Fungal-induced fossil biomineralization

•Abundant hydroxyapatite nanofibers in 1- to 2-million-year-old fossil hyena coprolites•A biomineralization process is proposed for nanofiber formation and fossil preservation•A geoactive fungus could precipitate nanofibers similar to those found in coprolites•Fungal-mediated biomineralization of soft tissues is involved in fossilization Exceptional preservation of fossils has often been attributed to the actions of bacteria that aid in the preservation of soft tissues that normally decay rapidly. However, it is well known that fungi play a major role in organic matter decomposition, biogeochemical cycling of elements, and metal-mineral transformations in modern ecosystems. Although the fungal fossil record can be traced back over a billion years, there are only a few recorded examples of fungal roles in fossilization. In this research, we have carried out a detailed geobiological investigation on early Pleistocene hyena coprolites (fossilized dung) in an attempt to ascertain possible fungal involvement in their formation. Using an advanced microscopic and mineralogical approach, we found that numerous hydroxyapatite nanofibers (25–34 nm on average), interwoven to form spheroidal structures, constituted the matrix of the coprolites in addition to food remains. These structures were found to be extremely similar in texture and mineral composition to biominerals produced during laboratory culture of a common saprophytic and geoactive fungus, Aspergillus niger, in the presence of a solid source of calcium (Ca) and phosphorus (P). This observation, and our other data obtained, strongly suggests that fungal metabolism can provide a mechanism that can result in fossil biomineralization, and we hypothesize, therefore, that this may have contributed to the formation of well-preserved fossils (Lagerstätten) in the geological record. The characteristic polycrystalline nanofibers may also have served as a potential biosignature for fungal life in early Earth and extraterrestrial environments. Exceptional preservation of fossils has often been attributed to the actions of bacteria that aid in the preservation of soft tissues that normally decay rapidly. However, it is well known that fungi play a major role in organic matter decomposition, biogeochemical cycling of elements, and metal-mineral transformations in modern ecosystems. Although the fungal fossil record can be traced back over a billion years, there are only a few recorded examples of fungal roles in fossilization. In this research, we have carried out a detailed geobiological investigation on early Pleistocene hyena coprolites (fossilized dung) in an attempt to ascertain possible fungal involvement in their formation. Using an advanced microscopic and mineralogical approach, we found that numerous hydroxyapatite nanofibers (25–34 nm on average), interwoven to form spheroidal structures, constituted the matrix of the coprolites in addition to food remains. These structures were found to be extremely similar in texture and mineral composition to biominerals produced during laboratory culture of a common saprophytic and geoactive fungus, Aspergillus niger, in the presence of a solid source of calcium (Ca) and phosphorus (P). This observation, and our other data obtained, strongly suggests that fungal metabolism can provide a mechanism that can result in fossil biomineralization, and we hypothesize, therefore, that this may have contributed to the formation of well-preserved fossils (Lagerstätten) in the geological record. The characteristic polycrystalline nanofibers may also have served as a potential biosignature for fungal life in early Earth and extraterrestrial environments. Fungi can grow in almost any environment on Earth, including many extreme ecosystems where other organisms cannot survive.1Drake H. Ivarsson M. Bengtson S. Heim C. Siljeström S. Whitehouse M.J. Broman C. Belivanova V. Åström M.E. Anaerobic consortia of fungi and sulfate reducing bacteria in deep granite fractures.Nat. Commun. 2017; 8: 55Crossref PubMed Scopus (66) Google Scholar,2Drake H. Ivarsson M. Heim C. Snoeyenbos-West O. Bengtson S. Belivanova V. Whitehouse M. 