Comment on Franz et al. 2023: A reinterpretation of the 1.5 billion year old Volyn &lsquo;biota&rsquo; of Ukraine, and discussion of the evolution of the eukaryotes

Head, Martin J.; Riding, James B.; O’Keefe, Jennifer M. K.; Jeiter, Julius; Gravendyck, Julia

doi:https://doi.org/10.5194/egusphere-2023-2748

Preprints

https://doi.org/10.5194/egusphere-2023-2748

Preprints

29 Nov 2023

| 29 Nov 2023

Comment on Franz et al. 2023: A reinterpretation of the 1.5 billion year old Volyn ‘biota’ of Ukraine, and discussion of the evolution of the eukaryotes

Martin J. Head, James B. Riding, Jennifer M. K. O’Keefe, Julius Jeiter, and Julia Gravendyck

Abstract. Franz et. al. (Biogeosciences 20, 1901–1924, 2023) report a diverse and three-dimensionally preserved suite of mid-Proterozoic microfossils from miarolotic cavities within the granitic Volyn pegmatite field, a major granitic plutonic complex in NW Ukraine. The biota is dated at between ~1.76 Ga and ~1.5 Ga and includes fungus-like objects. This biota is reported as evidence of organisms living within the continental lithosphere, illuminating part of a ~1.8–0.8 billion year interval of the Proterozoic Eon characterised by relatively low climatic variability and slow biological evolution. We show that at least some of this putative diversity represents modern contamination including plant hairs, a distinctive pollen grain assignable to the extant conifer genus Pinus, and likely later fungal growth. Comparable diversity is shown to exist in modern museum dust, this calling into question whether any part of the Volyn ‘biota’ is biological in origin while emphasising the need for scrupulous care in collecting, analysing, and identifying Precambrian microfossils.

Received: 19 Nov 2023 – Discussion started: 29 Nov 2023

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09 Apr 2024

Comment on “The Volyn biota (Ukraine) – indications of 1.5 Gyr old eukaryotes in 3D preservation, a spotlight on the `boring billion' ” by Franz et al. (2023)

Martin J. Head, James B. Riding, Jennifer M. K. O'Keefe, Julius Jeiter, and Julia Gravendyck

Biogeosciences, 21, 1773–1783, https://doi.org/10.5194/bg-21-1773-2024,https://doi.org/10.5194/bg-21-1773-2024, 2024

Short summary

Martin J. Head, James B. Riding, Jennifer M. K. O’Keefe, Julius Jeiter, and Julia Gravendyck

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2023-2748', Anonymous Referee #1, 02 Dec 2023

Head et al. present a timely cautionary note regarding Franz and colleagues’ report of eukaryotic fossils preserved within 1.5-1.7 billion year old pegmatite. As Head et al. make clear, the fact that microorganisms occur in cracks, vesicles or other spaces spaces within 1.5 billion year old rocks does not mean that the microbes themselves are 1.5 billion years old. As studies of Earth’s deep biosphere document, living microorganisms can be found today in many subterranean environments, including igneous and metamorphic substrates. We can debate questions of fungal antiquity, but Head et al. are unambiguously correct that the reported microstrcutures include vascular plant hairs and pine pollen, which must be much younger than the rocks in which they occur. The filaments with apparent sporangia are probably fungi, but as they are very likely the same age as the plant materials, they, too, are almost certainly much younger than the pegmatites.
Head et al. helpfully place the reported fossils within a broader physical and biological context. Whether the assembly of Rodinia records the onset of modern plate tectonics is hotly debated; recent papers by Chris Spencer may provide a more nuanced sense of the tectonic state of the mid-Proterozoic Earth. Also, note in line 66 that Gibson et al. dated the oldest known red algae and not the advent of photosynthesis within eukaryotes, which must have evolved earlier in stem group archaeplastids. The fossil record of fungi themselves is fraught. Loron et al. discovered interesting filaments in billion year old rocks, but the presence of sporangia at the termini of filaments does not make the fossils unambiguously fungal. (Neither does the inferred wall chemistry.) They could be, but calling them “possible” fungi would be recommended. And Head et al should note that Bengtson et al.’s report of mycelia in 2.4 billion year old basalt vesicles is subject to the same issues that surround Franz et al.’s structures. It might be noted that no molecular clock for fungal evolution predicts anything close to 1.5 billion year old fungi. While one might argue that the fungal fossil record is poorly sampled and may therefore yield erroneously young estimates of divergence time, molecular clocks for animals – close relatives of fungi – also hover around 700-800 million years, consistent, at least, with published clocks for fungi.
These are all details, but Head et al.’s identification of vascular plant remains in the Volyn rocks unambiguously shows that Volyn pegmatites contains biological materials much younger than the rocks themselves. The hypothesis that Volyn structures could have been emplaced as dust permeating museum drawers is both reasonable and testable. The description of what turn out to be modern remains in ancient rocks was common in the early days of Precambrian paleontology, but is relatively rare today. Nonetheless, Head et al. provide a timely reminder that care is always advisable, especially when reporting stratigraphically unexpected remains from geologically unusual substrates.

