Dietary options and behavior suggested by plant biomarker evidence in an early human habitat

Significance

Humans evolved in response to the availability of plant and water resources over space and through time. Their influence on our species’ evolution is debated, though, because archives of their spatial distribution are scarce at early human (hominin) localities. Meter-scale vegetation patterns are revealed from sedimentary plant biomarkers across an archaeological horizon at Olduvai Gorge (FLK Zinj). Biomarkers evince a varied local landscape with a woodland patch near a small freshwater wetland, surrounded by an open grassland landscape. Biomarkers from the wetland indicate diverse edible plants near potable water. The coexistence of butchered large animal bones and hominin remains, including juveniles, within an isolated biomarker-delineated wooded microhabitat at FLK Zinj provide support for early provisioning behaviors by our ancestors.

Abstract

The availability of plants and freshwater shapes the diets and social behavior of chimpanzees, our closest living relative. However, limited evidence about the spatial relationships shared between ancestral human (hominin) remains, edible resources, refuge, and freshwater leaves the influence of local resources on our species’ evolution open to debate. Exceptionally well-preserved organic geochemical fossils—biomarkers—preserved in a soil horizon resolve different plant communities at meter scales across a contiguous 25,000 m² archaeological land surface at Olduvai Gorge from about 2 Ma. Biomarkers reveal hominins had access to aquatic plants and protective woods in a patchwork landscape, which included a spring-fed wetland near a woodland that both were surrounded by open grassland. Numerous cut-marked animal bones are located within the wooded area, and within meters of wetland vegetation delineated by biomarkers for ferns and sedges. Taken together, plant biomarkers, clustered bone debris, and hominin remains define a clear spatial pattern that places animal butchery amid the refuge of an isolated forest patch and near freshwater with diverse edible resources.

Spatial patterns in archaeological remains provide a glimpse into the lives of our ancestors (1–5). Although many early hominin environments are interpreted as grassy or open woodlands (6–8), fossil bones and plant remains are rarely preserved together in the same settings. As a result, associated landscape reconstructions commonly lack coexisting fossil evidence for hominins and local-scale habitat (microhabitat) that defined the distribution of plant foods, refuge, and water (7). This problem is exacerbated by the discontinuous nature and low time resolution often available across ancient soil (paleosol) horizons, including hominin archaeological localities. One notable exception is well-time-correlated 1.8-million-y-old paleosol horizons exposed at Olduvai Gorge. Associated horizons contain exceptionally preserved plant biomarkers along with many artifacts and fossilized bones. Plant biomarkers, which previously revealed temporal patterns in vegetation and water (8), are well preserved in the paleosol horizon and document plant-type spatial distributions that provide an ecosystem context (9, 10) for resources that likely affected the diets and behavior of hominin inhabitants.

Plant biomarkers are delivered by litter to soils and can distinguish plant functional type differences in standing biomass over scales of 1–1,000 m² (11). Trees, grasses, and other terrestrial plants produce leaf waxes that include long-chain n-alkanes such as hentriacontane (nC₃₁), whereas aquatic plants and phytoplankton produce midchain homologs (e.g., nC₂₃) (12, 13). The ratio of shorter- versus long-chain n-alkane abundances distinguish relative organic matter inputs from aquatic versus terrestrial plants to sediments (13):

$$P_{\mathrm{aq}}=\left(n{\mathrm{C}}_{23}+n{\mathrm{C}}_{25}\right)/\left(n{\mathrm{C}}_{23}+n{\mathrm{C}}_{25}+n{\mathrm{C}}_{29}+n{\mathrm{C}}_{31}\right).$$

Sedges and ferns are prolific in many tropical ecosystems (14). These plants both have variable and therefore nondiagnostic n-alkane profiles. However, sedges produce distinctive phenolic compounds [e.g., 5-n-tricosylresorcinol (ⁿR₂₃)] and ferns produce distinctive midchain diols [e.g., 1,13-dotriacontanediol (C₃₂-diol)] (SI Discussion).

