Yuta Isaji

Detection of endogenous biomolecules in vertebrate fossils remains controversial because labile compounds degrade quickly and are highly susceptible to contamination. Here, we present data on the preservation of heme-derived tetrapyrroles, and vascular canal-constrained ellipsoidal microstructures, inferred to be red blood cells, in limb bones of Aratasaurus museunacionali , a 115-million-year-old theropod dinosaur. High-performance liquid chromatography–mass spectrometry identified parent and diagenetically altered heme B as the principal tetrapyrroles of bone extract, contrasting the chlorophyll and bacteriochlorophyll porphyrin signatures of the surrounding matrix supporting an endogenous origin. Separate spatially resolved analyses by Raman spectroscopy and transmission electron microscopy confirmed the ellipsoid microstructure localization of i) porphyrin signals of heme and ii) key elements of heme (Fe, N, and C) and mineral stabilizing hausmannite (Mn ₃ O ₄ ), respectively. These results demonstrate blood-derived biomolecules in mineralized red blood cells of a dinosaur.

Eukaryotes are theorized to have an origin associated with the archaeal phylum Promethearchaeota (formerly Asgard archaea) ^1–3 . The first cultured representatives revealed valuable insights ^4,5 but are still distantly related to eukaryotes, leaving many unknowns regarding eukaryogenesis. Here we report the isolation of two strains, HC1 and SC1, that belong to the archaeal lineage that branches closest to eukaryotes (Heimdallarchaeia); ^6–8 these isolates are members of a pure co-culture and a tri-culture with methanogenic partners. Both are obligately anaerobic, syntrophic, peptide-degrading and mesophilic archaea that produce protrusions and vesicles but lack persistent internal membrane structures, like Promethearchaeales ^4,5 . All Promethearchaeota isolates demonstrate behaviour focused on cell construction and membrane structures rather than division, in contrast to typical prokaryotes ⁹ . Strain HC1 possesses genes associated with aerobic lifestyles (such as cytochrome c oxidase (Cox)) but co-cultures cannot grow under (micro)oxic conditions. Meanwhile, the strain demonstrates growth under microoxic conditions when in the presence of aerobes, suggesting that Cox supports oxygen detoxification rather than respiration. Comparative genomics provides evidence that, like HC1, Cox-possessing Heimdallarchaeia studied to date probably do not respire oxygen. The wide conservation of anaerobiosis, cell structure without persistent intracellular membranes and membrane protrusions across Promethearchaeota suggest that the archaeal ancestor of eukaryotes may have had such physiological features. In this cultivation-informed scenario for eukaryogenesis, the archaea–eukaryote transition would involve a steep transition in cell structure and physiological shift from anaerobiosis to (facultative) aerobiosis ¹⁰ , with early interactions between the archaeal host and mitochondrial ancestor probably occurring under low-oxygen conditions and at least partially mediated by oxygen detoxification.

Coenzyme F430, a nickel-containing tetrapyrrole, serves as a cofactor for methyl-coenzyme M reductase (MCR), which catalyzes both the final step of methanogenesis and the initial step of anaerobic oxidation of methane (AOM). Due to its central role in these metabolic processes, coenzyme F430 can be used as a biomarker to investigate the distribution and activity of methanogens and anaerobic methane-oxidizing archaea (ANMEs) in various environments. In the northwestern Black Sea, chimney-like microbial mats dominated by a subcluster ANME-1 in the internal pink mat and ANME-2 in the outer black mat provide a sufficient quantity of coenzyme F430 for both stable carbon and nitrogen isotope analyses. In this study, we report the stable carbon and nitrogen isotopic compositions of coenzyme F430 and its derivatives from ANME-1 and ANME-2, as well as those of cell components, including bulk mat, amino acids, and membrane lipids. The carbon isotopic compositions of coenzyme F430 exhibit significant ¹³ C-depletion, reflecting the incorporation of methane-derived carbon. In contrast, coenzyme F430 from ANME-1 is enriched in ¹³ C and depleted in ¹⁵ N compared to that from ANME-2. These isotopic differences between ANME-1 and ANME-2 cannot be explained solely by biosynthetic isotope effects and likely reflect sequential methane and ammonium consumption within the microbial mat, which is influenced by surrounding seawater with low sulfate concentrations.

