Technique to deduce the time in prehistory when two or more life forms diverged

The molecular clock is a figurative term for a technique that uses the mutation rate of biomolecules to deduce the time in prehistory when two or more life forms diverged. The biomolecular data used for such calculations are usually nucleotide sequences for DNA, RNA, or amino acid sequences for proteins. The benchmarks for determining the mutation rate are often fossil or archaeological dates. The molecular clock was first tested in 1962 on the hemoglobin protein variants of various animals, and is commonly used in molecular evolution to estimate times of speciation or radiation. It is sometimes called a gene clock or an evolutionary clock.

The notion of the existence of a so-called "molecular clock" was first attributed to mile Zuckerkandl and Linus Pauling who, in 1962, noticed that the number of amino acid differences in hemoglobin between different lineages changes roughly linearly with time, as estimated from fossil evidence.[1] They generalized this observation to assert that the rate of evolutionary change of any specified protein was approximately constant over time and over different lineages (known as the molecular clock hypothesis).

The genetic equidistance phenomenon was first noted in 1963 by Emanuel Margoliash, who wrote: "It appears that the number of residue differences between cytochrome c of any two species is mostly conditioned by the time elapsed since the lines of evolution leading to these two species originally diverged. If this is correct, the cytochrome c of all mammals should be equally different from the cytochrome c of all birds. Since fish diverges from the main stem of vertebrate evolution earlier than either birds or mammals, the cytochrome c of both mammals and birds should be equally different from the cytochrome c of fish. Similarly, all vertebrate cytochrome c should be equally different from the yeast protein."[2] For example, the difference between the cytochrome c of a carp and a frog, turtle, chicken, rabbit, and horse is a very constant 13% to 14%. Similarly, the difference between the cytochrome c of a bacterium and yeast, wheat, moth, tuna, pigeon, and horse ranges from 64% to 69%. Together with the work of Emile Zuckerkandl and Linus Pauling, the genetic equidistance result directly led to the formal postulation of the molecular clock hypothesis in the early 1960s.[3]

Similarly, Vincent Sarich and Allan Wilson in 1967 demonstrated that molecular differences among modern Primates in albumin proteins showed that approximately constant rates of change had occurred in all the lineages they assessed.[4] The basic logic of their analysis involved recognizing that if one species lineage had evolved more quickly than a sister species lineage since their common ancestor, then the molecular differences between an outgroup (more distantly related) species and the faster-evolving species should be larger (since more molecular changes would have accumulated on that lineage) than the molecular differences between the outgroup species and the slower-evolving species. This method is known as the relative rate test. Sarich and Wilson's paper reported, for example, that human (Homo sapiens) and chimpanzee (Pan troglodytes) albumin immunological cross-reactions suggested they were about equally different from Ceboidea (New World Monkey) species (within experimental error). This meant that they had both accumulated approximately equal changes in albumin since their shared common ancestor. This pattern was also found for all the primate comparisons they tested. When calibrated with the few well-documented fossil branch points (such as no Primate fossils of modern aspect found before the K-T boundary), this led Sarich and Wilson to argue that the human-chimp divergence probably occurred only ~46 million years ago.[5]

The observation of a clock-like rate of molecular change was originally purely phenomenological. Later, the work of Motoo Kimura[6] developed the neutral theory of molecular evolution, which predicted a molecular clock. Let there be N individuals, and to keep this calculation simple, let the individuals be haploid (i.e. have one copy of each gene). Let the rate of neutral mutations (i.e. mutations with no effect on fitness) in a new individual be {displaystyle mu } . The probability that this new mutation will become fixed in the population is then 1/N, since each copy of the gene is as good as any other. Every generation, each individual can have new mutations, so there are {displaystyle mu } N new neutral mutations in the population as a whole. That means that each generation, {displaystyle mu } new neutral mutations will become fixed. If most changes seen during molecular evolution are neutral, then fixations in a population will accumulate at a clock-rate that is equal to the rate of neutral mutations in an individual.

The molecular clock alone can only say that one time period is twice as long as another: it cannot assign concrete dates. For viral phylogenetics and ancient DNA studiestwo areas of evolutionary biology where it is possible to sample sequences over an evolutionary timescalethe dates of the intermediate samples can be used to more precisely calibrate the molecular clock. However, most phylogenies require that the molecular clock be calibrated against independent evidence about dates, such as the fossil record.[7] There are two general methods for calibrating the molecular clock using fossil data: node calibration and tip calibration.[8]