Lignin monomers provide evidence for woody and nonwoody plants. This refractory biopolymer occurs in both leaves and wood, serves as a structural tissue, and accounts for up to half of the total organic carbon in modern vegetation (11). Lignin is composed of three phenolic monomer types that show distinctive distributions in woody and herbaceous plant tissues. Woody tissues from dicotyledonous trees and shrubs contain syringyl (S) and vanillyl (V) phenols (12), whereas cinnamyl (C) phenols are exclusively found in herbaceous tissues (12). The relative abundance of C versus V phenols (C/V) is widely used to distinguish between woody and herbaceous inputs to sedimentary and soil organic matter (15).

Plant biomarker ¹³C/¹²C ratios (expressed as δ¹³C values) are sensitive indicators of community composition, ecosystem structure, and climate conditions (8). Most woody plants and forbs in eastern Africa use C₃ photosynthesis (6), whereas arid-adapted grasses use C₄ photosynthesis (8, 14). These two pathways discriminate differently against ¹³C during photosynthesis, resulting in characteristic δ¹³C values for leaf waxes derived from C₃ (about –36.0‰) and C₄ (–21.0‰) plants (16). Carbon isotopic abundances of phenolic monomers of lignin amplify the C₃–C₄ difference and range between ca. –34.0‰ (C₃) and –14.0‰ (C₄) in tropical ecosystems (15). Terrestrial C₃ plant δ¹³C values decrease with increased exposure to water, respired CO₂, and shade (8), with lowest values observed in moist regions with dense canopy (17). Although concentration and δ¹³C values of atmospheric CO₂ can affect C₃ plant δ¹³C values (17), this influence is not relevant to our work here, which focuses on a single time window (SI Discussion). The large differences in leaf-wax δ¹³C values between closed C₃ forest to open C₄ grassland are consistent with soil organic carbon isotope gradients across canopy-shaded ground surfaces (6) and serve as a quantitative proxy for woody cover (f_woody) in savannas (8).

As is observed for nonhuman primates, hominin dietary choices were likely shaped by ecosystem characteristics over habitat scales of 1–1,000 m² (3–5). To evaluate plant distributions at this small spatial scale (9), we excavated 71 paleosol samples from close-correlated trenches across a ∼25,000-m² area that included FLK Zinjanthropus Level 22 (FLK Zinj) at Olduvai Gorge (Fig. 1). Recent excavations (18–21) at multiple trenches at four sites (FLK^NN, FLK^N, FLK, and FLK^S, Fig. 1D) exposed a traceable thin (5–50 cm), waxy green to olive-brown clay horizon developed by pedogenic alterations of playa lake margin alluvium (22). Weak stratification and irregular redox stains suggest initial soil development occurred during playa lake regression (18, 22), around 1.848 Ma (ref. 23 and SI Discussion). To date, craniodental remains from at least three hominin individuals (18–20), including preadolescent early Homo and Paranthropus boisei, were recovered from FLK Zinj. Fossils and artifacts embedded in the paleosol horizon often protrude into an overlying airfall tuff (18, 19), which suggests fossil remains were catastrophically buried in situ under volcanic ash. Rapid burial likely fostered the exceptional preservation of both macrofossils (10) and plant biomarkers across the FLK Zinj land surface.

Fig. 1.

Location and map of FLK Zinj paleosol excavations. (A and B) Location of FLK Zinj as referenced to reconstructed depositional environments at Olduvai Gorge during the early Pleistocene (18, 22) and the modern gorge walls. The perennial lake contained shallow saline–alkaline waters that frequently flooded the surrounding playa margin (i.e., floodplain) flats. (C) Outline of FLK Zinj paleosol excavation sites used for our spatial biomarker reconstructions. (D) Concentric (5 m) gridded distribution map of FLK Zinj paleosol excavations relative to previous archaeological trenches (18–21). Major aggregate complexes (FLK^NN, FLK^N, FLK, and FLK^S) are color-coded to show excavation-site associations.

