Footnotes and extra appendixes transferred away from the PhD manuscript for the lack of space
Appendix: On the usage of the term Mryr, millions of radiometric years
“I have published arguments for my usage of the abbreviation Mryr (millions of radiometric years) contra Myr (millions of years) or MA (mega-annum, according to the SI-units), in the World Wide Web: http://www.helsinki.fi/~pjojala/Mryr.htm. The term appears to me more moderate and self-explaining, than the ‘absolute’ Myr. The unortodox terminology is defended on the basis of discrepancy between the ‘rocks’ and the (molecular) ‘clocks’ (racemization etc.). Also, the possibility of the survival of ancient biological macromolecules, or even alleged 250 Myr old bacillus spores popularized as a proof of panspermia at the turn of the Millennium, begs the issue. I have published a case study concerning the consensus problem associated with the evolutionary molecular clock hypothesis: http://www.helsinki.fi/~pjojala/Molecular_Clock.htm. These appendixes have not gone through a peer review process and should be evaluated with extra caution.”
Past can not be reproduced. In this thesis the abbreviation Mryr, millions of radiometric years, is used when referring to the time scales extrapolated from the established paradigm of uniformitarianism and actualism. This is in order to clarify that the numbers are not absolute but rely, ultimately, on the inorganic radioactive clocks that generally are calibrated by vulcanism. Fossil clocks and radioclocks rely on each other to begin with, which makes the dating game prone to circular reasoning. Also the phrase ”geochronological years” is rather accurate.
The reliability of the various direct organic clocks is far from being widely accepted. Their tendency seems to be to yield orders of magnitude smaller ages compared to the eons of geological time.
Half-lives for the racemization of a sample of amino acids in different
temperatures is given in the Table II. Usually only the concentrations of amino
acids is measured after extracting the residues in strong acids in the
quantitative amino acid analysis. When used as a clock, also the chirality
remaining is quantified, however. L-amino acids equilibrate to their D-forms
with a half-lifes between 400-50,000 years in
The dozen or more articles describing PCR-work on 10-130 Mryr old fossils have been abandoned in the mainstream community and in the same high profile journals they were originally peer reviewed and published in, referring to the contamination risk and painstaking reproducability. It must be emphasized, that despite its widespread usage, PCR is a very recent method. On the other hand, the articles published in 1990-1996 were done on valuable samples that sometimes were consumed in the process, and the (necessary) demand for reproduction is particularly expensive here. In the case of the 25-30 Mryr old termite-inclusion in the anhydrous amber matrix, it was not only the multicopy mitochondrial DNA, but also nuclear DNA that matched (DeSalle et al 1992). Cano et al (1994) had already published phylogenetics on the bacillus extracted from bee-inclusion. Chemically DNA lasts only for 10,000 years (Lindahl 1993), or 50,000 years at the very most (Svante Pääbo, personal communication).
When Bada, Pääbo and others joined the forces in 1994 (Bada et al) to study the amino acids in insects entombed in fossilized tree resins ranging in age from <100 years to 130 Mryrs, the residues in 40-130 Mryr old amber-entombed insects resembled those in a modern fly. They concluded that the racemization rate in amber insect inclusions is retarded by a factor of >104, and suggested that
"…in amber insect inclusions DNA depurination rates would also likely be retarded in comparison to aqueous solution measurements, and thus DNA fragments containing many hundreds of base pairs should be preserved. This conclusion is consistent with the reported successful retrieval of DNA sequences from amber-entombed organisms."
In other words, at the time these result were still trusted, the tabulated and empirical data on rasemization etc. was rather discarded, than the radiometric timescales to be questioned. In their Science-article the same experts concluded (Poinar et al 1996):
"Paleontological finds from which DNA sequences purportedly millions of years old have been reported show extensive racemization, and the amino acids present are mainly contaminates. An exception is the amino acids in some insects preserved in amber."