Sometimes referred to as node dating, node calibration is a method for phylogeny calibration that is done by placing fossil constraints at nodes. A node calibration fossil is the oldest discovered representative of that clade, which is used to constrain its minimum age. Due to the fragmentary nature of the fossil record, the true most recent common ancestor of a clade will likely never be found.[8] In order to account for this in node calibration analyses, a maximum clade age must be estimated. Determining the maximum clade age is challenging because it relies on negative evidencethe absence of older fossils in that clade. There are a number of methods for deriving the maximum clade age using birth-death models, fossil stratigraphic distribution analyses, or taphonomic controls.[9] Alternatively, instead of a maximum and a minimum, a prior probability of the divergence time can be established and used to calibrate the clock. There are several prior probability distributions including normal, lognormal, exponential, gamma, uniform, etc.) that can be used to express the probability of the true age of divergence relative to the age of the fossil;[10] however, there are very few methods for estimating the shape and parameters of the probability distribution empirically.[11] The placement of calibration nodes on the tree informs the placement of the unconstrained nodes, giving divergence date estimates across the phylogeny. Historical methods of clock calibration could only make use of a single fossil constraint (non-parametric rate smoothing),[12] while modern analyses (BEAST[10] and r8s[13]) allow for the use of multiple fossils to calibrate the molecular clock. Simulation studies have shown that increasing the number of fossil constraints increases the accuracy of divergence time estimation.[14]

Sometimes referred to as tip dating, tip calibration is a method of molecular clock calibration in which fossils are treated as taxa and placed on the tips of the tree. This is achieved by creating a matrix that includes a molecular dataset for the extant taxa along with a morphological dataset for both the extinct and the extant taxa.[9] Unlike node calibration, this method reconstructs the tree topology and places the fossils simultaneously. Molecular and morphological models work together simultaneously, allowing morphology to inform the placement of fossils.[8] Tip calibration makes use of all relevant fossil taxa during clock calibration, rather than relying on only the oldest fossil of each clade. This method does not rely on the interpretation of negative evidence to infer maximum clade ages.[9]

Demographic changes in populations can be detected as fluctuations in historical coalescent effective population size from a sample of extant genetic variation in the population using coalescent theory.[15][16][17] Ancient population expansions that are well documented and dated in the geological record can be used to calibrate a rate of molecular evolution in a manner similar to node calibration. However, instead of calibrating from the known age of a node, expansion calibration uses a two-epoch model of constant population size followed by population growth, with the time of transition between epochs being the parameter of interest for calibration.[18][19] Expansion calibration works at shorter, intraspecific timescales in comparison to node calibration, because expansions can only be detected after the most recent common ancestor of the species in question. Expansion dating has been used to show that molecular clock rates can be inflated at short timescales[18] (< 1 MY) due to incomplete fixation of alleles, as discussed below[20][21]

This approach to tip calibration goes a step further by simultaneously estimating fossil placement, topology, and the evolutionary timescale. In this method, the age of a fossil can inform its phylogenetic position in addition to morphology. By allowing all aspects of tree reconstruction to occur simultaneously, the risk of biased results is decreased.[8] This approach has been improved upon by pairing it with different models. One current method of molecular clock calibration is total evidence dating paired with the fossilized birth-death (FBD) model and a model of morphological evolution.[22] The FBD model is novel in that it allows for "sampled ancestors", which are fossil taxa that are the direct ancestor of a living taxon or lineage. This allows fossils to be placed on a branch above an extant organism, rather than being confined to the tips.[23]

Bayesian methods can provide more appropriate estimates of divergence times, especially if large datasetssuch as those yielded by phylogenomicsare employed.[24]

Sometimes only a single divergence date can be estimated from fossils, with all other dates inferred from that. Other sets of species have abundant fossils available, allowing the hypothesis of constant divergence rates to be tested. DNA sequences experiencing low levels of negative selection showed divergence rates of 0.70.8% perMyr in bacteria, mammals, invertebrates, and plants.[25] In the same study, genomic regions experiencing very high negative or purifying selection (encoding rRNA) were considerably slower (1% per 50Myr).

In addition to such variation in rate with genomic position, since the early 1990s variation among taxa has proven fertile ground for research too,[26] even over comparatively short periods of evolutionary time (for example mockingbirds[27]). Tube-nosed seabirds have molecular clocks that on average run at half speed of many other birds,[28] possibly due to long generation times, and many turtles have a molecular clock running at one-eighth the speed it does in small mammals, or even slower.[29] Effects of small population size are also likely to confound molecular clock analyses. Researchers such as Francisco J. Ayala have more fundamentally challenged the molecular clock hypothesis.[30][31][32] According to Ayala's 1999 study, five factors combine to limit the application of molecular clock models:

Molecular clock users have developed workaround solutions using a number of statistical approaches including maximum likelihood techniques and later Bayesian modeling. In particular, models that take into account rate variation across lineages have been proposed in order to obtain better estimates of divergence times. These models are called relaxed molecular clocks[33] because they represent an intermediate position between the 'strict' molecular clock hypothesis and Joseph Felsenstein's many-rates model[34] and are made possible through MCMC techniques that explore a weighted range of tree topologies and simultaneously estimate parameters of the chosen substitution model. It must be remembered that divergence dates inferred using a molecular clock are based on statistical inference and not on direct evidence.

