Editing Molecular clock

{{short description|Technique to deduce the time in prehistory when two or more life forms diverged}}
{{distinguish|Chemical clock|Biological clock (disambiguation){{!}}Biological clock}}
{{Use dmy dates|date=January 2020}}
{{Evolutionary biology}}
The '''molecular clock ''' is a figurative term for a technique that uses the [[mutation rate]] of [[biomolecule]]s to [[Chronological dating|deduce the time]] in [[prehistory]] when two or more [[life form]]s [[Genetic divergence|diverged]]. The biomolecular data used for such calculations are usually [[nucleotide]] [[DNA sequence|sequences]] for [[DNA]], [[RNA]], or [[amino acid]] sequences for [[protein]]s.

==Early discovery and genetic equidistance==
The notion of the existence of a so-called "molecular clock" was first attributed to [[Emile Zuckerkandl|Émile Zuckerkandl]] and [[Linus Pauling]] who, in 1962, noticed that the number of [[amino acid]] differences in [[hemoglobin]] between different lineages changes roughly [[Linear function|linearly]] with time, as estimated from fossil evidence.<ref name=Zuckerkand62>{{cite book | vauthors = Zuckerkandl E, Pauling | author-link1 = Emile Zuckerkandl | author-link2 = Linus Pauling | year = 1962 | title = Horizons in Biochemistry | chapter-url = https://archive.org/details/horizonsinbioche0000kash| chapter-url-access = registration| chapter = Molecular disease, evolution, and genic heterogeneity |editor=Kasha, M. |editor2=Pullman, B| pages = [https://archive.org/details/horizonsinbioche0000kash/page/189 189–225] | publisher = Academic Press, New York}}</ref> They generalized this observation to assert that the rate of [[evolution]]ary 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."<ref>{{cite journal | vauthors = Margoliash E | title = Primary Structure and Evolution of Cytochrome C | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 50 | issue = 4 | pages = 672–679 | date = October 1963 | pmid = 14077496 | pmc = 221244 | doi = 10.1073/pnas.50.4.672 | doi-access = free | bibcode = 1963PNAS...50..672M }}</ref> 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 led directly to the formal postulation of the molecular clock hypothesis in the early 1960s.<ref>{{cite journal | vauthors = Kumar S | title = Molecular clocks: four decades of evolution | journal = Nature Reviews. Genetics | volume = 6 | issue = 8 | pages = 654–662 | date = August 2005 | pmid = 16136655 | doi = 10.1038/nrg1659 | s2cid = 14261833 }}</ref>

Similarly, [[Vincent Sarich]] and [[Allan Wilson (biologist)|Allan Wilson]] in 1967 demonstrated that molecular differences among modern [[primate]]s in [[albumin]] proteins showed that approximately constant rates of change had occurred in all the lineages they assessed.<ref>{{cite journal | vauthors = Sarich VM, Wilson AC | title = Rates of albumin evolution in primates | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 58 | issue = 1 | pages = 142–148 | date = July 1967 | pmid = 4962458 | pmc = 335609 | doi = 10.1073/pnas.58.1.142 | doi-access = free | bibcode = 1967PNAS...58..142S }}</ref> 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 (''[[Common chimpanzee|Pan troglodytes]]'') albumin immunological cross-reactions suggested they were about equally different from [[New World monkey|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 [[Cretaceous–Paleogene boundary|K-T boundary]]), this led Sarich and Wilson to argue that the human-chimp divergence probably occurred only ~4–6 million years ago.<ref>{{cite journal | vauthors = Sarich VM, Wilson AC | title = Immunological time scale for hominid evolution | journal = Science | volume = 158 | issue = 3805 | pages = 1200–1203 | date = December 1967 | pmid = 4964406 | doi = 10.1126/science.158.3805.1200 | s2cid = 7349579 | bibcode = 1967Sci...158.1200S | jstor = 1722843 }}</ref>

==Relationship with neutral theory==

The observation of a clock-like rate of molecular change was originally purely [[phenomenology (philosophy)|phenomenological]]. Later, the work of [[Motoo Kimura]]<ref name=Kimura68>{{cite journal | vauthors = Kimura M | title = Evolutionary rate at the molecular level | journal = Nature | volume = 217 | issue = 5129 | pages = 624–626 | date = February 1968 | pmid = 5637732 | doi = 10.1038/217624a0 | s2cid = 4161261 | bibcode = 1968Natur.217..624K }}</ref> 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 [[Ploidy|haploid]] (i.e. have one copy of each gene). Let the rate of neutral [[mutation]]s (i.e. mutations with no effect on [[Fitness (biology)|fitness]]) in a new individual be <math>\mu</math>. The probability that this new mutation will become [[Fixation (population genetics)|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 <math>\mu</math>N new neutral mutations in the population as a whole. That means that each generation, <math>\mu</math> new neutral mutations will become fixed. If most changes seen during [[molecular evolution]] are neutral, then [[Fixation (population genetics)|fixations]] in a population will accumulate at a clock-rate that is equal to the rate of neutral [[mutation]]s in an individual.