The urgent need for HGT to cover the lack of coherent phylogenetic signal is met with the transformability of bacteria. There is a theoretical threat, however, that a fox is put in the guard of the henhouse in the topic. Regarding the surprising speed of HGT amongst the eubacteria, it must be recalled that Martin accepts the established timescales when he summarized in 1999:
10% of the current E. coli genome consists of genes
that were acquired in over 200 events of lateral gene transfer, which occurred
subsequent to the divergence of E. coli and Salmonella some 100 million years
ago. Overall, the data suggest that no less than 18%
of E. coli's genes might be relatively recent foreign acquisitions, and that
the average rate of acquisition may be close to about 16 kb per million years.
These quantitative estimates of comparatively recent genome flux have profound
impact on evolutionary genome comparisons. They tend to suggest that a search
should be on to identify prin
ciples that might ultimately govern gene distribution patterns across prokaryotic genomes."
What is left of the "Creighton-Spielberg" years? It is still held, that despite exogenous contaminants, the ancient bones do occasionally contain trace amounts of endogenous material (Bada et al 1999). There are still the reports of proteins past the Jurassic (65 Mryr). This list of endeavours includes e.g. dentin, kollagen, gelatin, albumin, osteocalcin, enamel, and different glycoproteins. Fossil organic material with preserved antigenic determinants are reported for other proteins, too (Ho 1965; Wyckoff & Davidson 1976; Weiner et al 1976; Gurley et al 1991; Sansom 1994 etc.). 25 Mryr old chitin maintains its oily colours and its presence can be affirmed by mass spectroscopy (Stankiewicz et al 1997). The ratio between calcium and phosphorus in 80 Mryr bones may be identical to fresh bones (Barreto et al 1993).
On the other hand, it is disturbing that kollagen from a less than 20,000 year old mammoth may have already disappeared (Science (1978) 200, 1275). Proteins as a class are chemically unstable. Unlike them, amino acids are stable compounds that can be recovered both from fossil shells and some ancient sediments. Wyckoff has written a book entitled The biochemistry of animal fossils (1972), and in http://www.helsinki.fi/~pjojala/FossilBiochemistry I reproduce his tables and statistics. According to Wyckoff, it had long been taken for granted that proteins could persist for geological periods of time and the first microscope photos of intact collagen fibers came as a surprise (p. 53). The first conclusions from these first studies was, that the amino acids have not been altered in more than 100 millions of radiometric years and that the proteins were not anymore simpler than those now being produced. Wyckoff writes (p. 81):
"It is significant for the future study of still older material that many of the Jurassic bones have retained as much proteins as the younger Cretaceous. These analyses support and extend the indications obtained with the younger Pliocene and Miocene specimens that the amount of protein in old fossils declines only slowly with age… Though invertebrate shells rather quickly lose a large part of their original protein, the oldest yet examined have retained sufficient quantities for study. It is a noteworthy fact that after an initial fall the amounts retained in both vertebrate and invertebrate fossils do not show a futher steady decline with age; clearly these residues are so stable that they may confidently be sought in the ooldest obtainable specimes." (Emphasis added.)
250 Mryr old and 25-40 Mryr old
bacteria have been revived to grow and divide from their spore stages
The finding, by itself, was nothing new and such been observed multiple times - before the one that made the headlines at the era of expensive Mars sojourney together with the Antarctica-meteorite sensation. Grant et al (1998), McGenity et al (2000), and Vreeland & Rosenzweig (2002) have written reviews of these "extremophiles" from diverse populations of halobacteria still living after 196-600 Mryr. This is despite the fact that the current culture techniques of recover only 0.1-10% of the total number of microbe kinds in non-ancient samples.