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We therefore propose that fungi may have played a vital role in the fossilization of coprolites and are probably of significance in the fossilization of certain Lagerstätten through geological history. The studied coprolites were collected from the Tuozidong (TZD) Cave in the Tangshan area in Nanjing, South China. Abundant vertebrate fossil skeletons were found in this karst cave during the 1990s, showing a fauna (called the TZD fauna) dominated by Artiodactyla, Carnivora, and Perissodactyla.33Dong W. Liu J.Y. Fang Y.S. The large mammals from Tuozidong (eastern China) and the Early Pleistocene environmental availability for early human settlements.Quat. Int. 2013; 295: 73-82Crossref Scopus (13) Google Scholar,34Fang Y.S. Dong W. The Early Pleistocene Mammalian Fauna at Tuozi Cave, Nanjing, China. Science Press, 2007Google Scholar According to the composition of the TZD fauna and its correlation with the fauna of adjacent areas, an early Pleistocene age was concluded34Fang Y.S. Dong W. The Early Pleistocene Mammalian Fauna at Tuozi Cave, Nanjing, China. Science Press, 2007Google Scholar of 2.58–1.8 million years ago. Coprolites included in the same strata as the TZD fauna were therefore also dated as early Pleistocene. Coprolites were concentrated in a layered (<30 cm) deposit. A total of 128 coprolites were collected and examined for morphological features, with 54 samples by computed tomography (CT) scanning (Table S1). The coprolites show various morphologies, such as conical, short to long-oval shaped, rounded to drop-shaped forms (Figures 1 and S1). Most coprolites are complete and show no signs of abrasion on their surfaces. This suggests that the aggregated coprolites were not caused by transportation but preserved in situ, suggesting the gregarious behavior of their producers. The largest specimen size had a width of 40.1 mm and length up to 55.8 mm. Coprolites show a two-layered structure from cut slabs and X-ray micro-CT (Figures 1 and S1). The outer layer (1–2 mm in thickness) is light in color and dominated by hydroxyapatite with quartz and clay minerals as subordinate components. The inner dark part of the coprolite consists mainly of hydroxyapatite and subordinate calcite forming septarian veins filling pore areas (Figure S2). The mineral composition was determined by X-ray diffraction (XRD) analyses on powdered samples and in situ Raman spectrometry on the inner area of the coprolites (Figure 3; Table S1). The coprolites mainly comprise a hydroxyapatite matrix plus food remains. Food remains usually include fossil hairs and fragmented fossil bones, which commonly occur inside many coprolites (Figures 1C, 1E–1H, and S1). Fossil hairs were preserved as either grooved casts or filled tubes (Figures 1G and 1H). Hair scale pattern was poorly preserved. When preserved as filled tubes, the fossil hair had an elliptical shape in cross section and was composed of Ca phosphate (Figure 1H). Similar preservation forms of fossil hairs have been reported in Paleogene and Pleistocene coprolites.35Meng J. Wyss A.R. Multituberculate and other mammal hair recovered from Palaeogene excreta.Nature. 1997; 385: 712-714Crossref PubMed Scopus (56) Google Scholar,36Backwell L. Pickering R. Brothwell D. Berger L. Witcomb M. Martill D. Penkman K. Wilson A. Probable human hair found in a fossil hyaena coprolite from Gladysvale cave, South Africa.J. Archaeol. Sci. 2009; 36: 1269-1276Crossref Scopus (37) Google Scholar,37Chin K. Analyses of coprolites produced by carnivorous vertebrates.in: Kowalewski M. Kelley P.H. Predation in the Fossil Record. Cambridge University Press, 2002: 43-50Crossref Google Scholar The relatively large size and phosphatic composition of the coprolites suggests that they were produced by carnivores.38Zatoń M. Niedźwiedzki G. Marynowski L. Benzerara K. Pott C. Cosmidis J. Krzykawski T. Filipiak P. 