Plant biomarker signatures reveal distinct types of vegetation juxtaposed across the FLK Zinj land surface (Figs. 2–4 and Fig. S1). In the northwest, FLK^NN trenches show high nC₂₃ δ¹³C values (Fig. 2B) as well as high C/V and P_aq values (Figs. 3 and 4A). They indicate floating or submerged aquatic plants (macrophytes) in standing freshwater (13), a finding that is consistent with nearby low-temperature freshwater carbonates (tufa), interpreted to be deposited from spring waters (22). Adjacent FLK^N trenches have lower P_aq values (Fig. 4A) with occurrences of fern-derived C₃₂-diol and sedge-derived ⁿR₂₃ (Fig. 2 C and D). These biomarker distributions indicate an abrupt (around 10 m) transition from aquatic to wetland vegetation. Less than 100 m away (Fig. 1C), low nC₃₁ δ¹³C values (Fig. 2A) and low C/V and very low P_aq values (Figs. 3 and 4A) collectively indicate dense woody cover (Fig. 4B). In the farthest southeastern (FLK^S) trenches, high C/V values and high δ¹³C values for C lignin phenols (Fig. 3) indicate open C₄ grassland.

Fig. 2.

Spatial distributions and δ¹³C values for plant biomarkers across FLK Zinj. Measured and modeled δ¹³C values (large and smaller circles, respectively) are shown for (A) nC₃₁ from terrestrial plants, (B) nC₂₃ from (semi)aquatic plants, (C) C₃₂-diol from ferns, and (D) ⁿR₂₃ from sedges (see refs. 12 and 13 and SI Discussion). Modeled values [inverse distance-weighted (9)] account for spatial autocorrelation (15-m radius) in standing biomass (35) over scales of soil organic matter accumulation (11). Black dots represent paleosols with insufficient plant biomarker concentrations for isotopic analysis.

Fig. 4.

Spatial relationships shared between local plant resources and hominin remains. Measured and modeled values (large and smaller circles, respectively) are shown for (A) P_aq (13) and (B) f_woody (8). Modeled values [inverse distance-weighted (9)] account for spatial autocorrelation (15-m radius) in standing biomass (35) over scales of soil organic matter accumulation (11). (C) Kernel density map of cut-marked bones (18–21) across the FLK Zinj land surface (Fig. S4). High estimator values indicate hotspots of hominin butchery (Fig. S5). A shaded rectangle captures the area (ca. 0.68 probability mass) with highest cut-marked bone densities and is shown in A and B for reference.

Fig. S1.

Total ion chromatograms for saturated hydrocarbons in representative paleosols at (A) FLK^NN, (B) FLK^N, (C) FLK, and (D) FLK^S. C₂₃, tricosane; C₂₅, pentacosane; C₂₉ nonacosane; C₃₁, hentriacontane.

Fig. 3.

Molecular and isotopic signatures for lignin phenols across FLK Zinj. Bivariate plots are shown for diagnostic lignin compositional parameters (see refs. 12 and 15 and Table S1) associated with aggregate excavation complexes (Fig. 1C). Symbols are colored according to respective δ¹³C values for the C lignin phenol, p-coumaric acid. FLK symbols are uncolored due to insufficient p-coumaric acid concentrations for isotopic analysis. Representative lignin compositional parameters (12, 15) are shown for monocotyledonous herbaceous tissues (G), dicotyledonous herbaceous tissues (H), cryptogams (N), and dicotyledonous woody tissues (W).

Biomarkers define a heterogeneous landscape at Olduvai and suggest an influence of local resources on hominin diets and behavior. It is recognized (2, 24–26) that early Homo species and P. boisei had similar physiological characteristics. These similarities in physical attributes suggest behavioral differences were what allowed for overlapping ranges and local coexistence (sympatry) of both hominins. For instance, differences in seasonal subsistence strategies or different behavior during periods of drought and limited food could have reduced local hominin competition and fostered diversification via niche specialization (27–29).