Regarding other walking things, the apparent maternal and paternal inheritance of mitochondrial genomes and Y-chromosome, respectively, lays out the basis for the empirical determination of mutation rates. In the cell division only ~dozen mitochondria are inherited as a bottleneck population out of the hundreds of copies. Also the absence of introns and histones and disputed lack of recombination adds to the specialities of mtDNA. [For evidence of recombination, see Eyre-Walker et al (1999) and Awadalla et al (1999), from the same group. The linkage studies of hereditary mtDNA diseases seem to be the main argument for the absence of it.]
In this field the "post-genomic era" has already continued nearly a quarter of a century, but only during this thesis have the first real mutation rates published from extensive screens. Before this, the rates had been derived from the sequence differences divided by geological time, and the build-in presumption does not seem to have bothered. The swedish-estonian Svante Pääbo wrote even before the onslaught (1996):
"…the high mutation rate represents an impressive discrepancy with the received wisdom among evolutionists".
The first large-scale maps of paternal gene pool proove an exponential increase of population, that started less than 30,000 years ago, and Shen et al (2000) had to reject the distribution expected under the established constant population size model. In a pairwise paper Thomson et al (2000) state: "the expected time to the most recent common ancestor is remarkably short, on the order of 50,000 years… the spread of Y chromosomes out of Africa is much more recent than previously was thought". In 1995, the time back to a MRCA was estimated to be 188,000 years, with a "95% confidence interval from 51,000 to 411,000 years." Even this was titled as "A recent common ancestry for human Y chromosomes" and found justification for publishing in Nature (Hammer 1995).
Like the TV-news of 250 Myr bacteria and panspermia, also Pääbo's own mtDNA sequencing from neanderthalians was exhaustively popularized (Krings et al 2001). What went unnoticed from the larger public, was the sequencing of remnants from a morphologically modern human that was even more diverged. In a strict sense, the results of Adcock et al (2001) would mean that our ancestors came from the recently populated Australia.
The repair mechanisms of mtDNA were known to be rare compared to the nuclear one even before. Nevertheless, when the MRCA of (wo)mankind (the equivocated mitochondrial "Eve") was extrapolated to have lived only ~6500 years ago based on 134 families and 327 generation events (Parsons et al 1997), it came as an anathema. Parsons et al studied the most variable 614 bp "control region" in the mtDNA, and got a result of 1/33 changes in generation, or 1.2-4.0 substitutions per site in a million year (95% confidence interval, 2.5 as mean value). The mutation distribution did not match the most polymorphic sites (mutational "hotspots") in the region, and Siegfrid Scherer comments this one of the first direct rate measurements in TREE by stating: "Even if the last common mitochondrial ancestor is younger than the last common real ancestor, it remains enigmatic how the known distribution of human populations and genes could have arisen in the past few thousand years." (Scherer 1997.) He refers to consistent studies with cattle, domestic dogs and wolves, and human minisatellite and mtDNA coding regions, albeit in smaller samples.
From 272 individuals related by 705 mtDNA transmission events, Sigurgardottir et al (2000) got a result 3/705 or 0.0043 per generation, or 0.32/site/1 million years. Altogether the estimates of the mutation rate in this non-stocastic region have spanned two orders of magnitude.
Denver et al (2000) cultivated
The comprehensive mapping of the whole mtDNA from human tribes, as done by the swedish team of Ingmann et al (2000) bargained the "out-of-Africa" date a bit down to 52,000± 28,000 years and MRCA to 171,500± 50,000 yr BP. The authors state that "our mtDNA data… provide a concurrent view on human evolution with respect to the age of modern humans", but it is very regretable that their rates seem to have been deduced by the established figure of divergence of human and chimps 5 Mryrs ago, and not by the actual differences in the maternal lineage of life. The detail is, namely, the very point where the question is. If the nuclear mutations are hours, and the mtDNA a minute hand, then the conclusion of the work is to dismiss the swift "second hand" D-loop covering 7% studied by Parsons et al and others. It is hard to understand, how specimens being hanged on deleterious mutations, get a new chance every 60th units. Ingmann et al nail down the number of bottleneck populations survaving into about three matriarchal populations.