Citation: https://doi.org/10.5194/egusphere-2023-2748-RC1
- CC2: 'Reply on RC1', Gerhard Franz, 14 Dec 2023
  
  I have a question to your comment on molecular clocks (this field is absolutely new to me, I have no experience and can only quote what I read about this subject!).
  You wrote “It might be noted that no molecular clock for fungal evolution predicts anything close to 1.5 billion year old fungi.” We quoted Wang et al. (1999) for an age of 1.58±9 Ga (type-o in our paper, Ma instead of Ga! But the text makes it clear that it should have been Ga) and Krings et al. (2013) with 1.2-1.4 Gyr ago. Berbee et al. (2020) placed the origin of fungi at 1.3 Ga. What is wrong with these data?
  
  Citation: https://doi.org/10.5194/egusphere-2023-2748-CC2
- AC1: 'Reply on RC1', Julia Gravendyck, 02 Feb 2024
  
  We are most grateful to the anonymous reviewer (R1) for their constructive remarks.
  
  We note that R1, together with all reviewers, is supportive of our publication.
  This reviewer agrees unambiguously with our claim that the pine pollen and plant hairs are much later contaminants. Providing a more nuanced view of Rodinia assembly and mid-Proterozoic plate tectonics, as suggested by R1, supports our concerns about the Volyn “biota” and we will follow R1’s recommendation to include reference to articles by C.J. Spencer including Spencer et al. (2021).
  R1 does pick up our error in stating that “Gibson et al. dated the oldest known red algae and not the advent of photosynthesis within eukaryotes, which must have evolved earlier in stem group archaeplastids”. We agree and can replace the following text:
  “Biologically the interval is significant because eukaryotes had evolved by at least 1.7 Ga (Javaux and Lepot, 2018; Miao et al., 2019) and questionably as far back as 2.4 Ga (Barlow et al., 2023), with the appearance of photosynthesizing eukaryotes estimated at 1.25 Ga (Gibson et al., 2018).”
  With:
  “Biologically the interval is significant because eukaryotes had evolved by at least 1.7 Ga (Javaux and Lepot, 2018; Miao et al., 2019) and questionably as far back as 2.4 Ga (Barlow et al., 2023), with the appearance of photosynthesizing eukaryotes dating at least to ~2.4 Ga (Buick, 2008) and fungi between 887 and 816 Ma (Chang et al., 2015; Berbee et al., 2020).”
  We accept R1’s advice in considering the fossils described by Loron et al. (2019) as “possible fungi” for the reasons given, and will be more circumspect regarding the Bengtson et al. (2017) report of mycelia in 2.4 billion year old basalt vesicles. R1 makes a very good point about molecular clocks for animals, and we will incorporate this in our comment, as follows:
  “We note that molecular clocks for fungal evolution do not predict an age close to 1.5 billion years (Berbee et al., 2020). It might be claimed that the fossil record of fungi is poorly sampled, yielding erroneously young estimates of divergence time. However, molecular clocks for animals, which are close relatives of fungi, also approximate 700–800 million years, largely consistent with published clocks for fungi.”
  R1 endorses our call for greater care when dealing with very old “fossils” in unlikely geological settings, but misunderstands the purpose of our study of museum dust. We are not necessarily suggesting that museum dust is responsible for the contamination, but more broadly that contamination occurring anywhere and at any step in the process (we happened to choose museum dust) must be considered carefully before claims of ancient biotas are made. We will adjust our text if necessary to make this point entirely clear. Finally, we want to thank Reviewer 1 again for very constructive comments.
  
  Citation: https://doi.org/10.5194/egusphere-2023-2748-AC1
CC1:
'Comment on egusphere-2023-2748', Gerhard Franz, 13 Dec 2023

