knoll et al., 1995;Nagovitsin et al., 2015). To the east and southwest, however, limestone beds exhibiting hummocky cross-stratification become more abundant and progressively thicker (e.g., Berkekit River, Kersyuke River, upper Khorbusuonka River, Kyutingde River basin), suggesting higher hydrodynamic energies . Further to the south, the Khatyspyt Formation correlates with thick-bedded limestones and dolomites (Kyutingde River basin, Ulakhan-Uettyakh and Balagannakh tributaries) . Thus, environments to the east and southwest were likely slightly shallower than in the type area, and the depositional system deepened gradually to the northwest. The overlying Turkut Formation consists of evaporitic carbonates (Knoll et al., 1995;Nagovitsin et al., 2015;Yakshin, 1987).
Because of the presence of ubiquitous organic matter, it was inferred that the Khatyspyt Formation was deposited under anoxic conditions beneath a stratified water column (Knoll et al., 1995;Pelechaty, Kaufman, et al., 1996). Indeed, stable carbon and sulfur isotopes suggest the initial presence of a stratified water body with episodes of euxinic deeper water that shifted to non-euxinic conditions (Cui et al., 2016). This is fairly similar to the Shibantan Member in South China (Duda, 2014;Duda, Zhu, & Reitner, 2015) and, possibly, parts of the Ara Group in south Oman (see Schröder & Grotzinger, 2007).

| Petrography and bulk analyses
The analyzed samples were stored in collections of the Trofimuk Institute of Petroleum Geology and Geophysics, Siberian Branch of the Russian Academy of Sciences (Novosibirsk, Russia). Thin sections (vertical cross sections) were prepared for all samples. Petrographic observation of thin sections (transmitted and reflected light) was conducted with a Zeiss SteREO Discovery.V8 stereomicroscope linked to an AxioCam MRc5 5-megapixel camera.
Contents of total organic carbon (TOC), sulfur (S tot ), and nitrogen (N tot ) were determined with a Hekatech Euro EA elemental analyzer and a Leco RC612 temperature programmable carbon analyzer. All values are reported in weight % (wt.%) of bulk sedimentary rock.
Rock-Eval pyrolysis was performed on unextracted sample powders with a Rock-Eval 6 instrument following the method described by Espitalié et al. (1977). Briefly, the temperature was ramped from 300°C (held for 3 min) to 650°C at 25°C/min. Pyrolysis products and released CO 2 were analyzed with a flame ionization detector (FID) and an infrared cell, respectively. The measurements included quantities of free hydrocarbons (S1, mg HC/g rock) and hydrocarbons yielded from the labile kerogen (S2, mg HC/g rock), temperatures at maximum yields of S2 hydrocarbons (T max , °C), as well as the CO 2 generated from organic carbon at higher temperature up to 650°C (S3, mg CO 2 /g rock). All these data were used to calculate the hydrogen index (HI; S2/TOC * 100), oxygen index (OI; S3/TOC * 100), and production index (PI; S1/(S1 + S2)) for each sample.

| Sample preparation for organic biomarker analysis
Selected samples of different Khatyspyt facies were analyzed for lipid biomarkers using established methodology (Duda, 2014; F I G U R E 1 Working area and composite section of the Khatyspyt Formation (modified after Nagovitsin et al., 2015 U-Pb zircon age of 543.9 ± 0.2 Ma from Bowring et al., 1993). Please note that the sample positions are only approximate and not to scale. Samples in bold were analyzed for organic biomarkers. Ch, Chuskuna Formation; Kh, Khatyspyt Formation; Ms, Maastakh Formation; Mt, Mattaia Formation; Sy, Syhargalakh Formation; Tr, Turkut Formation. FAD, First appearance datum. Asterisks mark samples from intervals with abundant and well-preserved fossils of Ediacara-type organisms (*) and mega-algae (**) Duda et al., 2016) (Figure 1). All glassware and other laboratory materials were heated to 500°C for 3 hr and/or extensively rinsed with acetone to circumvent trace organic contamination transfer to rocks and rock powders. Full procedural blanks (pre-combusted sea sand) were prepared and analyzed in parallel to monitor laboratory contaminations.
Exterior parts of all samples (≥5 mm) were removed with a pre-cleaned precision saw (Buehler IsoMet 1000). The exterior and interior parts were then crushed and powdered using a pebble mill (Retsch MM 301). Ground sample material (ca. 20 g) was extracted in the following steps: (a) dichloromethane (DCM), (b) DCM/n-hexane (1/1; v/v), and (c) n-hexane (20 min ultrasonication, respectively). The resulting extracts were combined, desulfurized with activated copper, and gently concentrated using a pre-cleaned rotary evaporator and N 2 to avoid a major loss of low-boiling compounds (Ahmed & George, 2004). The isolates were then fractionated by column chromatography (1.5 cm in diameter, 8 cm in height; 7 g of dry silica gel). The saturated fraction (F1) was eluted with n-hexane (27 ml), the aromatic fraction (F2) with n-hexane/DCM (1/1; v/v; 32 ml), and a polar residue (F3) with Our results reported are for the interior rock portions which are much less prone to containing contaminant contributions.
Comparisons between outer and inner portions were performed ( Figure S1) and showed very similar compound profiles and negligible contamination of outer portions in all cases.