I think that one of the most fascinating approaches to test the degeneration hypothesis would be a systematic and statistical analysis of gene loss/transfer between nucleus and mitochondria or chloroplasts. Another set of methodology could be the plotting of nuclear GC-content as a function of generations. Rodríguez-Trelles et al (2000), for instance, report that the most recent common ancestor of Drosophila had an elevated GC content.
Lastly, when a Coelacanth population was found near the coast of South
Africa in 1938 (Smith 1939), the fish quickly earned the place of the most
famous living fossil. It is thought to be a crucial intermediate link on the
way to land living animals. At the beginning of this PhD project, a second
population was reported in Indonesia at a distance of
Non multa sed multum. As a conclusion, I am not claiming that dating game for an uninitiated biochemist looks like ‘Heads, I win. Tails, you lose.’ It would be important, however, to confess the level of uncertainty and impact of assumptions in the popularization to layman. The ±-sign is required in even much more trivial panels than these ones. My own experience is, that many students of biology refer to the "absolute" time scales of physics, if confronted with critiscism coming from their own field. I hope the students of physics do not look upon biology with the same escapism in mind.
I have compiled some quotes related to the problem of sample bias in the physical radiometric clocks themselves in http://www.helsinki.fi/~pjojala/Dating_game.
Adcock GJ, Dennis ES, Easteal S, Huttley GA,
Jermiin LS, Peacock WJ & Thorne A (2001) Mitochondrial DNA sequences in
ancient Australians: Implications for modern human origins. PNAS 98,
Awadalla P, Eyre-Walker A & Smith JM (1999) Linkage disequilibrium and recombination in hominid mitochondrial DNA. Science 286, 2524-5
Bada JL (1982) Racemization of Amino Acids in Nature. Int Disc Sci Rev 7, 30-46
Bada JL, Wang XS, Poinar HN, Paabo S & Poinar GO (1994) Amino acid racemization in amber-entombed insects: implications for DNA preservation. Geochim Cosmochim Acta 58, 3131-5
Bada JL, Wang XS & Hamilton H (1999) Preservation of key biomolecules in the fossil record: current knowledge and future challenges. Philos Trans R Soc Lond B Biol Sci 354, 77-87
Barreto C, Albrecht RM, Bjorling DE, Horner JR & Wilsman NJ (1993) Evidence of the growth plate and the growth of long bones in juvenile dinosaurs. Science 262, 2020-3
Cano RJ, Borucki MK, Higby-Schweitzer M, Poinar HN, Poinar GO Jr & Pollard KJ (1994) Bacillus DNA in fossil bees: an ancient symbiosis? Appl Environ Microbiol 60, 2164-7
Cano RJ & Borucki MK (1995) Revival and identification of bacterial spores in 25- to 40-million-year-old Dominican amber. Science 268, 1060-4
Denver DR, Morris K, Lynch M, Vassilieva LL & Thomas WK (2000) High Direct Estimate of the Mutation Rate in the Mitochondrial Genome of Caenorhabditis elegans Science 289, 2342-4
DeSalle R, Gatesy J, Wheeler W & Grimaldi D (1992) DNA Sequences from a Fossil Termite in Oligo-Miocene Amber and Their Phylogenetic Implications. Science 257, 1933-6
Eyre-Walker A, Smith NH & Smith JM (1999) How clonal are human mitochondria? Proc R Soc London Ser B Biol Sci 266, 477-83
Grant WD, Gemmell RT & McGenity TJ (1998) Halobacteria: the evidence for longevity. Extremophiles 2, 279-87