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New Mexico Museum of Natural History and Science, 2012: 369-377Google Scholar Combined with the faunal composition of the TZD fauna and the evidence listed above, the coprolites were most likely produced by hyenas in the TZD fauna. Scanning electron microscopy (SEM) imaging showed that the coprolite matrix was composed of numerous tightly packed mineral spheroids (Figures 2A, 2B, S1, and S2). These spheroids either formed aggregates stacking together or were present as individuals that were adjacent to each other. Diameters of these spheroids ranged from 0.20 to 5.88 μm, with an average of 1.53 μm based on measurements of 1,220 individuals (Table S2). Further high-magnification examination of the spheroids showed that they were composed of numerous interwoven nanofibers that were fused together. In some regions, two spheroids were bridged by rod-like nanofiber aggregates (Figures 2B, 2C, S1, and S2). Individual nanofibers were rod-shaped and usually straight or slightly curved. The diameters of individual fibers were identical, and the surface of single nanofibers was granular to smooth (Figure 2C). Several rod-like nanofibers were aligned together, fusing to form larger anhedral rods or euhedral hexagonal rod crystals (Figure S2). The nanofibers possessed diameters ranging from 9.7 to 62.0 nm, based on measurements from various specimens (Tables S1 and S2), with averages ranging from 25.4 to 34.1 nm. The nanofibers showed maximum lengths of up to 1 μm. Cross-sections of the nanofibers were circular when observed from their terminal ends (Figures 2 and S2). Energy-dispersive X-ray spectroscopy (EDS) analyses showed that the spheroids were composed of Ca, P, and oxygen (O) (Figures 5C and S2). Nanofibers and their assembled spheroidal microstructures were found in numerous coprolite samples, suggesting that this was not an accidental phenomenon formed through post-depositional diagenetic alteration. The Raman spectrum of the coprolite matrix comprising spheroids exhibited a strong peak at 963.6 cm−1 over a scan from 200 to 1,200 cm−1 (Figure 3), which corresponds to the ν1 stretching vibration of P–O from PO43−.41Cosmidis J. Benzerara K. Guyot F. Skouri-Panet F. Duprat E. Férard C. Guigner J.M. Babonneau F. Coelho C. Calcium-phosphate biomineralization induced by alkaline phosphatase activity in Escherichia coli: localization, kinetics, and potential signatures in the fossil record.Front. Earth Sci. 2015; 3: 84Crossref Scopus (27) Google Scholar A strong fluorescent signal prevented band identification at 1,200 cm−1. Fourier transform infrared (FTIR) spectroscopy revealed strong peaks at 559.3 and 599.7 cm−1, which are interpreted to result from anti-symmetrical deformation of PO43−.42Rodrigues M.I.C. da Silva J.H. Santos F.E.P. Dentzien-Dias P. Cisneros J.C. de Menezes A.S. Freire P.T.C. Viana B.C. Physicochemical analysis of Permian coprolites from Brazil.Spectrochim. 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To test whether some fungal species have the potential to induce precipitation of hydroxyapatite minerals with a similar morphology and texture as those observed in the coprolites, we conducted incubation experiments using Aspergillus niger as the model fungus. We selected A. niger as it is a saprotrophic fungus that is widespread in the terrestrial environment, including soil, decaying organic matter, and animal dung.44Ganesh Kumar C.G. Mongolla P. Joseph J. Nageswar Y.V.D. Kamal A. Antimicrobial activity from the extracts of fungal isolates of soil and dung samples from Kaziranga National Park, Assam, India.J. Mycol. Med. 2010; 20: 283-289Crossref Scopus (19) Google Scholar In addition, the host environment of the hyena coprolites was a paleoarts cave, which is dark, cool, and humid, conditions that are ideal for many bacteria and fungi, including A. niger.45Zhang Z.F. Zhou S.Y. Eurwilaichitr L. Ingsriswang S. Raza M. Chen Q. Zhao P. Liu F. Cai L. Culturable mycobiota from Karst Caves in China II, with descriptions of 33 new species.Fungal Divers. 2021; 106: 29-136Crossref Scopus (36) Google Scholar A. niger also possesses significant geoactive properties and can mediate the biomineralization of a range of secondary minerals, including Ca carbonate, oxides, hydroxides, phosphates, and a series of metal carbonates and oxalates.17Gadd G.M. Raven J.A. Geomicrobiology of eukaryotic microorganisms.Geomicrobiol. J. 2010; 27: 491-519Crossref Scopus (80) Google Scholar,24Ferrier J. Csetenyi L. Gadd G.M. Selective fungal bioprecipitation of cobalt and nickel for multiple-product metal recovery.Microb. Biotechnol. 