| Gas chromatography-mass spectrometry
Gas chromatography-mass spectrometry (GC-MS) analyses were carried out with a Thermo Scientific Trace 1300 Series GC coupled to a Thermo Scientific Quantum XLS Ultra MS. The GC was equipped with a Phenomenex Zebron ZB-5 column (30 m length, 0.25 mm inner diameter, 0.25 µm film thickness). Saturated and aromatic fractions were injected into a splitless injector and transferred to the GC column at 300°C. Helium was used as carrier gas with a flow rate of 1.5 ml/min. The GC oven temperature was ramped from 80°C (held for 1 min) to 310°C (held for 20 min) at 5°C/min. Electron ionization mass spectra were recorded in full-scan mode at an electron energy of 70 eV with a mass range of m/z 50-600 and scan time of 0.42 s.

| Metastable reaction monitoring-gas chromatography-mass spectrometry
Metastable reaction monitoring-gas chromatography-mass spectrometry (MRM-GC-MS) was conducted on a Waters Autospec Premier MS coupled to an Agilent 7890A GC. The GC was equipped with a DB-1MS column (60 m length, 0.25 mm inner diameter, 0.25 μm film thickness). 1-2 µl of the saturated hydrocarbon fractions dissolved in n-hexane were injected onto the GC column in splitless injection mode and transferred to the GC column at 320°C.
Helium was used as carrier gas with a flow rate of 1.0 ml/min. The GC oven temperature was ramped from 60°C (held for 2 min) to 150°C at 10°C/min and then to 320°C at 3°C/min (held for 22.5 min).
Formulae for all other molecular indices are provided in the text.

| Petrographic features
The Khatyspyt Formation comprises finely laminated bituminous limestones, marls, and shales ( Figure 1). Most of the observed samples contain abundant µm-sized pyrite crystals.
Fossils of macroscopic, complex organisms are most abundant and best preserved in two distinct stratigraphic intervals ( Figure 1, Table 2). Mega-algae assemblages tend to occur in laminated limestones consisting of an alternation of thin (<1 mm) micritic to finegrained calcite layers (k605-11.3-11.5a,b; Figure 2a). Fossils of Ediacara-type organisms, in contrast, are preserved in facies that additionally exhibit interwoven networks of very thin, dark-brownish laminae (k601c-13.5-15.0a,b; Figure 2b). Notably, some of the samples from this interval are recrystallized to an extent that any other primary features such as grains are lost (k601c-13.5-15.0b; Figure 2c).
In samples from other parts of the Khatyspyt Formation, primary sedimentary features such as the original bedding and lamination appear to be disturbed (e.g., k701b-4.25; Figure 2d). In addition, the formation comprises bituminous limestones that are not laminated on the microfacies scale (e.g., k601c-6.0; Figure 2e). Grainy layers in undisturbed and well-preserved facies commonly exhibit sparry calcite cements ( Figure 2f). These cements are very prominent and can even be recognized in hand specimens by the unaided eye.
In cross section, samples from the interval with Ediacara-type fossils locally comprise small (<1 mm) circular to oval structures that are filled with grains or carbonate cements (k601c-13.5-15.0a;  Figure S2). Some of these might be remains of shelly organisms, but this requires further investigation. Therefore, the components are classified as incertae sedis.