The molecular clock runs into particular challenges at very short and very long timescales. At long timescales, the problem is saturation. When enough time has passed, many sites have undergone more than one change, but it is impossible to detect more than one. This means that the observed number of changes is no longer linear with time, but instead flattens out. Even at intermediate genetic distances, with phylogenetic data still sufficient to estimate topology, signal for the overall scale of the tree can be weak under complex likelihood models, leading to highly uncertain molecular clock estimates.[35]

At very short time scales, many differences between samples do not represent fixation of different sequences in the different populations. Instead, they represent alternative alleles that were both present as part of a polymorphism in the common ancestor. The inclusion of differences that have not yet become fixedleads to a potentially dramatic inflation of the apparent rate of the molecular clock at very short timescales.[21][36]

The molecular clock technique is an important tool in molecular systematics, the use of molecular genetics information to determine the correct scientific classification of organisms or to study variation in selective forces. Knowledge of approximately constant rate of molecular evolution in particular sets of lineages also facilitates estimation of the dates of phylogenetic events, including those not documented by fossils, such as the divergences between living taxa. In these casesespecially over long stretches of timethe limitations of the molecular clock hypothesis (above) must be considered; such estimates may be off by 50% or more.

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Molecular clock - Wikipedia

1953 scientific paper on the helical structure of DNA by James Watson and Francis Crick

"Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid" was the first article published to describe the discovery of the double helix structure of DNA, using X-ray diffraction and the mathematics of a helix transform. It was published by Francis Crick and James D. Watson in the scientific journal Nature on pages 737738 of its 171st volume (dated 25 April 1953).[1][2]

This article is often termed a "pearl" of science because it is brief and contains the answer to a fundamental mystery about living organisms. This mystery was the question of how it is possible that genetic instructions are held inside organisms and how they are passed from generation to generation. The article presents a simple and elegant solution, which surprised many biologists at the time who believed that DNA transmission was going to be more difficult to deduce and understand. The discovery had a major impact on biology, particularly in the field of genetics, enabling later researchers to understand the genetic code.

The application of physics and chemistry to biological problems led to the development of molecular biology, which is particularly concerned with the flow and consequences of biological information from DNA to proteins. The discovery of the DNA double helix made clear that genes are functionally defined parts of DNA molecules, and that there must be a way for cells to translate the information in DNA to specific amino acids, which are used in order to make proteins.

Linus Pauling was a chemist who was very influential in developing an understanding of the structure of biological molecules. In 1951, Pauling published the structure of the alpha helix, a fundamentally important structural component of proteins. In early 1953, Pauling published a triple helix model of DNA, which subsequently turned out to be incorrect.[3] Both Crick, and particularly Watson, thought that they were racing against Pauling to discover the structure of DNA.

Max Delbrck was a physicist who recognized some of the biological implications of quantum physics. Delbruck's thinking about the physical basis of life stimulated Erwin Schrdinger to write, What Is Life? Schrdinger's book was an important influence on Crick and Watson. Delbruck's efforts to promote the "Phage Group" (exploring genetics by way of the viruses that infect bacteria) was important in the early development of molecular biology in general and the development of Watson's scientific interests in particular.[4]

Crick, Watson, and Maurice Wilkins who won the Nobel Prize for Medicine in recognition of their discovery of the DNA double helix.

It is not always the case that the structure of a molecule is easy to relate to its function. What makes the structure of DNA so obviously related to its function was described modestly at the end of the article: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material".

The "specific pairing" is a key feature of the Watson and Crick model of DNA, the pairing of nucleotide subunits.[5] In DNA, the amount of guanine is equal to cytosine and the amount of adenine is equal to thymine. The A:T and C:G pairs are structurally similar. In particular, the length of each base pair is the same and they fit equally between the two sugar-phosphate backbones. The base pairs are held together by hydrogen bonds, a type of chemical attraction that is easy to break and easy to reform. After realizing the structural similarity of the A:T and C:G pairs, Watson and Crick soon produced their double helix model of DNA with the hydrogen bonds at the core of the helix providing a way to unzip the two complementary strands for easy replication: the last key requirement for a likely model of the genetic molecule.

Indeed, the base-pairing did suggest a way to copy a DNA molecule. Just pull apart the two sugar-phosphate backbones, each with its hydrogen bonded A, T, G, and C components. Each strand could then be used as a template for assembly of a new base-pair complementary strand.