Eukaryotes are theorized to have originated from an archaeal phylum Promethearchaeota (formerly ‘Asgard’ archaea) ^1,2 . The first cultured representatives revealed valuable insight ^3,4 but are distantly related to the first eukaryotic common ancestor (FECA), leaving many unknowns regarding this archaeon’s biology. Here, we report isolation of two strains belonging to the order proposed as FECA’s closest relative, ‘Hodarchaeales’, ⁵ as members of a pure co- and tri-culture with methanogenic partners. Both are obligately anaerobic, syntrophic, peptide-degrading, and mesophilic archaea that have simple internal cell structure and produce protrusions and vesicles, like Promethearchaeales ^3,4 . All strains demonstrate behavior focused on cell construction rather than division, unlike typical prokaryotes ⁶ . Strain HC1 possesses genes associated with aerobic lifestyles but lacks complete pathways for aerobic respiration and co-cultures cannot grow under (micro)aerobic conditions, suggesting the genes support oxygen detoxification rather than respiration. Reflecting this, HC1 can survive and grow under microaerobic conditions only when aerobic organisms are present. Phylogenetic analyses indicate FECA may have possessed these genes and thus some aerotolerance. Physiological, genomic, and phylogenetic observations indicate FECA was a simple-celled anaerobic syntrophic peptide-degrading archaeon with a non-growth-centric lifestyle and potential adaptations towards an oxygenated planet—the archaea-eukaryote transition was steep in both cell structure and aerobiosis.

Heme B is an iron-coordinated porphyrin cofactor that facilitates essential biochemical reactions. As a major iron component in almost all life forms, the abundance of heme B in the ocean provides novel insights into iron biogeochemistry. In this study, we investigated the distribution of heme B in suspended particulate material collected from the surface mixed layer of the eastern Indian Ocean and the western North Pacific Ocean. Within the photic zone of the regions studied, particulate heme B concentrations ranged 1.24–8.39 pmol L⁻¹and were positively correlated with particulate organic carbon and chlorophyll a concentrations, consistent with the biologically ubiquitous nature of heme B. Profiles of heme B normalized to particulate organic carbon (heme B/POC) and chlorophyll a (heme B/chl a) revealed a complex response of the microbial heme B pool to environmental factors. In the eastern Indian Ocean, heme B/POC increased in response to enhanced iron bioavailability. Notably, a sharp increase in heme B/POC, up to 3.04 µmol mol⁻¹ in the Bay of Bengal, was attributed to the alleviation of iron stress due to substantial iron inputs from monsoonal aeolian dust and riverine sources. Conversely, heme B/POC as low as 0.88 µmol mol⁻¹ in the South Indian Ocean was consistent with the previous incubation experiments indicating iron limitation. In the western North Pacific Ocean, relatively low heme B/POC values in both the iron-limited subarctic and nitrogen-limited subtropical regions highlighted the influence of factors beyond iron bioavailability. In the subarctic region, an elevation in dissolved iron concentrations due to seasonal deepening of the surface mixed layer was counterbalanced by greater iron investment in photosynthetic proteins to acclimate to low light intensities. On the other hand, microbial communities in the subtropical western North Pacific Ocean were less likely to have experienced iron stress. However, a reduction in the intracellular abundance of heme B-containing photosynthetic proteins and nitrate reductase under nitrogen-limited conditions may have resulted in heme B/POC values comparable to those observed in the iron-limited regions. Based on our particulate heme B measurements, we estimated the global particulate biogenic iron pool in the surface ocean, which showed consistency with model-simulated estimates. This study highlights the utility of heme B as a valuable parameter for understanding iron biogeochemistry, which is critical for elucidating the links between marine iron and carbon cycles.

We developed a new method for stable carbon and nitrogen isotopic (δ¹³C and δ¹⁵N) analysis of underivatized amino acid (AA) enantiomers simultaneously, based on high-performance liquid chromatography (HPLC) separation and off-line isotopic measurement. l- and d-Enantiomers of each AA were isolated using a ReproSil Chiral-AA column, purified by wet chemical procedure, and analyzed for δ¹³C and δ¹⁵N values with a nanomol-scale elemental analyzer/isotope-ratio mass spectrometry (nano-EA/IRMS) system. We successfully achieved the separation of l- and d-enantiomers of 15 proteinogenous AAs, with all l-enantiomers eluting before respective d-enantiomers. The δ¹³C and δ¹⁵N values of AA enantiomers were consistent before and after HPLC separation, demonstrating that this analytical method conserves isotopic information. By coupling this column with a multidimensional HPLC system for isolating individual AAs, we analyzed l- and d-AAs in a natural sample, peptidoglycan isolated from Gram-positive bacterium Bacillus subtilis. Results show a surprisingly large ¹⁵N-depletion, up to 20‰, in d-glutamic acid relative to its l-counterpart. The first example, to our knowledge, of δ¹³C and δ¹⁵N analyses of underivatized AA enantiomers is expected to contribute to various research areas in the future.