| Organic geochemistry-bulk characteristics
Bulk sedimentary organic matter data are listed in Table 3. TOC contents of the analyzed samples range from very low to high values (0.12-2.22 wt.%). Sulfur and nitrogen contents are low (0.01-0.08 and 0.01-0.04 wt.%, respectively). Rock-Eval data are only considered totally robust here if TOC contents are ≥0.2 wt.% and S2 values are >0.5 mg HC/g rock (see Peters, 1986;Peters & Cassa, 1994

| Geobiology of the Khatyspyt Lagerstätteinferences from sedimentary facies
The alternating micritic to fine-grained layers (Figure 2a) representallochthonous event deposits (Table 2). Organic laminae that interweave sedimentary grains (Figure 2b,f), in contrast, are remnants of microbial mats that thrived in the Khatyspyt environment (Table 2).
It should be noted, however, that these microbial laminae are easily confused with diagenetic pressure solution seams that can be  (Table 2). Such traces were likely produced by burrowers that exploited microbial mats for resources such as food and oxygen (Gehling, 1999;Gingras et al., 2011;Seilacher & Pflüger, 1994).
Vertically extending structures exhibiting fishhook-and antler-like shapes in cross section (Figure 3b,c) are typically produced by organisms that exploit deeper sediment levels ( Table 2) Table 2) and add to findings of meniscate burrows in the Khatyspyt ecosystem (Rogov et al., 2012.
This highlights the presence of diverse burrowing strategies in the terminal Ediacaran, including vertical exploitation of sediment as known from the Phanerozoic.

F I G U R E 7
Background-subtracted mass spectra of gammacerane (left) and the putative 2-methylgammacerane (right). The methylgammacerane has a characteristic main fragment ion of m/z 205 instead of 191 due to the presence of an additional methyl group (likely at position C-2). Note the absence of fragment ions at m/z 369 or 383 which are characteristics of the hopane and methylhopane series, respectively