==Calibration==
To use molecular clocks to estimate divergence times, molecular clocks need to be "calibrated". This is because molecular data alone does not contain any information on absolute times. For viral phylogenetics and [[ancient DNA]] studies—two areas of evolutionary biology where it is possible to sample sequences over an evolutionary timescale—the dates of the intermediate samples can be used to calibrate the molecular clock. However, most phylogenies require that the molecular clock be [[calibration|calibrated]] using independent evidence about dates, such as the [[fossil]] record.<ref name=Benton01>{{cite journal | vauthors = Benton MJ, Donoghue PC | title = Paleontological evidence to date the tree of life | journal = Molecular Biology and Evolution | volume = 24 | issue = 1 | pages = 26–53 | date = January 2007 | pmid = 17047029 | doi = 10.1093/molbev/msl150 | name-list-style = amp | doi-access = free }}</ref> There are two general methods for calibrating the molecular clock using fossils: node calibration and tip calibration.<ref name=Donoghue02>{{cite journal | vauthors = Donoghue PC, Yang Z | title = The evolution of methods for establishing evolutionary timescales | journal = Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences | volume = 371 | issue = 1699 | page = 20160020 | date = July 2016 | pmid = 27325838 | pmc = 4920342 | doi = 10.1098/rstb.2016.0020 | name-list-style = amp }}</ref>

===Node calibration===
Sometimes referred to as node dating, node calibration is a method for time-scaling [[phylogenetic tree]]s by specifying time constraints for one or more nodes in the tree. Early methods of clock calibration only used a single fossil constraint (e.g. non-parametric rate smoothing),<ref>{{Cite journal| vauthors = Sanderson M |date=1997|title=A nonparametric approach to estimating divergence times in the absence of rate constancy|journal=Molecular Biology and Evolution|volume=14|issue=12|pages=1218–1231|doi=10.1093/oxfordjournals.molbev.a025731|s2cid=17647010|doi-access=free}}</ref> but newer methods (BEAST<ref name="ReferenceA"/> and [[sourceforge:projects/r8s/|r8s]]<ref>{{cite journal | vauthors = Sanderson MJ | title = r8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock | journal = Bioinformatics | volume = 19 | issue = 2 | pages = 301–302 | date = January 2003 | pmid = 12538260 | doi = 10.1093/bioinformatics/19.2.301 | doi-access = free }}</ref>) allow for the use of multiple fossils to calibrate molecular clocks. The oldest fossil of a [[clade]] is used to constrain the minimum possible age for the node representing the most recent common ancestor of the clade. However, due to incomplete fossil preservation and other factors, clades are typically older than their oldest fossils.<ref name="Donoghue02" /> In order to account for this, nodes are allowed to be older than the minimum constraint in node calibration analyses. However, determining how much older the node is allowed to be is challenging. There are a number of strategies for deriving the maximum bound for the age of a clade including those based on birth-death models, fossil [[stratigraphy|stratigraphic]] distribution analyses, or [[taphonomy|taphonomic]] controls.<ref name="O'Reilly03">{{cite journal | vauthors = O'Reilly JE, Dos Reis M, Donoghue PC | title = Dating Tips for Divergence-Time Estimation | journal = Trends in Genetics | volume = 31 | issue = 11 | pages = 637–650 | date = November 2015 | pmid = 26439502 | doi = 10.1016/j.tig.2015.08.001 | hdl-access = free | name-list-style = amp | hdl = 1983/ba7bbcf4-1d51-4b74-a800-9948edb3bbe6 | url = https://research-information.bris.ac.uk/en/publications/ba7bbcf4-1d51-4b74-a800-9948edb3bbe6 }}</ref> Alternatively, instead of a maximum and a minimum, a [[probability density]] can be used to represent the uncertainty about the age of the clade. These calibration densities can take the shape of standard probability densities (e.g. [[Normal distribution|normal]], [[Log-normal distribution|lognormal]], [[Exponential distribution|exponential]], [[Gamma distribution|gamma]]) that can be used to express the uncertainty associated with divergence time estimates. <ref name="ReferenceA">{{cite journal | vauthors = Drummond AJ, Suchard MA, Xie D, Rambaut A | title = Bayesian phylogenetics with BEAUti and the BEAST 1.7 | journal = Molecular Biology and Evolution | volume = 29 | issue = 8 | pages = 1969–1973 | date = August 2012 | pmid = 22367748 | pmc = 3408070 | doi = 10.1093/molbev/mss075 }}</ref> Determining the shape and parameters of the probability distribution is not trivial, but there are methods that use not only the oldest fossil but a larger sample of the fossil record of clades to estimate calibration densities empirically.<ref name="Claramunt2022">{{cite journal | last=Claramunt | first=S | title=CladeDate : Calibration information generator for divergence time estimation | journal=Methods in Ecology and Evolution | publisher=Wiley | volume=13 | issue=11 | date=2022 | issn=2041-210X | doi=10.1111/2041-210x.13977 | pages=2331–2338| s2cid=252353611 | doi-access=free | bibcode=2022MEcEv..13.2331C }}</ref> Studies have shown that increasing the number of fossil constraints increases the accuracy of divergence time estimation.<ref name=Zheng04>{{cite journal | vauthors = Zheng Y, Wiens JJ | title = Do missing data influence the accuracy of divergence-time estimation with BEAST? | journal = Molecular Phylogenetics and Evolution | volume = 85 | issue = 1 | pages = 41–49 | date = April 2015 | pmid = 25681677 | doi = 10.1016/j.ympev.2015.02.002 | bibcode = 2015MolPE..85...41Z | s2cid = 3895351 | name-list-style = amp }}</ref>