Physical and isotopic properties of fossil teeth indicate P. boisei was more water-dependent [low enamel δ¹⁸O values (24)] and consumed larger quantities of abrasive, ¹³C-enriched foodstuffs [flat-worn surfaces (25) and high enamel δ¹³C values (26)] than coexisting early Homo species. Although ¹³C-enriched enamel is commonly attributed to consumption of C₄ grasses or meat from grazers (14), this was not likely, because P. boisei craniodental features are inconsistent with contemporary gramnivores (24, 25) or extensive uncooked flesh mastication (26). Numerous scholars have proposed the nutritious underground storage organs (USOs) of C₄ sedges were a staple of hominin diets (14, 24, 26, 27). Consistent with this suggestion, occurrences of ⁿR₂₃ attest to the presence of sedges at FLK^NN and FLK^N (Fig. 2D). However, the low δ¹³C values measured for ⁿR₂₃ at these same sites (Fig. 2D and Fig. S2) indicate C₃ photosynthesis (12, 16), a trait common in modern sedges that grow in alkaline wetlands and lakes (30) (Fig. S3). Thus, biomarker signatures support the presence of C₃ sedges in the wetland area of FLK Zinj.

Fig. S2.

Total ion chromatogram [TIC (A)] and selected ion chromatograms for derivatized 5-n-alkylresorcinols [m/z 268 (●)] and midchain diols [m/z 369 (○)] from a representative paleosol at FLK^N. Also shown are δ¹³C values for homologous (B) 5-n-alkylresorcinols and (C) midchain diols. C₃₂-diol, dotriacontanediol; ⁿR₂₃, tricosylresorcinol.

Fig. S3.

Summary phyogenetic consensus tree of Cyperaceae (sedges) based on nucleotide (rcbL and ETS1f) sequence data (50–54, 95, 96). Important taxonomic distinctions discussed in SI Discussion, Fern Alkyldiols are shown explicitly. Triangle-enclosed digits represent the number of additional branches at different levels of taxonomic classification. CEFA, Cypereae Eleocharideae Fuireneae Abildgaardieae; CSD, Cariceae Scirpeae Dulichieae.

Alternative foodstuffs with abrasive, ¹³C-enriched biomass include seedless vascular plants (cryptogams), such as ferns and lycophytes [e.g., quillworts (27, 30)]. Ferns are widely distributed throughout eastern Africa in moist and shaded microhabitats (31) and are often found near dependable sources of drinking water (32). Today, ferns serve as a dietary resource for humans and nonhuman primates alike (27), and fiddlehead consumption is consistent with the inferred digestive physiology [salivary proteins (33)] and the microwear on molars (34) of P. boisei in eastern Africa (25, 26). Ferns were present at FLK^N, based on measurements of C₃₂-diol (Fig. 2D). Further, the high δ¹³C values measured for these compounds are consistent with significant fern consumption by P. boisei at Olduvai Gorge.

Ferns and grasses were not the only plant foods present during the time window documented by FLK Zinj. Further, the exclusive reliance on a couple of dietary resources was improbable for P. boisei, because its fossils occur in diverse localities (24–26). Aquatic plants are an additional candidate substrate, as evidenced by high P_aq values at FLK^NN and FLK^N (Fig. 4A). Floating and submerged plants proliferate in wetlands throughout eastern Africa today (13, 14), and many produce nutritious leaves and rootstock all year long (27, 28). Although C₄ photosynthesis is rare among modern macrophytes (30), they can assimilate bicarbonate under alkaline conditions, which results in C₄-like isotope signatures in their biomass (30). Their leaf waxes, such as nC₂₃ (13), are both present and carry ¹³C-enriched signatures at FLK^NN and FLK^N (Fig. 2B). It is also likely that aquatic macrophytes sustained invertebrates and fish with comparably ¹³C-enriched biomass, as they do in modern systems (14), and we suggest aquatic animal foods could have been important in P. boisei diets (27, 28).

Biomarkers across the FLK Zinj soil horizon resolve clear patterns in the distribution of plants and water and suggest critical resources that shaped hominin existence at Olduvai Gorge. The behavioral implications of local conditions require understanding of regional climate and biogeography (3–5, 7), because hominin species likely had home ranges much larger than the extent of excavated sites at FLK Zinj. Lake sediments at Olduvai Gorge include numerous stacked tuffs with precise radiometric age constraints (23). These tephrostratigraphic correlations (21) tie the FLK Zinj landscape horizon to published records of plant biomarkers in lake sediments that record climate cycles and catchment-scale variations in ecology. Correlative lake sediment data indicate the wet and wooded microhabitats of FLK Zinj sat within a catchment dominated by arid C₄ grassland (8). Under similarly arid conditions today, only a small fraction of landscape area (ca. 0.05) occurs within 5 km of either forest or standing freshwater (35). Given a paucity of shaded refuge and potable water in the catchment, the concentration of hominin butchery debris (18–21) exclusively within the forest microhabitat and adjacent to a freshwater wetland (Fig. 4) is notable. We suggest the spatial patterns defined by both macro- and molecular fossils reflect hominins engaged in social transport of resources (1–5), such as bringing animal carcasses and freshwater-sourced foods from surrounding grassy or wetland habitats to a wooded patch that provided both physical protection and access to water.