Head et al. question the interpretation that the Volyn biota are an example of Precambrian fossils. Instead they claim that the investigated samples were contaminated by recent organisms what they described as “museum dust”. We appreciate their comment, because this question of contamination was not raised before, neither in our papers from 2017, 2022, and 2023, nor in any of the previous publications about kerite who either described kerite as an a-biogenic material (Ginzburg et al., 1987; Luk’yanova et al., 1992; Yushkin 1996, 1998) or as fossilized cyanobacteria (Gorlenko et al., 2000; Zhmur, 2003).
We will prepare a detailed reply to this comment. Here, we just want to mention that the only sample, which could potentially have been contaminated in a museum, is our sample kerite 0. This sample, however, shows only filamentous kerite, and if this should be a contamination, then the whole sample is contamination. This makes no sense, because it was sampled as kerite and was stored in the museum as such. All other samples kerite 1 to kerite 7 were sampled underground, put into firmly closed plastic sample bags (double ones with label in outer one), transported first to Luxemburg and then sent to Berlin. They were opened only in the electron microscopy laboratory of TU Berlin, which is a special building for electron microscopy with the appropriate arrangements to prevent contamination by dust. All rooms are equipped with airlocks for climatization and in addition water-cooled ceilings minimizes airstream and dust movement in the rooms. Samples were preparation in an exhaust hood. Of course, we cannot completely rule out that some objects are contaminants, but the overwhelming majority of objects on the aluminum sample holders are original as recovered from underground.
Meanwhile, we would be happy to read more comments from the scientific community on this subject. As Head et al. pointed out, the occurrence of these fossils is of prime importance for the evolution history in the Precambrian, and therefore any doubts should be ruled out.

Citation: https://doi.org/10.5194/egusphere-2023-2748-CC1
- AC4: 'Reply on CC1', Julia Gravendyck, 02 Feb 2024
  
  We are most grateful to Gerhard Franz for his response to our comment (Head et al.) in which we question the biogenicity of the Volyn “biota” and provide illustrations of modern museum dust that point to contamination of the material presented by Franz et al. (2023).
  Our concerns are echoed by the three reviewers who find our identification of bisaccate pollen and plant trichomes in the presented Volyn “biota” to be compelling evidence of modern contamination. Reviewer 2 agrees that micro-FTIR does not provide proof of ancient fungal affinity, although we do not rule out later fungal contamination. We will revise our comment to incorporate the many helpful suggestions made by all three reviewers.
  We will clarify a point that seems to have been misunderstood by Gerhard Franz. Our study of museum dust is not to suggest that contamination has come from a museum. We are showing how airborne contamination is pervasive. It can affect any sample at any stage: before, during or after collection unless special care is taken. Even then, since sterile conditions can never be achieved, all objects reported should be carefully scrutinized before extraordinary claims are made, such as the presence of a diverse “biota” dated to ~1.5 billion years. Our study prompts new questions about the reported biogenicity of the entire Volyn “biota”, including the kerite itself, as reviewer 3 (Prof. Butterfield) eloquently states. We hope our comment will then serve as a cautionary note to those reporting extremely old putative biotas, especially when accepted molecular clocks are challenged, and that it will encourage critical future research on the Volyn deposits.
  
  Citation: https://doi.org/10.5194/egusphere-2023-2748-AC4
RC2:
'Comment on egusphere-2023-2748', Anonymous Referee #2, 13 Dec 2023

The comment by Head and colleagues presents a welcome reminder of the challenges of analysing early traces of life in rocks (syngenicity and biogenicity). The comment is well structured, clear to read and highlights the main issues arising from Franz et al 2023 study. I concur that the illustrated objects are unambiguous modern contaminants.
The section on FTIR spectroscopy is very pertinent. Possibly, the authors could emphase more on the fact that the reported bands (CH stretching and bending for example), are very general organic band that could not by themselves constitute a strong reference point. The general aspect of the original spectra is indeed very characteristic of spectra for bitumen, kerogen or mature sedimentary organic carbon in general. The high ratio between CH3 and CH2, the intense and broad absorption around 1660 cm-1, and the hydrothermal influence makes me think that this organic material is quite degraded, with short chain aliphatic and strong aromatic contribution.
I recommend this comment for publication.

Citation: https://doi.org/10.5194/egusphere-2023-2748-RC2
- AC2: 'Reply on RC2', Julia Gravendyck, 02 Feb 2024
  
  We are most grateful to the anonymous reviewer (R2) for their constructive remarks.
  Reviewer 2 also agrees unreservedly with our claim that at least some of the objects presented by Franz et al. (2023) represent modern contamination. R2 finds our discussion of FTIR spectroscopy to be very pertinent, and agrees that the spectra presented by Franz et al. (2023) in being characteristic of bitumen, kerogen or mature sedimentary organic carbon in general, should not therefore be used as evidence for fungal affinity. We will expand our discussion to take account of R2’s comments on FTIR spectroscopy, and propose the following addition (shown in bold) to Line 123:
  “Given this variation (Fig. 2), and that CH stretching and bending observed in the Volyn biota spectra are common in many kinds of thermally mature organic matter, including solid bitumen and vitrinite, and in settings impacted by intrusions and hydrothermal alteration (Lis et al., 2005; Presswood et al., 2016; Liu et al., 2019; Teng et al., 2020), micro-FTIR results should not be used as stand-alone evidence for the presence of fungi.”
  