| Biomarker data integrity (thermal maturity, biodegradation & syngeneity)
Rock-Eval pyrolysis is an established tool used to assess the thermal maturity of rocks (e.g., Espitalié et al., 1977;Peters, 1986;Tissot & Welte, 1984). Sample k601c-6.0 exhibits a PI >0.2 which might indicate the presence of migrated oil (see Peters, 1986 (Table 3). However, Rock-Eval parameters (T max , HI, OI) and lipid biomarker data cannot be considered absolutely valid for this sample and are therefore excluded.
Thus, the effect of biodegradation appears to be negligible for the analyzed samples.
The ratio of eukaryotic steranes to bacterial hopanes is a well-established measure for the relative importance of the two domains in ancient settings (e.g., Peters et al., 2005). Sterane/hopane ratios <1  Table 1).
Taken together, sterane/hopane ratios from all records with assemblages of Ediacara-type fossils are all well below 1, reflecting higher relative inputs by bacteria as compared to eukaryotes. This suggests that not all communities with Ediacara-type organisms inhabited algal-rich environments, as proposed elsewhere (Bobrovskiy et al., 2020). Furthermore, only the highest of these values fall into the lowermost average range of post-Ediacaran rocks and oils (i.e., 0.5-2.0, mean = 1.0: Cao et al., 2009;Peters et al., 2005), calling the proposed similarity to nutrient-replete Phanerozoic ecosystems (Bobrovskiy et al., 2020) into question. All lipid biomarker data available for successions with Ediacara-type fossils suggest rather oligotrophic environments dominated by bacteria. It remains to be tested whether these baseline conditions also prevailed elsewhere in other ecosystems with Ediacara-type organisms.
In the samples analyzed herein, 24-ipc and 26-mes are only present in minor traces (≤0.4% and ≤0.2% of the total C 27 -C 30 regular steranes, respectively) ( Figure 6). The calculated relative abundances are probably even overestimated due to low signal-to-noise ratios and cross talk effects. Given the low thermal maturity of organic matter in the Khatyspyt Formation  results of this study), a preferential loss due to thermal stress ("thermal taphonomy": Mißbach et al., 2016) appears unlikely. At the same time, low quantities of these compounds might form through diagenetic side chain modification of C 29 sterols, although this requires further investigation. In any case, organisms capable of directly synthesizing 24-ipc and 26-mes were not widespread in the Khatyspyt Lagerstätte.
Late Ediacaran strata from Baltica contain similarly low amounts of these source-specific biomarkers, with 24-ipc being ≤0.6% relative to the total C 27 -C 30 sterane ratios and below detection limits in over half the sample set (Pehr et al., 2018). This is at least an order of magnitude lower than in Neoproterozoic rocks and oils from other localities such as South Oman, where 24-ipc and 26-mes typically account for 1%-4% of the total C 27 -C 30 steranes Love et al., 2009;McCaffrey et al., 1994;Zumberge, 2019;Zumberge et al., 2018Zumberge et al., , 2019. This suggests that Ediacara-type organisms and mega-algae occupied different niches than demosponges (oligotrophic vs eutrophic, respectively).
However, methylgammacerane can clearly be distinguished from 3β-methylhopanes, C 31 βα homohopanes (S + R), and gammacerane based on distinct mass spectral characteristics (i.e., main fragment ion at m/z 205, absence of fragment ions at m/z 369 or 383, and a molecular ion at m/z 426: Figure 7). For the samples analyzed herein, this interpretation was further corroborated by MRM-GC-MS ( Figure S3).
The occurrence of methylated tetrahymanols in extant organisms strongly suggests a primary origin of methylgammacerane in ancient sedimentary rocks. The decoupled gammacerane and methylgammacerane abundances (MGI = 1.7-5.6: Table 2) potentially indicate different biological sources for both compounds.
By analogy to A-ring methylated steranes, diagenetic alkylation of tetrahymanol may constitute an alternative source for the observed methylgammacerane. A-ring methylated steroids form via alkylation of ∆ 2 -sterene intermediates-therefore resulting in 2-and 3-alkylsteranes (see Summons & Capon, 1988. Alternatively, a diagenetic alkylation of the hydroxyl group in tetrahymanol (i.e., However, methylgammacerane in Oman is suspected to be mainly alkylated at C-2 (Grosjean et al., 2012), and potential precursors are well known from extant organisms-in contrast to 2-and 3-methylsteranes (Brocks & Summons, 2003). Furthermore, methylgammacerane abundances seem to be decoupled from gammacerane (see above). Therefore, a biological origin of methylgammacerane appears likely, although a diagenetic origin cannot completely be excluded.

| Paleoenvironmental conditions
CPI values of ~1 and the predominance of short-chain n-alkanes with maxima at ≤n-C 19 indicate a marine environment (Peters et al., 2005) ( Figure 4, Table 1). This is in good accordance with Ediacaran records from South China Duda et al., 2015) and the Russian White Sea (Grazhdankin, 2003). Neoproterozoic-Cambrian sedimentary rocks in Oman were also mostly deposited in marine environments (Gorin, Racz, & Walter, 1982), while parts of Baltica were possibly temporarily affected by freshwater conditions (Bojanowski et al., 2020).
In concert, these data suggest that organisms in the Khatyspyt Lagerstätte were confronted with dynamically changing environmental conditions. This is in good accordance with findings from the Shibantan Member (Duda, 2014;Duda et al., 2015;Xiao et al., 2020).

