New evidence for 250 Ma age of halotolerant bacterium from a Permian salt crystal: Comment and reply

COMMENT

Eske Willerslev* even 120-bp DNA fragments can be reproducibly obtained from samples Martin B. Hebsgaard ≥2 Ma (Willerslev et al., 2004a). Ancient DNA & Group, Centre for Ancient Genetics, Niels 3. Relative rate tests conducted on 16S ribosomal DNA and protein Bohr Institute & Biological Institute, , coding gene (recA and splB) sequences obtained from strain 2-9-3 Juliane Maries vej 30, DK-2100, strongly suggest that it is not geologically ancient (Graur and Pupko, 2001; Nickle et al., 2002; Maughan et al., 2002; Hebsgaard et al., 2005). This test Vreeland et al. (2000) reported the successful isolation of a spore- investigates the relative genetic distance from an outgroup to the postulated forming , Bacillus strain 2-9-3, from a brine inclusion within ancient organism and its closest contemporary relatives. One assumption a halite crystal recovered from a presumably 250 Ma Permian Salado of the relative rate test is that the mutation rate is similar and constant in the Formation in Carlsbad, New Mexico. The authors claim the bacterium DNA sequences for both the ancient organism and its contemporary rela- isolate to be as old as the formation itself, thereby nominating it to be tives. If Bacillus strain 2-9-3 is ancient, then a signifi cantly shorter genetic the oldest viable organism ever recovered. Since then, both the age of distance to the outgroup is expected as compared to its contemporary rela- the brine inclusion and the bacteria isolate itself have been questioned tives, as the latter should have an additional 250 m.y. to accumulate substi- (Hazen and Roedder, 2001; Graur and Pupko, 2001; Nickle et al., 2002; tutions. Although mutation rates are not always similar across organisms, Willerslev et al., 2004a; Hebsgaard et al., 2005). Recently, Satterfi eld et it is striking that not a single claim of geologically ancient DNA, including al. (2005) presented evidence that brine inclusions from the same layer that of Bacillus strain 2-9-3, has passed the rate test so far, strongly suggest- of salt that housed Bacillus strain 2-9-3 are composed of evaporated ing that contamination is involved (Hebsgaard et al., 2005). Late Permian seawater. The authors fi nd these results to be in strong Therefore, despite the recent results of Satterfi eld et al. (2005), it still support of the old age of strain 2-9-3. We strongly disagree with this remains highly controversial whether the Bacillus strain 2-9-3 is, indeed, conclusion. Although the age of the brine from where strain 2-9-3 was millions of years old. isolated might, indeed, be 250 Ma, it does not necessarily mean that Ba- cillus strain 2-9-3 is of similar age. In fact, there are a number of both REFERENCES CITED theoretical and empirical reasons to suggest otherwise. Graur, D., and Pupko, T., 2001, The Permian bacterium that isn’t: Molecular 1. The use of nonspecifi c media for the culturing of Bacillus strain Biology and Evolution, v. 18, p. 1143–1146. 2-9-3 makes the risk of false positive results extremely high. Although Hazen, R.M., and Roedder, E., 2001, How old are bacteria from the Permian Satterfi eld et al. (2005, p. 265) state that Bacillus strain 2-9-3 “has re- age?: Nature, v. 411, p. 155, doi: 10.1038/35075663. ceived signifi cant publicity because of the extreme sterilization techniques Hebsgaard, M.B., Phillips, M.J., and Willerslev, E., 2005, Geologically ancient DNA: Fact or artefact?: Trends in Microbiology, v. 13, p. 212–220, doi: used to avoid contamination by modern microorganisms,” Vreeland et al. 10.1016/j.tim.2005.03.010. (2000) did not follow some of the most basic authentication criteria, such Maughan, H., Birky, C.W., Jr., Nicholson, W.L., Rosenzweig, W.D., and as replication of their result in an independent laboratory. This criterion is Vreeland, R.H., 2002, The Paradox of the “ancient” bacterium which of great importance if laboratory based contamination is to be excluded contains “modern” protein-coding genes: Molecular Biology and Evolution, v. 19, p. 1637–1639. (i.e., all types of contamination related to the laboratory), as it’s unlikely Nickle, D.C., Learn, G.H., Rain, M.W., Mullins, J.I., and Mittler, J.E., 2002, that different laboratories would obtain the exact same result due to a Curiously modern DNA for a “250 million-year-old” bacterium: Journal of common lab contaminant (Willerslev and Cooper, 2005). Intriguingly, no Molecular Evolution, v. 54, p. 134–137. claims of geologically ancient cultures or DNA sequences published to Poinar, H.N., Höss, M., Bada, J.L., and Pääbo, S., 1996, Amino acid racemization date (i.e., claims >1 Ma) have followed this simple criterion of authentica- and the preservation of ancient DNA: , v. 272, p. 864–866. Satterfi eld, C.L., Lowenstein, T.K., Vreeland, R.H., Rosenzweig, W.D., and Powers, tion (Hebsgaard et al., 2005). D.W., 2005, New evidence for 250 Ma age of halotolerant bacterium from a 2. DNA is a relatively unstable molecule compared to other cellular Permian salt crystal: Geology, v. 33, p. 265–268, doi: 10.1130/G21106.1. components such as lignin and cutine, and will degrade with time if not Smith, C.I., Chamberlain, A.T., Riley, M.S., Cooper, A., Stringer, C.B., and repaired. The rate of degradation is known to be highly dependent on the Collins, M.J., 2001, Neanderthal DNA: Not just old but old and cold?: Nature, v. 410, p. 771–772, doi: 10.1038/35071177. environment, particularly the temperature (Smith et al., 2001; Willerslev Vreeland, R.H., Rosenzweig, W.D., and Powers, D.W., 2000, Isolation of a 250 et al., 2004b; Willerslev and Cooper, 2005). For example, calculations million-year-old halotolerant bacterium from a primary salt crystal: Nature, have shown that free DNA experiencing depurination damage will break v. 407, p. 897–900, doi: 10.1038/35038060. down to <100 base-pair (bp) fragments in less than 10 k.y. under warm Willerslev, E., and Cooper, A., 2005, Ancient DNA: Proceedings of the Royal and humid conditions, and in less than 100 k.y. under cold conditions Society of London, Series B, Biological , v. 272, p. 3–16, doi: 10.1098/rspb.2004.2813. (Poinar et al., 1996; Smith et al., 2001; Willerslev et al., 2004b). No meta- Willerslev, E., Hansen, A.J., Brand, T.B., Rønn, R., Barnes, I., Wiuf, C., Gilichinsky, bolic activity has yet been measured from like Bacillus strain D.A., Mitchell, D., and Cooper, A., 2004a, Long-term persistence of bacterial 2-9-3, excluding the possibility of active DNA repair prior to germination. DNA: Current Biology, v. 14, p. R9–R10, doi: 10.1016/j.cub.2003.12.012. Although endospores have special adaptations such as DNA binding α/β- Willerslev, E., Hansen, A.J., and Poinar, H.N., 2004b, Isolation of nucleic acids and cultures from ice and permafrost: Trends in Ecology & Evolution, v. 19, type small acid soluble proteins to reduce the rate of genomic modifi ca- p. 141–147, doi: 10.1016/j.tree.2003.11.010. tion, it still remains unlikely that they should be able to germinate after hundreds of millions of years of dormancy, particular under nonfrozen conditions. In support of this, a recent study of bacterial DNA in perma- REPLY frost—an environment considered the most promising for long-term DNA survival—has shown that DNA from -forming bacteria >600 Tim K. Lowenstein bp in size cannot be obtained from samples older than 0.5 Ma, and not Cindy L. Satterfi eld Department of Geological Sciences and Environmental Studies, State *E-mail: [email protected] University of New York, Binghamton, New York 13902-6000, USA

e93 Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/33/1/e93/3530487/i0091-7613-33-1-e94.pdf by guest on 23 September 2021 Russell H. Vreeland used as evidence against the Permian age of Virgibacillus strain 2-9-3. William D. Rosenzweig Willerslev and Hebsgaard state, “The rate of degradation (of DNA) is Department of Biology, West Chester University, West Chester, known to be highly dependent on the environment.” In this regard, fl uid Pennsylvania 19383, USA inclusions in halite from shallow burial environments (<1 km, <30 °C) offer a “friendly” setting for long-term preservation of microbial cells Dennis W. Powers and DNA. They are low in oxygen and their salty waters are known from Consulting Geologist, 140 Hemley Road, Anthony, Texas 79821, USA experiments to slow down the breakup of DNA by depurination (Lin- dahl and Nyberg, 1972). There has been no systematic research on DNA Willerslev and Hebsgaard’s comment on the evidence for the 250 Ma preservation in such oxygen-poor, saline systems at low temperatures, age of the halite, brine inclusion, and Virgibacillus strain 2-9-3 reported although that is changing. We are currently studying ancient microor- in Satterfi eld et al. (2005) comes at a time when earth scientists are busily ganisms and DNA in fl uid inclusions in halite from Death Valley and searching for signs of microscopic life in ancient samples of permafrost, Saline Valley salt cores, 103 to 105 years old. ice, deep-sea sediments, amber, salt, and chert. In the not too distant fu- 3. Relative rate tests, which suggest that Virgibacillus strain 2-9-3 is ture, sedimentary rocks may be returned from Mars. It is critically impor- not geologically ancient, are based on the assumption that evolution follows tant that the scientifi c community agree on the methods used for study of a predictable mutation rate and that the rate is known. The rates being used ancient microbes and ancient DNA and that issues of sample age, contam- by the researchers cited are based on nucleotide substitutions in laboratory- ination, sterilization, and replication of results be addressed now, not later. grown bacteria, which may not be realistic for all organisms. Furthermore, In this regard, Willerslev and Hebsgaard here question the age and origin growth rates of microorganisms in nature may, in some cases, be measured of Virgibacillus strain 2-9-3 cultured from a brine inclusion in halite from on time scales of centuries or longer, as illustrated by Parkes et al. (2000) for the Permian Salado salts. They do not take issue with the evidence for