  Citation: https://doi.org/10.5194/egusphere-2023-2748-AC2
RC3:
'Comment on egusphere-2023-2748', Nicholas J. Butterfield, 22 Dec 2023

Reports of well preserved, early Mesoproterozoic eukaryotes in pegmatite cavities from the Volyn region of Ukraine (Franz et al. 2023) are extraordinary, and demand a corresponding level of interrogation – for biogenicity, eukaryoticity, and of course syngenicity with the host deposit. Head et al. take an important first pass at scrutinizing these putative microfossils, drawing immediate attention to a specimen that looks very much like a bisaccate pollen grain. The comparison is compelling and legitimately introduces the likelihood of modern contamination. It’s possible, of course, that the similarity is entirely accidental – a pareidolia – but difficult to maintain alongside other constituents that look very much like modern plant trichomes and other airborne debris. In this context, there is little point in debating the nature of associated geochemical signatures or broader phylogenetic affiliations. The first order of business is identifying which, if any, of these structures is of Proterozoic age?
The host material is a fibrous bituminous-kerogenous ‘kerite’ deposit that was emplaced at some point prior to ~1500 Ma. Importantly, its dark/opaque appearance under white light (Franz et al. 2023, fig. 2a-c) clearly distinguishes its constituent fibres from ‘museum dust.’ That’s not to suggest that all of the recoverable fibres are Proterozoic – any more than filamentous microbes in the Nakhla meteorite necessarily come from Mars (cf., Toporski & Steele 2007) – but there are clearly indigenous structures in the Volyn kerite that warrant consideration. At the same time, it’s worth appreciating the likely biological origin of the carbon from which the kerite was formed. Whatever its particular mode of emplacement, this will almost certainly be transmitting inherited biogeochemical signatures. The real question then, is whether any of the demonstrably indigenous Volyn structures are demonstrably biogenic in terms of their current morphology?
Branching fibrous structures recovered from the Volyn kerite share a number of features with modern filamentous fungi (Franz et al. 2023, figs. 3a-i, 4-6), but none that are diagnostically fungal. At a more fundamental level, these filaments also fail to satisfy one of the key criteria for assessing first-order biogenicity – namely a hollow constitution that convincingly mirrors the distinction between a structural, carbon-rich, cell wall and its essentially water-filled lumen (Buick 1990). Early diagenetic mineralization commonly prevents microfossil collapse, but it remains to be seen how such a process could produce the massive kerogenous interiors of the Volyn fibres. The more likely origin of such structures is through abiological processing, associated with the emplacement and hydrothermal alteration of locally ponded bitumen. To be fair, Franz et al (2023, p. 1920) do address the possibility of abiological biomorphs, but with no serious interrogation of any carbon-based ‘chemical garden’ models (cf., Lukjanova & Lovzova 1994, McMahon 2019).

Buick, R. 1990. Microfossil recognition in Archean rocks: an appraisal of spheroids and filaments from a 3500 m.y. old chert-barite unit at North Pole, Western Australia. Palaios 5, 441–459.
Franz, G., Khomenko, V., Lyckberg, P., Chournousenko, V., Struck, U., Gernert, U. & Nissen, J. 2023. The Volyn biota (Ukraine) – indications of 1.5 Gyr old eukaryotes in 3D preservation, a spotlight on the “boring billion”. Biogeosciences 20, 1901–1924.
Lukjanova, V.T. & Lovzova, R.V. 1994. Carbon fibers in nature. Carbon 32, 777–783.
McMahon, S. 2019. Earth’s earliest and deepest purported fossils may be iron-mineralized chemical gardens. Proceedings of the Royal Society B, 286, 20192410.
Toporski, J. & Steele, A. 2007. Observations from a 4-year contamination study of a sample depth profile through martian meteorite Nakhla. Astrobiology 7, 389–401.

Citation: https://doi.org/10.5194/egusphere-2023-2748-RC3
- AC3: 'Reply on RC3', Julia Gravendyck, 02 Feb 2024
  
  Prof. Butterfield joins reviewers 1 and 2 in endorsing our concerns that the reported Volyn “biota” has been compromised by contamination. However, we are not suggesting that the fibres in our museum dust are analogues for the filaments of “kerite” as reported by Franz et al. (2023) and we will make this clear in our revised MS. Rather, museum dust as a whole presents a picture of the variety of airborne objects that potentially contaminate material such as the Volyn “biota” – it has significance beyond the specifics of the Franz et al. (2023) article. We did not address the “kerite” fibres directly other than raise the concern that perhaps none of the Volyn “biota” is of biological origin. Prof. Butterfield supports this concern, and we will expand our discussion to include the obvious inconsistency that the “kerite” filaments have no observed internal structure that would support a biological origin. We will add that without evidence for biogenicity, abiological processes including carbon-based ‘chemical garden’ models (Lukjanova and Lovzova, 1994; McMahon, 2019) should be given serious consideration. Prof. Butterfield accepts our claims of modern contaminating pine pollen and plant trichomes as compelling. Our claim is based on having personally seen thousands of such pine pollen and plant trichomes in modern preparations.
  We are especially grateful to Nicholas J. Butterworth, as R3, for his own detailed perspectives that give further weight to our concerns about the biogenicity of the Volyn “biota”.
  