When Watson and Crick produced their double helix model of DNA, it was known that most of the specialized features of the many different life forms on Earth are made possible by proteins. Structurally, proteins are long chains of amino acid subunits. In some way, the genetic molecule, DNA, had to contain instructions for how to make the thousands of proteins found in cells. From the DNA double helix model, it was clear that there must be some correspondence between the linear sequences of nucleotides in DNA molecules to the linear sequences of amino acids in proteins. The details of how sequences of DNA instruct cells to make specific proteins was worked out by molecular biologists during the period from 1953 to 1965. Francis Crick played an integral role in both the theory and analysis of the experiments that led to an improved understanding of the genetic code.[6]

Other advances in molecular biology stemming from the discovery of the DNA double helix eventually led to ways to sequence genes. James Watson directed the Human Genome Project at the National Institutes of Health.[7] The ability to sequence and manipulate DNA is now central to the biotechnology industry and modern medicine. The austere beauty of the structure and the practical implications of the DNA double helix combined to make Molecular structure of Nucleic Acids; A Structure for Deoxyribose Nucleic Acid one of the most prominent biology articles of the twentieth century.

Although Watson and Crick were first to put together all the scattered fragments of information that were required to produce a successful molecular model of DNA, their findings had been based on data collected by researchers in several other laboratories. For example, they drew on published research relating to the discovery of Hydrogen bonds in DNA by John Masson Gulland, Denis Jordan and their colleagues at University College Nottingham in 1947.[8][9][10] However the discovery of the DNA double helix also used a considerable amount of material from the unpublished work of Rosalind Franklin, A.R. Stokes, Maurice Wilkins, and H.R. Wilson at King's College London. Key data from Wilkins, Stokes, and Wilson, and, separately, by Franklin and Gosling, were published in two separate additional articles in the same issue of Nature with the article by Watson and Crick.[11][12] The article by Watson and Crick acknowledged that they had been "stimulated" by experimental results from the King's College researchers, and a similar acknowledgment was published by Wilkins, Stokes, and Wilson in the following three-page article.

In 1968, Watson published a highly controversial autobiographical account of the discovery of the double-helical, molecular structure of DNA called The Double Helix, which was not publicly accepted either by Crick or Wilkins.[13] Furthermore, Erwin Chargaff also printed a rather "unsympathetic review" of Watson's book in the 29 March 1968 issue of Science. In the book, Watson stated among other things that he and Crick had access to some of Franklin's data from a source that she was not aware of, and also that he had seenwithout her permissionthe B-DNA X-ray diffraction pattern obtained by Franklin and Gosling in May 1952 at King's in London. In particular, in late 1952, Franklin had submitted a progress report to the Medical Research Council, which was reviewed by Max Perutz, then at the Cavendish Laboratory of the University of Cambridge. Watson and Crick also worked in the MRC-supported Cavendish Laboratory in Cambridge whereas Wilkins and Franklin were in the MRC-supported laboratory at King's in London. Such MRC reports were not usually widely circulated, but Crick read a copy of Franklin's research summary in early 1953.[13][14]

Perutz's justification for passing Franklin's report about the crystallographic unit of the B-DNA and A-DNA structures to both Crick and Watson was that the report contained information which Watson had heard before, in November 1951, when Franklin talked about her unpublished results with Raymond Gosling during a meeting arranged by M.H.F. Wilkins at King's College, following a request from Crick and Watson;[15] Perutz said he had not acted unethically because the report had been part of an effort to promote wider contact between different MRC research groups and was not confidential.[16] This justification would exclude Crick, who was not present at the November 1951 meeting, yet Perutz also gave him access to Franklin's MRC report data. Crick and Watson then sought permission from Cavendish Laboratory head William Lawrence Bragg, to publish their double-helix molecular model of DNA based on data from Franklin and Wilkins.

By November 1951, Watson had acquired little training in X-ray crystallography, by his own admission, and thus had not fully understood what Franklin was saying about the structural symmetry of the DNA molecule.[14] Crick, however, knowing the Fourier transforms of Bessel functions that represent the X-ray diffraction patterns of helical structures of atoms, correctly interpreted further one of Franklin's experimental findings as indicating that DNA was most likely to be a double helix with the two polynucleotide chains running in opposite directions. Crick was thus in a unique position to make this interpretation because he had formerly worked on the X-ray diffraction data for other large molecules that had helical symmetry similar to that of DNA. Franklin, on the other hand, rejected the first molecular model building approach proposed by Crick and Watson: the first DNA model, which in 1952 Watson presented to her and to Wilkins in London, had an obviously incorrect structure with hydrated charged groups on the inside of the model, rather than on the outside. Watson explicitly admitted this in his book The Double Helix.[14]

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