Gurley LR, Valdez JG, Spall WD, Smith BF & Gillette DD (1991) Proteins in the fossil bone of the dinosaur, Seismosaurus. J Protein Chem 10, 75-90
Hammer MF (1995) A recent common ancestry for human Y chromosomes. Nature 378, 376-8
Ho TY (1965) The amino acid composition of bone and tooth proteins in late Pleistocene mammals. Proc Natl Acad Sci 54, 26-31
Hissmann K, Fricke HW & Schauer J (1998) Population monitoring of a living fossil: the coelacanth Latimeria chalumnae in decline? Conservation Biol 12, 759-76
Holder MT, Erdmann MV, Wilcox TP, Caldwell RL & Hillis DM (1999) Two living species of coelacanths? Proc Natl Acad Sci 96, 12616-20
Ingman M, Kaessmann H, Paabo S & Gyllensten U (2000) Mitochondrial genome variation and the origin of modern humans. Nature 408, 708-13
Krings M, Capelli C, Tschentscher F, Geisert H, Meyer S, von Haeseler A, Grossschmidt K, Possnert G, Paunovic M & Paabo S (2001) A view of Neandertal genetic diversity Nat Genet 26, 144-6
Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362, 709-15
Martin W (1999) Mosaic bacterial chromosomes: a challenge en route to a tree of genomes. Bioessays 21, 99-104
Parsons TJ et al (1997) A high observedsubstitution rate in the human mitochondrial control region. Nat Genet 15, 363-8
Poinar HN, Hoss M, Bada JL & Paabo S (1996) Amino acid racemization and the preservation of ancient DNA. Science 272, 864-6
Pääbö S (1996) Invited Editorial, Mutational Hot Spots in the Mitochondrial Microcosm. Am J Hum Genet 59, 493-6
McGenity TJ, Gemmell RT, Grant WD & Stan-Lotter H (2000) Origins of halophilic microorganisms in ancient salt deposits.Environ Microbiol 2, 243-50
Rodriguez-Trelles F, Tarrio R & Ayala FJ (2000) Evidence for a high ancestral GC content in Drosophila. Mol Biol Evol 17, 1710-7
Sansom IJ, Smith MP & Smith MM (1994) Dentine in conodonts. Nature 368, 591
Scherer S (1997) Mitochondrial Eve: the plot thickens. Trends Ecol Evol 12, 422-3
Shen P, Wang F, Underhill PA, Franco C, Yang WH, Roxas A, Sung R, Lin AA, Hyman RW, Vollrath D, Davis RW, Cavalli-Sforza LL & Oefner PJ (2000) Population genetic implications from sequence variation in four Y chromosome genes. Proc Natl Acad Sci 97, 7354-9
Sigurgardottir S, Helgason A, Gulcher JR, Stefansson K & Donnelly P (2000) The mutation rate in the human mtDNA control region. Am J Hum Genet 66, 1599-609
Smith JLB (1939) A living fish of mesozoic time. Nature 143, 455-6
Stankiewicz BA, Briggs DEG, Evershed RP, Flannery MB & Wuttke M (1997) Preservation of chitin in 25 million-year-old fossils. Science 276, 1541-3
Thomson R, Pritchard JK, Shen P, Oefner PJ & Feldman MW (2000) Recent common ancestry of human Y chromosomes: evidence from DNA sequence data. Proc Natl Acad Sci 97, 7360-5
Vreeland RH, Rosenzweig WD & Powers DW (2000) Isolation of a 250 million-year-old halotolerant bacterium from a primary salt
crystal. Nature 407, 897-900
Vreeland RH & Rosenzweig WD (2002) The question of uniqueness of ancient bacteria. J Ind Microbiol Biotech 28, 32 - 41
Weiner S, Lowenstam HA & Hood L (1976) Characterization of 80-million-year-old mollusk shell proteins. Proc Natl Acad Sci 73, 2541-5
Wyckoff R (1972) The biochemistry of animal fossils. Williams & Wilkins, Baltimore
Wyckoff RW & Davidson FD (1976) Pleistocene and dinosaur gelatins. Comp Biochem Physiol B. 55, 95-7