  Citation: https://doi.org/10.5194/egusphere-2023-2748-AC3

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2023-2748', Anonymous Referee #1, 02 Dec 2023

Head et al. present a timely cautionary note regarding Franz and colleagues’ report of eukaryotic fossils preserved within 1.5-1.7 billion year old pegmatite. As Head et al. make clear, the fact that microorganisms occur in cracks, vesicles or other spaces spaces within 1.5 billion year old rocks does not mean that the microbes themselves are 1.5 billion years old. As studies of Earth’s deep biosphere document, living microorganisms can be found today in many subterranean environments, including igneous and metamorphic substrates. We can debate questions of fungal antiquity, but Head et al. are unambiguously correct that the reported microstrcutures include vascular plant hairs and pine pollen, which must be much younger than the rocks in which they occur. The filaments with apparent sporangia are probably fungi, but as they are very likely the same age as the plant materials, they, too, are almost certainly much younger than the pegmatites.
Head et al. helpfully place the reported fossils within a broader physical and biological context. Whether the assembly of Rodinia records the onset of modern plate tectonics is hotly debated; recent papers by Chris Spencer may provide a more nuanced sense of the tectonic state of the mid-Proterozoic Earth. Also, note in line 66 that Gibson et al. dated the oldest known red algae and not the advent of photosynthesis within eukaryotes, which must have evolved earlier in stem group archaeplastids. The fossil record of fungi themselves is fraught. Loron et al. discovered interesting filaments in billion year old rocks, but the presence of sporangia at the termini of filaments does not make the fossils unambiguously fungal. (Neither does the inferred wall chemistry.) They could be, but calling them “possible” fungi would be recommended. And Head et al should note that Bengtson et al.’s report of mycelia in 2.4 billion year old basalt vesicles is subject to the same issues that surround Franz et al.’s structures. It might be noted that no molecular clock for fungal evolution predicts anything close to 1.5 billion year old fungi. While one might argue that the fungal fossil record is poorly sampled and may therefore yield erroneously young estimates of divergence time, molecular clocks for animals – close relatives of fungi – also hover around 700-800 million years, consistent, at least, with published clocks for fungi.
These are all details, but Head et al.’s identification of vascular plant remains in the Volyn rocks unambiguously shows that Volyn pegmatites contains biological materials much younger than the rocks themselves. The hypothesis that Volyn structures could have been emplaced as dust permeating museum drawers is both reasonable and testable. The description of what turn out to be modern remains in ancient rocks was common in the early days of Precambrian paleontology, but is relatively rare today. Nonetheless, Head et al. provide a timely reminder that care is always advisable, especially when reporting stratigraphically unexpected remains from geologically unusual substrates.

Citation: https://doi.org/10.5194/egusphere-2023-2748-RC1
- CC2: 'Reply on RC1', Gerhard Franz, 14 Dec 2023
  
  I have a question to your comment on molecular clocks (this field is absolutely new to me, I have no experience and can only quote what I read about this subject!).
  You wrote “It might be noted that no molecular clock for fungal evolution predicts anything close to 1.5 billion year old fungi.” We quoted Wang et al. (1999) for an age of 1.58±9 Ga (type-o in our paper, Ma instead of Ga! But the text makes it clear that it should have been Ga) and Krings et al. (2013) with 1.2-1.4 Gyr ago. Berbee et al. (2020) placed the origin of fungi at 1.3 Ga. What is wrong with these data?
  
  Citation: https://doi.org/10.5194/egusphere-2023-2748-CC2
- AC1: 'Reply on RC1', Julia Gravendyck, 02 Feb 2024
  
  We are most grateful to the anonymous reviewer (R1) for their constructive remarks.
  