SI Discussion

Sedge Alkylresorcinols.

Resorcinolic compounds have been isolated from plants, algae, and bacteria (47). Odd-numbered long-chained 5-n-alkylresorcinol homologs are characteristic of the monocotyledonous plant (sub)families Pooideae and Cyperaceae. In eastern Africa, extant genera of Pooideae are limited to high-altitude environments [>2,000 m (48)], well above the mean elevation of Olduvai Gorge (ca. 1,450 m). In contrast, extant genera of Cyperaceae are quite cosmopolitan, occurring in wetlands and seasonal floodplains from sea level to about 2,000 m (48, 49). Tropical Africa contains around 1,400 Cyperaceae species, of which ∼50% occur in just two genera: Cyperus and Carex (50–54). Recent DNA data suggest that C₄ Cyperus form a monophyletic clade nested within Cyperus sensu stricto (53, 54) and comprise nine segregate genera. Together with earlier descriptive studies about the resorcinolic compounds of C₄ Cyperus sedges (e.g., Cyperus longus) in northern and East Africa (55, 56), our new analyses of Cyperus papyrus (Fig. S2) suggest that 5-n-alkylresorcinols with saturated C₁₉–C₂₅ side chains occur in Cyperus sections of Rotundi Clarke and Papyrus (54). Earlier studies also indicate alkylresorcinols occur in taxa of the Cyperus segregate, Pycreus sedges (51, 55, 57) across northeast Africa (58). Importantly, ⁿR₂₃ δ¹³C values for C. papyrus are much higher [–21.5 ± 0.5‰ (n = 3)] than measured at FLK Zinj (Fig. S2).

Cyperus and Pycreus (54) are within the cardinal tribe Cypereae, which is one of two major clades of the subfamily Cyperoideae (Fig. S3). The other major clade of this subfamily possesses cardinal tribes called Cariceae and Scirpeae (50, 53), which include widespread African sedges of the genera Carex and Eriophorum (58), respectively. We found Carex trisperma contains significant ⁿR₂₃ with low δ¹³C values [–29.6 ± 1.1‰ (n = 4)], and this corroborates earlier data for cosmopolitan species of Carex and Eriophorum (38, 59, 60). Thus, 5-n-alkylresorcinols with saturated C₁₉–C₂₅ side chains occur in (sub)tropical tribes of Cyperoideae that include C₄ and C₃ clades (53, 54).

Fern Alkyldiols.

Midchain diols derive from a variety of marine, freshwater, and terrestrial photosynthetic organisms (40, 61) and can preserve in sediments and soils over geologic timescales (39). In general, longer midchain diols occur as a series of even-numbered homologs with characteristic isomer distributions (40). Previous studies identify terminal C₃₀–C₃₂ diols with ω20 hydroxyl positions (e.g., C₃₂-diol) as diagnostic molecular constituents in ferns (39, 61), and we identified the same major homologs in paleosols around FLK Zinj (Fig. S2).

Strict C₄ photosynthesis is not found in monilophytes [ferns (62)] or related lycophytes (63), but these common vascular cryptogams (64) often use alternative carbon-concentrating pathways [e.g., crassulacean acid metabolism (CAM)] that lead to similarly ¹³C-enriched lipids and biomass (30, 65–70). In subtropical Africa, ferns can show variable degrees of CAM (69–71), and their associated bulk δ¹³C values span at least 14‰, from C₃ (∼−34‰) to more C₄-like (−20‰) signatures (39, 71–73). Other edible vascular cryptogams with CAM [e.g., Isoëtes sp. (14, 65)] likewise are common in seasonal freshwater semiaquatic environments across southern and East Africa (64, 74) and have even more ¹³C-enriched C₄-like signatures (30, 65, 70, 73).