the bacteria isolated from subseafl oor sediments. Such long generation times Permian age of the brine inclusions discussed in Satterfi eld et al. (2005), might explain the similarities in 16S ribosomal DNA of Virgibacillus strain but they imply that Virgibacillus strain 2-9-3, reported by Vreeland et al. 2-9-3 and its contemporary relatives (Maughan et al., 2002). (2000), is a modern organism, presumably a laboratory contaminant. That As we continue to study microorganisms preserved in ancient halite, conclusion is based on (1) lack of replication of results in an independent perhaps others will participate or conduct the additional independent work laboratory, (2) degradation of relatively unstable DNA molecules over that Willerslev and Hebsgaard want to see. We agree that extreme care is geological time, and (3) “relative rate tests,” which suggest that Virgiba- required when analyzing small numbers of ancient cells or small amounts cillus strain 2-9-3 is not geologically ancient. of ancient DNA. As the fi eld of geobiology expands, the need for disci- 1. Willerslev and Cooper (2005) and Hebsgaard et al. (2005) re- pline-wide laboratory standards is greater than ever. viewed issues of contamination and proposed important guidelines for geobiological studies by offering criteria for the authentication of results ACKNOWLEDGMENTS for the study of ancient DNA and viable microbial cells. Vreeland et al. (2000) described their laboratory procedures and the sterilization tech- This research was supported by the NSF Geosciences Life in Ex- niques used to avoid contamination by modern organisms. They meet treme Environments and Biogeosciences Programs. the guidelines of Hebsgaard et al. (2005). Willerslev and Hebsgaard are correct that Vreeland et al. (2000) did not obtain replication of their results REFERENCES CITED by an independent laboratory before publication. Such verifi cation is im- Hebsgaard, M.B., Phillips, M.J., and Willerslev, E., 2005, Geologically ancient portant because it is not likely that separate laboratories would have com- DNA: Fact or artefact?: Trends in Microbiology, v. 13, p. 212–220, doi: mon microbial contaminants. We welcomed any geobiologists interested 10.1016/j.tim.2005.03.010. in studying the Salado halites and fl uid inclusions in the years since 2000. Lindahl, T., 1993, Instability and decay of the primary structure of DNA: Nature, None have so far taken up the offer. v. 362, p. 709–715, doi: 10.1038/362709a0. Lindahl, T., and Nyberg, B., 1972, Rate of depurination of native deoxyribonucleic Satterfi eld et al. (2005) showed that halites of the Permian Salado acid: Biochemistry, v. 11, p. 3610–3618, doi: 10.1021/bi00769a018. salts trapped surface brines as fl uid inclusions. This halite is ideal for the Lowenstein, T.K., Timofeeff, M.N., Brennan, S.T., Hardie, L.A., and Demicco, R.V., study of ancient DNA and microbial cells because samples of brine, once 2001, Oscillations in Phanerozoic seawater chemistry: Evidence from fl uid trapped inside crystals as fl uid inclusions, can remain completely sealed inclusions: Science, v. 294, p. 1086–1088, doi: 10.1126/science.1064280. Maughan, H., Birky, C.W., Jr., Nicholson, W.L., Rosenzweig, W.D., and and isolated from the environment for periods of more than 500 m.y. Such Vreeland, R.H., 2002, The paradox of the “ancient” bacterium which preservation of fl uid inclusions has allowed samples of ancient evaporated contains “modern” protein-coding genes: Molecular Biology and Evolution, seawater to be analyzed from halites going back to the late Precambrian v. 19, p. 1637–1639. (Lowenstein et al., 2001). Careful surface sterilization of similar halite Parkes, R.J., Cragg, B.A., and Wellsbury, P., 2000, Recent studies on bacterial crystals should also allow study of uncontaminated samples of Earth’s populations and processes in subseafl oor sediments: a review: Hydrogeology, v. 8, p. 11–28. ancient biosphere in fl uid inclusions. Satterfi eld, C.L., Lowenstein, T.K., Vreeland, R.H., Rosenzweig, W.D., and Powers, 2. Willerslev and Hebsgaard state, “DNA is a relatively unstable D.W., 2005, New evidence for 250 Ma age of halotolerant bacterium from a molecule compared to other cellular components…. and will degrade Permian salt crystal: Geology, v. 33, p. 265–268, doi: 10.1130/G21106.1. with time if not repaired.” Twelve years ago, it was thought that DNA, Vreeland, R.H., Rosenzweig, W.D., and Powers, D.W., 2000, Isolation of a 250- 4 million-year-old halotolerant bacterium from a primary salt crystal: Nature, as short fragments, may survive 10 years (Lindahl, 1993). Now it is v. 407, p. 897–900, doi: 10.1038/35038060. reported that, under certain conditions such as in permafrost, preserva- Willerslev, E., and Cooper, A., 2005, Ancient DNA: Proceedings of the Royal tion of DNA may approach 106 years (Hebsgaard et al., 2005). Clearly, Society of London, Series B, Biological Sciences, v. 272, p. 3–16, doi: the upper limit of DNA survival has changed and should not therefore be 10.1098/rspb.2004.2813.

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