  We note that R1, together with all reviewers, is supportive of our publication.
  This reviewer agrees unambiguously with our claim that the pine pollen and plant hairs are much later contaminants. Providing a more nuanced view of Rodinia assembly and mid-Proterozoic plate tectonics, as suggested by R1, supports our concerns about the Volyn “biota” and we will follow R1’s recommendation to include reference to articles by C.J. Spencer including Spencer et al. (2021).
  R1 does pick up our error in stating that “Gibson et al. dated the oldest known red algae and not the advent of photosynthesis within eukaryotes, which must have evolved earlier in stem group archaeplastids”. We agree and can replace the following text:
  “Biologically the interval is significant because eukaryotes had evolved by at least 1.7 Ga (Javaux and Lepot, 2018; Miao et al., 2019) and questionably as far back as 2.4 Ga (Barlow et al., 2023), with the appearance of photosynthesizing eukaryotes estimated at 1.25 Ga (Gibson et al., 2018).”
  With:
  “Biologically the interval is significant because eukaryotes had evolved by at least 1.7 Ga (Javaux and Lepot, 2018; Miao et al., 2019) and questionably as far back as 2.4 Ga (Barlow et al., 2023), with the appearance of photosynthesizing eukaryotes dating at least to ~2.4 Ga (Buick, 2008) and fungi between 887 and 816 Ma (Chang et al., 2015; Berbee et al., 2020).”
  We accept R1’s advice in considering the fossils described by Loron et al. (2019) as “possible fungi” for the reasons given, and will be more circumspect regarding the Bengtson et al. (2017) report of mycelia in 2.4 billion year old basalt vesicles. R1 makes a very good point about molecular clocks for animals, and we will incorporate this in our comment, as follows:
  “We note that molecular clocks for fungal evolution do not predict an age close to 1.5 billion years (Berbee et al., 2020). It might be claimed that the fossil record of fungi is poorly sampled, yielding erroneously young estimates of divergence time. However, molecular clocks for animals, which are close relatives of fungi, also approximate 700–800 million years, largely consistent with published clocks for fungi.”
  R1 endorses our call for greater care when dealing with very old “fossils” in unlikely geological settings, but misunderstands the purpose of our study of museum dust. We are not necessarily suggesting that museum dust is responsible for the contamination, but more broadly that contamination occurring anywhere and at any step in the process (we happened to choose museum dust) must be considered carefully before claims of ancient biotas are made. We will adjust our text if necessary to make this point entirely clear. Finally, we want to thank Reviewer 1 again for very constructive comments.
  
  Citation: https://doi.org/10.5194/egusphere-2023-2748-AC1
CC1:
'Comment on egusphere-2023-2748', Gerhard Franz, 13 Dec 2023

Head et al. question the interpretation that the Volyn biota are an example of Precambrian fossils. Instead they claim that the investigated samples were contaminated by recent organisms what they described as “museum dust”. We appreciate their comment, because this question of contamination was not raised before, neither in our papers from 2017, 2022, and 2023, nor in any of the previous publications about kerite who either described kerite as an a-biogenic material (Ginzburg et al., 1987; Luk’yanova et al., 1992; Yushkin 1996, 1998) or as fossilized cyanobacteria (Gorlenko et al., 2000; Zhmur, 2003).
We will prepare a detailed reply to this comment. Here, we just want to mention that the only sample, which could potentially have been contaminated in a museum, is our sample kerite 0. This sample, however, shows only filamentous kerite, and if this should be a contamination, then the whole sample is contamination. This makes no sense, because it was sampled as kerite and was stored in the museum as such. All other samples kerite 1 to kerite 7 were sampled underground, put into firmly closed plastic sample bags (double ones with label in outer one), transported first to Luxemburg and then sent to Berlin. They were opened only in the electron microscopy laboratory of TU Berlin, which is a special building for electron microscopy with the appropriate arrangements to prevent contamination by dust. All rooms are equipped with airlocks for climatization and in addition water-cooled ceilings minimizes airstream and dust movement in the rooms. Samples were preparation in an exhaust hood. Of course, we cannot completely rule out that some objects are contaminants, but the overwhelming majority of objects on the aluminum sample holders are original as recovered from underground.
Meanwhile, we would be happy to read more comments from the scientific community on this subject. As Head et al. pointed out, the occurrence of these fossils is of prime importance for the evolution history in the Precambrian, and therefore any doubts should be ruled out.

Citation: https://doi.org/10.5194/egusphere-2023-2748-CC1
- AC4: 'Reply on CC1', Julia Gravendyck, 02 Feb 2024
  
  We are most grateful to Gerhard Franz for his response to our comment (Head et al.) in which we question the biogenicity of the Volyn “biota” and provide illustrations of modern museum dust that point to contamination of the material presented by Franz et al. (2023).
  Our concerns are echoed by the three reviewers who find our identification of bisaccate pollen and plant trichomes in the presented Volyn “biota” to be compelling evidence of modern contamination. Reviewer 2 agrees that micro-FTIR does not provide proof of ancient fungal affinity, although we do not rule out later fungal contamination. We will revise our comment to incorporate the many helpful suggestions made by all three reviewers.
  We will clarify a point that seems to have been misunderstood by Gerhard Franz. Our study of museum dust is not to suggest that contamination has come from a museum. We are showing how airborne contamination is pervasive. It can affect any sample at any stage: before, during or after collection unless special care is taken. Even then, since sterile conditions can never be achieved, all objects reported should be carefully scrutinized before extraordinary claims are made, such as the presence of a diverse “biota” dated to ~1.5 billion years. Our study prompts new questions about the reported biogenicity of the entire Volyn “biota”, including the kerite itself, as reviewer 3 (Prof. Butterfield) eloquently states. We hope our comment will then serve as a cautionary note to those reporting extremely old putative biotas, especially when accepted molecular clocks are challenged, and that it will encourage critical future research on the Volyn deposits.
  