Sedimentation and Landscape Relief During Paleosol Formation.

Recent landscape reconstructions disagree on interpreted sediment facies and stratigraphic correlations around FLK Zinj (see refs. 18–21). These differences largely reflect different interpretations of the origin of local topographic relief. Here, we adopt the approach of Uribelarrea et al. (21), which links sediment facies analyses with geomorphic models (Fig. S4) for stratigraphic correlations and landscape reconstruction. An alternative model that FLK Zinj was developed by gradational fluvial incision or surface erosion (20) is not supported by this approach. Instead, cumulative evidence suggests low-energy processes dominated during sediment deposition at FLK Zinj and that a paleosol surface developed during lake regression from tuffaceous clays and was catastrophically overlain with volcanic ash [Tuff I^C (21)].

Fig. S4.

Schematic representation of the associated Tuff I^B to Tuff I^C stratigraphic sequences at FLK Zinj paleosol excavations after geometric correction (0.42°) for tectonic deformation and sediment compaction (21). The FLK Zinj soil horizon is shown in brown and traced between excavation sites. Excavation complexes are color-coded to match Figs. 1, 2, and 4). Distances shown under each excavation complex represent their associated (horizontal) transect lengths.

Fig. S5.

Bivariate kernel density estimation with paired values (n = 22) for woody cover and cut-marked bone density. Contours envelope estimations of 2D (axis-aligned) kernel density. Bandwidths were selected using the direct plug-in approach (97) over binned (5% and 0.125) grid space. Warmer colors are indicative of higher densities.

The exact timespan represented by paleosols around FLK Zinj is debated but constrained by high-precision radiometric ages and taphonomic data. Although Tuff I^C lies about 100 cm above Tuff I^B, they have indistinguishable argon–argon ages of 1.848 ± 0.008 Ma and 1.848 ± 0.003 Ma (23), which set the maximum duration of this entire sequence at 0.011 Ma (75). Taphonomic studies indicate archaeological materials at FLK Zinj accumulated during a time window with a duration that was somewhere between several successive seasons up to a century (4, 18–20, 76, 77). These short durations are estimated from an accumulation span of 50–100 y derived from the sedimentation rate (0.1–0.4 mm⋅y⁻¹) during paleosol formation at FLK Zinj (75, 78), the ca. 50-y residence time of organic carbon in modern (sub)tropic surface soil horizons [0–10 cm (6, 79–81)], and our average sample depth resolution (∼0–20 mm underneath Tuff I^C).

Prior Constraints on P. boisei Dietary Behavior.

Nonspecific plant and animal fossils complement plant biomarker signatures to provide heuristic constraints on the local distributions and availability of food resources around FLK Zinj. Scattered leaf impressions bearing parallel longitudinal striations occur at southern FLK^NN and FLK^N (19, 20, 82) alongside Heterocephalus [African mole-rat (83)] fossils (18, 76) and corroborate plant biomarker signatures in suggesting that C₃ graminoids [e.g., Typha (19, 82)] were locally abundant around FLK Zinj (Fig. 2 and Fig. S1). Further, silicified culms in sediments underlying FLK Zinj (17, 82) resemble the edible USOs (e.g., corms) of African cryptogams [e.g., Isoëtes (84, 85)], which often show lower δ¹³C values due to C₃–C₄ intermediate photosynthesis (30, 65, 69, 70). Proximate playa lakeshore vegetation was an unlikely source for edible C₄ grasses and sedges, because saline-alkaline water bodies [e.g., paleolake Olduvai (18)] are characteristically devoid of aquatic and emergent C₄ macrophytes (86).