Nitrogen isotope analysis of chloropigments provides information on the sources of nitrogenous nutrients assimilated by phytoplankton. The abundant, ubiquitous chlorophyll a records nitrogen isotopic compositions (δ¹⁵N) of eukaryotic phytoplankton and cyanobacteria, whereas more source‐specific chloropigments, such as the divinylchlorophylls exclusively possessed by the marine picocyanobacterium Prochlorococcus, can potentially resolve isotopic variability within the phytoplankton community. In this study, we analyzed the δ¹⁵N of both chlorophyll a and divinylchlorophyll a isolated from suspended particulate material collected at the subsurface chlorophyll maximum (SCM) along a meridional transect at 88°E in the oligotrophic eastern Indian Ocean. The nitrogen isotopic compositions of Prochlorococcus (δ¹⁵N_PRO) and the combined biomass of eukaryotic phytoplankton and Synechococcus (δ¹⁵N_EU+SYN) estimated from the δ¹⁵N of divinylchlorophyll a and chlorophyll a, respectively, revealed systematic variations that were not apparent from bulk isotope measurements. Whereas consistently low δ¹⁵N_PRO indicated reliance of Prochlorococcus on regenerated nitrogen throughout the transect, elevation in δ¹⁵N_EU+SYN values at several stations was interpreted to reflect assimilation of subsurface NO₃⁻ by eukaryotic phytoplankton. The δ¹⁵N distributions revealed subtle differences in NO₃⁻ availability at the SCM along the transect, which were consistently explained by the occurrence of mesoscale eddies in the Bay of Bengal, deepening of the mixed layer induced by a seasonal Wyrtki Jet in the equatorial region, and substantial deepening of the nutricline in the South Indian Ocean gyre. Our results highlight the utility of compound‐specific isotopic measurements of multiple species of chlorophylls in obtaining essential non‐incubation‐based biogeochemical constraints on the primary production in the ocean.

Heme B is an iron-coordinated tetrapyrrole molecule that acts as a cofactor in hemoproteins. It is expected to be ubiquitous in the environment, as b-type hemoproteins catalyze a variety of essential biochemical reactions. In this study, we developed an analytical method to quantify heme B in biological and environmental samples using high-performance liquid chromatog-raphy (HPLC) coupled to a photodiode array detector. The applicability of our method was further extended by the use of liquid chromatography/mass spectrometry (LC/MS; detection limit: ~1 fmol), which enabled the quantification of trace amount of dissolved heme B in filtered seawater, and sedimentary heme B coexisting with an abundant interfering organic matrix. For compound-specific carbon and nitrogen isotopic measurements, heme B was successfully isolated and purified from biological and environmental samples by a combination of anion-exchange column chromatography, methyl esterification, and dual-step HPLC. While carbon and nitrogen isotopic compositions of heme B in phototrophs were mostly comparable to those of chlorophyll a, heme B in suspended particulate materials in coastal water and an intertidal sediment was ¹³C-depleted and ¹⁵N-enriched relative to chlorophyll a, suggesting that non-phototrophic microorganisms are also significant source of heme B in natural environments.

Compound-specific isotope analysis of geoporphyrin, which are derivatives of chloropigments possessed by phototrophs, provide essential records of the biogeochemical cycle of past marine environments. Here, we evaluated uncertainties in carbon and nitrogen isotopic compositions (δ¹³C and δ¹⁵N) associated with high-performance liquid chromatography (HPLC) purifi-cation and isotopic measurements of geoporphyrins. Evaluation of total blank carbon and nitrogen for the HPLC and our sensitivity improved elemental analyzer/isotope ratio mass spectrometer (nano-EA/IRMS) analysis confirmed that blank car-bon can be corrected and that blank nitrogen is negligible compared to the mass of geoporphyrins required for the isotopic measurement. While geoporphyrins exhibited substantial in-peak carbon and nitrogen isotopic fractionations, no systematic changes in δ¹³C and δ¹⁵N values were observed during reversed- and normal phase HPLC isolation of Ni- and VO-porphyrin standards, with the changes in δ¹³C and δ¹⁵N values within ±0.6‰ and ±1.2‰ (2σ), respectively. These values are compara-ble to the instrumental precision of the nano-EA/IRMS system (±1.3‰ for 0.70 µgC and ±1.1‰ for 0.08 µgN, 2σ), con-firming that no substantial artifact in the δ¹³C and δ¹⁵N value would be expected during the reversed- and normal-phase HPLC purification. The sensitivity and precision of our method enable us to determine δ¹³C and δ¹⁵N of both major and minor geoporphyrins found in ancient sediments, which would provide detailed information on the photosynthetic primary producers and the carbon and nitrogen cycles in the past.