  Citation: https://doi.org/10.5194/egusphere-2023-2748-AC4
RC2:
'Comment on egusphere-2023-2748', Anonymous Referee #2, 13 Dec 2023

The comment by Head and colleagues presents a welcome reminder of the challenges of analysing early traces of life in rocks (syngenicity and biogenicity). The comment is well structured, clear to read and highlights the main issues arising from Franz et al 2023 study. I concur that the illustrated objects are unambiguous modern contaminants.
The section on FTIR spectroscopy is very pertinent. Possibly, the authors could emphase more on the fact that the reported bands (CH stretching and bending for example), are very general organic band that could not by themselves constitute a strong reference point. The general aspect of the original spectra is indeed very characteristic of spectra for bitumen, kerogen or mature sedimentary organic carbon in general. The high ratio between CH3 and CH2, the intense and broad absorption around 1660 cm-1, and the hydrothermal influence makes me think that this organic material is quite degraded, with short chain aliphatic and strong aromatic contribution.
I recommend this comment for publication.

Citation: https://doi.org/10.5194/egusphere-2023-2748-RC2
- AC2: 'Reply on RC2', Julia Gravendyck, 02 Feb 2024
  
  We are most grateful to the anonymous reviewer (R2) for their constructive remarks.
  Reviewer 2 also agrees unreservedly with our claim that at least some of the objects presented by Franz et al. (2023) represent modern contamination. R2 finds our discussion of FTIR spectroscopy to be very pertinent, and agrees that the spectra presented by Franz et al. (2023) in being characteristic of bitumen, kerogen or mature sedimentary organic carbon in general, should not therefore be used as evidence for fungal affinity. We will expand our discussion to take account of R2’s comments on FTIR spectroscopy, and propose the following addition (shown in bold) to Line 123:
  “Given this variation (Fig. 2), and that CH stretching and bending observed in the Volyn biota spectra are common in many kinds of thermally mature organic matter, including solid bitumen and vitrinite, and in settings impacted by intrusions and hydrothermal alteration (Lis et al., 2005; Presswood et al., 2016; Liu et al., 2019; Teng et al., 2020), micro-FTIR results should not be used as stand-alone evidence for the presence of fungi.”
  
  Citation: https://doi.org/10.5194/egusphere-2023-2748-AC2
RC3:
'Comment on egusphere-2023-2748', Nicholas J. Butterfield, 22 Dec 2023

Reports of well preserved, early Mesoproterozoic eukaryotes in pegmatite cavities from the Volyn region of Ukraine (Franz et al. 2023) are extraordinary, and demand a corresponding level of interrogation – for biogenicity, eukaryoticity, and of course syngenicity with the host deposit. Head et al. take an important first pass at scrutinizing these putative microfossils, drawing immediate attention to a specimen that looks very much like a bisaccate pollen grain. The comparison is compelling and legitimately introduces the likelihood of modern contamination. It’s possible, of course, that the similarity is entirely accidental – a pareidolia – but difficult to maintain alongside other constituents that look very much like modern plant trichomes and other airborne debris. In this context, there is little point in debating the nature of associated geochemical signatures or broader phylogenetic affiliations. The first order of business is identifying which, if any, of these structures is of Proterozoic age?
The host material is a fibrous bituminous-kerogenous ‘kerite’ deposit that was emplaced at some point prior to ~1500 Ma. Importantly, its dark/opaque appearance under white light (Franz et al. 2023, fig. 2a-c) clearly distinguishes its constituent fibres from ‘museum dust.’ That’s not to suggest that all of the recoverable fibres are Proterozoic – any more than filamentous microbes in the Nakhla meteorite necessarily come from Mars (cf., Toporski & Steele 2007) – but there are clearly indigenous structures in the Volyn kerite that warrant consideration. At the same time, it’s worth appreciating the likely biological origin of the carbon from which the kerite was formed. Whatever its particular mode of emplacement, this will almost certainly be transmitting inherited biogeochemical signatures. The real question then, is whether any of the demonstrably indigenous Volyn structures are demonstrably biogenic in terms of their current morphology?
Branching fibrous structures recovered from the Volyn kerite share a number of features with modern filamentous fungi (Franz et al. 2023, figs. 3a-i, 4-6), but none that are diagnostically fungal. At a more fundamental level, these filaments also fail to satisfy one of the key criteria for assessing first-order biogenicity – namely a hollow constitution that convincingly mirrors the distinction between a structural, carbon-rich, cell wall and its essentially water-filled lumen (Buick 1990). Early diagenetic mineralization commonly prevents microfossil collapse, but it remains to be seen how such a process could produce the massive kerogenous interiors of the Volyn fibres. The more likely origin of such structures is through abiological processing, associated with the emplacement and hydrothermal alteration of locally ponded bitumen. To be fair, Franz et al (2023, p. 1920) do address the possibility of abiological biomorphs, but with no serious interrogation of any carbon-based ‘chemical garden’ models (cf., Lukjanova & Lovzova 1994, McMahon 2019).