Dietary behavior in P. boisei is further informed by enamel δ¹³C values for contemporaneous animals in eastern and southern Africa during the early Pleistocene. Enamel δ¹³C values of P. boisei are markedly high relative to modern and fossil Heterocephalus species with specialized USO-rich diets (87), suggesting limited USO consumption by P. boisei. Besides, most wild USOs are innutritious without extensive extraoral processing or roasting (88). Rather, P. boisei shows enamel δ¹³C values intermediate between fossil Hippopotamus (hippos) and Phacochoerus (pigs) in eastern Africa (24, 89). Based on combined isotopic (89) and craniodental (24) evidence from individual fossils at Olduvai Gorge, both taxa consumed tough browse-graze vegetation (90) such as ferns (91–94), suggesting similar dietary and characteristics for P. boisei.

Materials and Methods

Plant Biomarker Extraction and Isolation.

Freeze-dried and powdered paleosol samples (10–20 g dry weight, n = 71) were extracted by accelerated solvent extraction (Dionex ASE 200 system) with 90:10 dichloromethane (DCM) to methanol by volume. Total lipid extracts were separated into fractions over activated silica gel by elution with hexane (apolar), DCM, and methanol. Apolar fractions were further separated over silver-impregnated alumina by elution with hexane (saturated apolar). Then, n-alkanes were separated from saturated apolar fractions by zeolitic (5 Å) sieve. Once extracted, residual paleosols were oxidized under alkaline conditions and acidified with hydrochloric acid. Lignin phenols were recovered by liquid extraction with diethyl ether. Additional details are provided in SI Materials and Methods.

Molecular and Isotopic Analysis.

Molecular signatures were characterized by GC-MS (SI Materials and Methods). Polar fractions and lignin phenols were derivatized with N,O-bis(trimethylsilyl)trifluoracetamide (BSTFA) in pyridine. Isotopic signatures were characterized by gas chromatography-combustion-isotope-ratio monitoring mass spectrometry and expressed in standard permil (‰) notation relative to Vienna Pee Dee Belemnite (VPDB):

$${\mathit{\delta }}^{13}\mathrm{C}=\mathrm{1,000}\left(R_{\mathrm{sample}}/R_{\mathrm{standard}}\mathrm{-}1\right),\mathit{R}\mathrm{=}{}^{13}\mathrm{C}/{}^{12}\mathrm{C}.$$

SI Materials and Methods

Plant Biomarker Extraction.

Freeze-dried and powdered paleosols (∼20–40 g) were extracted by accelerated solvent extraction (Dionex ASE 200 system) with DCM and methanol (90:10 by volume) in three cycles of 5 min at 100 °C and 1,500 psi (10.3 MPa). Then, extracted paleosols were oxidized under alkaline conditions with cupric oxide at 155 °C for 180 min in stainless steel pressure vessels (36) and then acidified with hydrochloric acid. Oxidation products (lignin phenols) were recovered by extraction with diethylether (36).

Lipid Biomarker Isolation.

Total lipid extracts were separated into apolar (hydrocarbon), intermediate-polarity, and polar fractions over activated silica gel by elution with hexane, DCM, and methanol, respectively. Hydrocarbons were separated further into saturated and unsaturated fractions over activated silver-impregnated (5% by weight) silica gel by elution with hexane and DCM, respectively (37). In turn, saturated hydrocarbons were separated further into unbranched (n-alkanes) and branched fractions via a zeolitic (5 Å) sieve.

Molecular Characterization.

Plant biomarkers were characterized by GC-MS with a Hewlett-Packard 6890 series GC and Hewlett-Packard 5973 mass selective detector. Samples were injected in splitless mode onto a 60-m DB5 fused-silica column (0.32 mm × 0.25 µm) via a Hewlett-Packard 7683 series autosampler. GC temperature was programmed to 60 °C for 1 min then ramped to 320 °C at 6 °C⋅min⁻¹ and held at final temperature for 20 min. Injector and detector temperatures were held at 320 °C. Functionalized biomarkers were derivatized with BSTFA and detected as trimethylsilyl (TMS) derivatives, and 5-n-alkylresorcinols were identified based on characteristic mass spectral fragment ions at m/z 268 (base peak) and m/z 281 (38). Midchain (1,ω20) diols were identified based on m/z 369 (base peak) and m/z 359 (39, 40).

Isotopic Characterization.