2021; 14: 1747-1756Crossref PubMed Scopus (3) Google Scholar,25Li Q. Liu F.X. Li M. Chen C.M. Gadd G.M. Nanoparticle and nanomineral production by fungi.Fungal Biol. Rev. 2022; 41: 31-44Crossref Scopus (10) Google Scholar,28Suyamud B. Ferrier J. Csetenyi L. Inthorn D. Gadd G.M. 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The most commonly occurring secondary mineral was Ca oxalate, which is characterized by its bipyramidal crystal polymorphs (Figure S3). The Ca oxalate crystals were scattered on or adjacent to fungal hyphae, either as single crystals or aggregates. EDS data showed that the elemental composition of these minerals comprised Ca, C, and O. In particular, the atomic ratio between Ca and C from EDS spectra was close to 1:2, confirming that these bipyramidal crystals were Ca oxalates. In addition, another needle-like mineral was also observed under SEM imaging (Figures 2D–2F and S4). These nanofiber precipitates were attached to, or enveloped by, the filamentous hyphae (Figure 2D). The diameters of these precipitated nanofibers ranged from 14.9 to 58.5 nm, with an average at 33.3 nm (based on 921 individual measurements; Table S2). Such a size range is very similar to that observed for nanofibers in the TZD coprolites. An independent samples t test was conducted to compare the means and distributions of nanofibers between the incubations and coprolites (Table S2). We found that of the seven examined coprolite samples, mean values of nanofibers from four were statistically similar to those obtained from fungal incubation experiments (Table S2). EDS spectra showed that these nanofibers consisted of Ca, O, and P (Figure 4F), which is consistent with the composition of nanofibers from the TZD coprolites (Figures 2C and 2F). In the incubation experiments using A. niger, we also observed nanofiber precipitation in association with Ca oxalate (Figure S5). Such an association excluded the possibility that the observed nanofibers were contaminants arising from coprolite substrates during the fungal solubilization process. Elemental composition through EDS analysis showed that these newly precipitated nanofibers in association with Ca oxalate were composed of Ca, P, and O (Figure 5F). The nature of the hydroxyapatite nanofibers within the TZD coprolites is enigmatic. Interestingly, Ca carbonate nanofibers of a similar morphology, but different mineralogy (Ca carbonate), have been commonly found in soil sediments and were interpreted to represent fossilizing bacteria.48Phillips S.E. Self P.G. Morphology, crystallography and origin of needle-fiber calcite in Quaternary pedogenic calcretes of South-Australia.Soil Res. 1987; 25: 429-444Crossref Scopus (100) Google Scholar,49Verrecchia E.P. Verrecchia K.E. Needle-fiber calcite: a critical review and a proposed classification.J. Sediment Res. 1994; 64: 650-664Google Scholar,50Loisy C. Verrecchia E.P. Dufour P. Microbial origin for pedogenic micrite associated with a carbonate paleosol (Champagne, France).Sediment. Geol. 1999; 126: 193-204Crossref Scopus (67) Google Scholar This conclusion was based on the morphological similarities between rod-shaped nanofibers and nano-bacteria. However, several lines of evidence argue against this interpretation. First, the diameters of nanofibers in the TZD coprolites (9.7–62.0 nm, with averages from 25.4 to 34.1 nm; Table S2) are clearly smaller than the lower size limit for nano-bacteria (200 nm).51Nealson K. Discussion.in: Steering G. In Size Limits of Very Small Microorganisms: Proceedings of a Workshop. National Academies Press, 1999: 39-42Google Scholar,52Nealson K.H. Stahl D.A. Microorganisms and biogeochemical cycles: what can we learn from layered microbial communities?.in: Banfield J.F. Nealson K.H. Volumn 35: Geomicrobiology, Interactions between Microbes and Minerals. Mineralogical Communications Society of America, 1997: 5-34Crossref Google Scholar,53Maniloff J. Nealson K.H. Psenner R. Loferer M. Folk R.L. Nannobacteria: size limits and evidence.Science. 1997; 276: 1773-1776Crossref Google Scholar On the other hand, the observed nanofibers were occasionally fused together to form wider rods. Individual nanofibers were also found to be interwoven, forming micron-sized, hexagonal hydroxyapatite cr