| Geobiology of the Khatyspyt Lagerstätte-a synthesis
The Khatyspyt Formation was deposited in marine carbonate ramp environments, very similar to the contemporaneous Shibantan Member in South China (see Duda, 2014;Duda et al., 2015). Such environments react sensitively on abiotic and biological influences. It is therefore not surprising that organisms in both systems were confronted with frequently changing chemical conditions and nutrient availabilities. The resulting fluctuating selection pressures likely promoted the development of pioneering physiological strategies and innovative lifestyles in different organism groups. It is tempting to speculate that these innovations eventually provided evolutionary advantages in times of global environmental change.
Occurrences of macroscopic complex fossils appear to be linked to specific ecological conditions. For instance, the most diverse assemblages of Ediacara-type fossils occur in facies characterized by prominent microbial mats and horizontal traces (k601c-13.5-15.0a, b; Figures 2b and 3a). Lipid biomarkers indicate a stratified marine environment, normal to moderately elevated salinities, and low oxygen levels (GI = 2.3-5.7; C 35 homohopane index = 0.1-0.2; equally important αββ R + S and ααα S + R sterane isomers: Figure 5, Table 2). Such conditions are widely considered problematic for macroscopic complex organisms. However, certain Avalon-type macro-organisms (rangeomorphs, arboreomorphs) may develop tolerances and/or life strategies that allowed them to cope with these ecological stressors (e.g., symbiotic lifestyles, interaction with microbial mat communities; see also Bykova et al., 2017).
Mega-algae, in contrast, are most abundant and diverse in facies that lack evidence for the presence of prominent microbial mats but are dominated by layers that represent allochthonous event deposits (samples k605-11.3-11.5a, b: Figure 2a). Lipid biomarkers in these samples indicate a non-stratified marine environment and normal salinities during deposition (GI = 0.6-0.8; C 35 homohopane index = 0.1; dominant ααα S + R sterane isomers: Figure 5, Table 2; see also Duda et al., 2016). In contrast to the environments described above, such conditions appear to be favorable for macroscopic complex organisms. It seems conceivable that mega-algae proliferated in times of intermittent event deposition, but this remains to be further tested in futures studies.
The Khatyspyt Lagerstätte shares some striking similarities with the Shibantan Member, including sedimentary facies and occurrences of Ediacara-type fossils. Unfortunately, however, lipid biomarkers are not well preserved in the Shibantan Member . A variety of lipid biomarker characteristics of the Khatyspyt Lagerstätte are similar to the Baltica record (e.g., low sterane/hopane ratios, scarcity of C 30 steranes; Goryl et al., 2018, Pehr et al., 2018. These lipid biomarker records, as well as reported low sterane/hopane ratios from the Russian White Sea (Bobrovskiy et al., 2020), suggest that the ecosystems were oligotrophic and dominated by bacteria. Furthermore, organisms capable of directly synthesizing the C 30 steranes 24-ipc and 26-mes (most likely candidates demosponges) appear not to have been widespread in these environments (Table 2).
Despite these similarities, the Khatyspyt Lagerstätte is special in that it exhibits assemblages of carbonaceous compression fossils (mega-algae), ichnofabrics produced by vertical burrowers, and very low to high TOC contents (0.12-2.22 wt.%; Table 3). In Baltica, TOC contents are mostly ≤0.5 wt.% and rarely exceed 1.0 wt.% (Goryl et al., 2018;Pehr et al., 2018). In South Oman, in contrast, TOC contents are mostly >1.0 wt.% and range up to 11.0 wt.% Love et al., 2009). The Khatyspyt record thus ecologically bridges between Baltica and South Oman. At the same time, the unique features of the Khatyspyt Lagerstätte demonstrate that Ediacaran ecosystems were more highly diverse than previously realized. Furthermore, it emphasizes the impact of sedimentary processes and various environmental controls (water column stratification, oxygen, and possibly salinity levels) on the distribution of Avalon-type macro-organisms and mega-algae (Table 2).

| CON CLUS IONS
Bituminous facies of the Khatyspyt Formation were deposited in marine carbonate ramp environments. Consistently low sterane/hopane ratios reflect relatively low inputs of eukaryotic organisms to the preserved organic matter, suggesting oligotrophic marine depositional environments in which bacteria were dominant. This is similar to lipid biomarker findings from other successions with Ediacara-type fossils (Baltica, including the Russian White Sea). C 30 steranes such as 24-isopropylcholestane and 26-methylstigmastane are present in minor traces but do not appear to be completely absent. Thus, Avalon-type organisms occupied different niches than organisms capable of directly synthesizing C 30 sterane precursors among their major lipids. Notably, some of the analyzed samples contain a putative methylgammacerane (possibly 2-methylgammacerane), which was likely sourced by organisms (ciliates, anoxygenic phototrophs, and/or purple sulfur bacteria) and may be a good proxy for strati-