Isotopic signatures were characterized by gas chromatography-combustion-isotope-ratio monitoring mass spectrometry with a Varian 3400 model GC connected to a Thermo MAT 252. Samples were injected in splitless mode onto a 60-m DB5 fused-silica column (0.32 mm × 0.25 µm) before combustion over nickel and platinum wire with oxygen in helium at 1,000 °C. Values were determined relative to reference gas calibrated to VPDB and expressed in permil (‰) units:

$${\mathit{\delta }}^{13}\mathrm{C}=\mathrm{1,000}\left(R_{\mathrm{sample}}/R_{\mathrm{standard}}\mathrm{-}1\right),\mathit{R}\mathrm{=}{}^{13}\mathrm{C}/{}^{12}\mathrm{C}.$$

Within-run precision (1σ) and accuracy were determined by coinjected internal standards and are equal, respectively, to 0.14‰ and 0.09‰ (n-alkanes, n = 136), to 0.49‰ and 0.17‰ (diols; n = 48), and to 0.66‰ and 0.21‰ (lignin phenols; n = 40). Lignin phenol δ¹³C values are reported for vanillic, syringic, and p-coumaric acids (Table S1). Alcoholic and acidic functional groups of lignin phenols were derivatized using BSTFA. Isotopic corrections for carbons added by BSTFA were made via measurements of the δ¹³C of benzene-1,2-dicarboxylic acid [commonly called phthalic acid (Schimmelmann standards)] after derivatization and then correcting for the mass-balanced δ¹³C value of derivative carbon (15).

Table S1.

Summary of plant biomarker signatures around FLK Zinj

			δ13CBiomarker
Plant biomarker	Source vegetation	Complex	Average	SD	n	[Biomarker], μg⋅g−1 C
nC23	Aquatic plants	FLKNN	−23.4	0.6	27	4
		FLKN	−24.7	1.2	12	3
nC31	Terrestrial plants	FLKNN	−26.6	1.3	27	4
		FLKN	−29.7	1.1	12	4
		FLK	−33.8	0.9	19	7
		FLKS	−21.9	0.6	12	12
nR23	Sedges	FLKNN	−33.8	0.3	2	1
		FLKN	−32.0	0.9	5	2
C32-diol	Ferns	FLKNN	−26.7	0.5	5	2
		FLKN	−27.1	0.5	8	6
		FLK	−30.3	0.7	4	2
V	Vascular plants	FLKNN	−24.4*	1.0	4	182
		FLKN	−24.0*	—	1	207
		FLK	−33.6*	0.2	3	111
		FLKS	−17.5*	0.4	2	159
S	Angiosperms	FLKNN	−23.0†	1.2	4	184
		FLKN	−24.9†	—	1	93
		FLK	−33.2†	0.2	3	84
		FLKS	−14.5†	0.2	2	167
C	Herbaceous	FLKNN	−17.9‡	1.3	4	55
		FLKN	−19.4‡	—	1	47
		FLKS	−14.2‡	0.1	2	92

C, [p-coumaric acid + ferulic acid]; S, [syringaldehyde + acetosyringone + syringic acid]; V, [vanillin + acetovanillone + vanillic acid].

^*Vanillic acid (TMS derivative).

^†Syringic acid (TMS derivative).

^‡p-coumaric acid (TMS derivative).

Spatial Analysis.

Paleosol excavations were referenced to concentric (5 m) quadrats after geometric correction (0.42°) for tectonic deformation and sediment compaction (21). This approach accounts for uneven spatial distributions in point density (41), enabling accurate distance representations in 2D interpolations (9).

Because plant biomarker reconstructions represent the sum of complex biogeochemical processes across space (9), we use a data-based (deterministic) spatial model based on inverse distance-weighted interpolations of all points within a fixed radius (see ref. 42). Based on combined theory (43) and modern observation (43–45), we calibrate our model with a 15-m radius to account for spatial autocorrelation in standing biomass over scales of soil organic matter accumulation (11). We converted spatially explicit point interpolations into probability surfaces assuming a unimodal (Gaussian) distribution (9, 41, 42). Derivative kernel density maps accurately represent primary spatial patterns (46) and facilitate comparison between diverse datasets with dissimilar resolution.