Buick, R. 1990. Microfossil recognition in Archean rocks: an appraisal of spheroids and filaments from a 3500 m.y. old chert-barite unit at North Pole, Western Australia. Palaios 5, 441–459.
Franz, G., Khomenko, V., Lyckberg, P., Chournousenko, V., Struck, U., Gernert, U. & Nissen, J. 2023. The Volyn biota (Ukraine) – indications of 1.5 Gyr old eukaryotes in 3D preservation, a spotlight on the “boring billion”. Biogeosciences 20, 1901–1924.
Lukjanova, V.T. & Lovzova, R.V. 1994. Carbon fibers in nature. Carbon 32, 777–783.
McMahon, S. 2019. Earth’s earliest and deepest purported fossils may be iron-mineralized chemical gardens. Proceedings of the Royal Society B, 286, 20192410.
Toporski, J. & Steele, A. 2007. Observations from a 4-year contamination study of a sample depth profile through martian meteorite Nakhla. Astrobiology 7, 389–401.

Citation: https://doi.org/10.5194/egusphere-2023-2748-RC3
- AC3: 'Reply on RC3', Julia Gravendyck, 02 Feb 2024
  
  Prof. Butterfield joins reviewers 1 and 2 in endorsing our concerns that the reported Volyn “biota” has been compromised by contamination. However, we are not suggesting that the fibres in our museum dust are analogues for the filaments of “kerite” as reported by Franz et al. (2023) and we will make this clear in our revised MS. Rather, museum dust as a whole presents a picture of the variety of airborne objects that potentially contaminate material such as the Volyn “biota” – it has significance beyond the specifics of the Franz et al. (2023) article. We did not address the “kerite” fibres directly other than raise the concern that perhaps none of the Volyn “biota” is of biological origin. Prof. Butterfield supports this concern, and we will expand our discussion to include the obvious inconsistency that the “kerite” filaments have no observed internal structure that would support a biological origin. We will add that without evidence for biogenicity, abiological processes including carbon-based ‘chemical garden’ models (Lukjanova and Lovzova, 1994; McMahon, 2019) should be given serious consideration. Prof. Butterfield accepts our claims of modern contaminating pine pollen and plant trichomes as compelling. Our claim is based on having personally seen thousands of such pine pollen and plant trichomes in modern preparations.
  We are especially grateful to Nicholas J. Butterworth, as R3, for his own detailed perspectives that give further weight to our concerns about the biogenicity of the Volyn “biota”.
  
  Citation: https://doi.org/10.5194/egusphere-2023-2748-AC3

Peer review completion

AR: Author's response | RR: Referee report | ED: Editor decision | EF: Editorial file upload

ED: Publish subject to minor revisions (review by editor) (06 Feb 2024) by Cindy De Jonge

AR by Julia Gravendyck on behalf of the Authors (19 Feb 2024) Author's response Author's tracked changes

EF by Polina Shvedko (20 Feb 2024) Manuscript

ED: Publish as is (23 Feb 2024) by Cindy De Jonge

AR by Julia Gravendyck on behalf of the Authors (25 Feb 2024)

Journal article(s) based on this preprint

09 Apr 2024

Comment on “The Volyn biota (Ukraine) – indications of 1.5 Gyr old eukaryotes in 3D preservation, a spotlight on the `boring billion' ” by Franz et al. (2023)

Martin J. Head, James B. Riding, Jennifer M. K. O'Keefe, Julius Jeiter, and Julia Gravendyck

Biogeosciences, 21, 1773–1783, https://doi.org/10.5194/bg-21-1773-2024,https://doi.org/10.5194/bg-21-1773-2024, 2024

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Martin J. Head, James B. Riding, Jennifer M. K. O’Keefe, Julius Jeiter, and Julia Gravendyck

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A diverse suite of 'fossils' associated with the ~1.5 Ga Volyn (Ukraine) kerite was published recently. We show that at least some of them represent modern contamination including plant hairs, pollen, and likely later fungal growth. Comparable diversity is shown to exist in modern museum dust, this calling into question whether any part of the Volyn biota is of biological origin while emphasising the need for scrupulous care in collecting, analysing, and identifying Precambrian microfossils.


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