===Tip calibration===
Sometimes referred to as [[tip dating]], tip calibration is a method of molecular clock calibration in which fossils are treated as [[taxon|taxa]] and placed on the tips of the tree. This is achieved by creating a matrix that includes a [[molecular phylogenetics|molecular]] dataset for the [[neontology|extant taxa]] along with a [[morphology (biology)|morphological]] dataset for both the extinct and the extant taxa.<ref name="O'Reilly03" /> 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.<ref name="Donoghue02" /> 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.<ref name="O'Reilly03" />

=== Expansion calibration ===
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.<ref>{{cite journal | vauthors = Rogers AR, Harpending H | title = Population growth makes waves in the distribution of pairwise genetic differences | journal = Molecular Biology and Evolution | volume = 9 | issue = 3 | pages = 552–569 | date = May 1992 | pmid = 1316531 | doi = 10.1093/oxfordjournals.molbev.a040727 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Shapiro B, Drummond AJ, Rambaut A, Wilson MC, Matheus PE, Sher AV, Pybus OG, Gilbert MT, Barnes I, Binladen J, Willerslev E, Hansen AJ, Baryshnikov GF, Burns JA, Davydov S, Driver JC, Froese DG, Harington CR, Keddie G, Kosintsev P, Kunz ML, Martin LD, Stephenson RO, Storer J, Tedford R, Zimov S, Cooper A | display-authors = 6 | title = Rise and fall of the Beringian steppe bison | journal = Science | volume = 306 | issue = 5701 | pages = 1561–1565 | date = November 2004 | pmid = 15567864 | doi = 10.1126/science.1101074 | bibcode = 2004Sci...306.1561S | s2cid = 27134675 | url = http://summit.sfu.ca/item/15088 }}</ref><ref>{{cite journal | vauthors = Li H, Durbin R | title = Inference of human population history from individual whole-genome sequences | journal = Nature | volume = 475 | issue = 7357 | pages = 493–496 | date = July 2011 | pmid = 21753753 | pmc = 3154645 | doi = 10.1038/nature10231 }}</ref> 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.<ref name=":0">{{cite journal | vauthors = Crandall ED, Sbrocco EJ, Deboer TS, Barber PH, Carpenter KE | title = Expansion dating: calibrating molecular clocks in marine species from expansions onto the Sunda Shelf Following the Last Glacial Maximum | journal = Molecular Biology and Evolution | volume = 29 | issue = 2 | pages = 707–719 | date = February 2012 | pmid = 21926069 | doi = 10.1093/molbev/msr227 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Hoareau TB | title = Late Glacial Demographic Expansion Motivates a Clock Overhaul for Population Genetics | journal = Systematic Biology | volume = 65 | issue = 3 | pages = 449–464 | date = May 2016 | pmid = 26683588 | doi = 10.1093/sysbio/syv120 | doi-access = free | hdl = 2263/53371 | hdl-access = free }}</ref> 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<ref name=":0" /> (< 1 MY) due to incomplete fixation of alleles, as discussed below<ref>{{cite journal | vauthors = Ho SY, Tong KJ, Foster CS, Ritchie AM, Lo N, Crisp MD | title = Biogeographic calibrations for the molecular clock | journal = Biology Letters | volume = 11 | issue = 9 | pages = 20150194 | date = September 2015 | pmid = 26333662 | pmc = 4614420 | doi = 10.1098/rsbl.2015.0194 }}</ref><ref name=":1" />

===Total evidence dating===
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.<ref name="Donoghue02" /> 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.<ref name=Heath05>{{cite journal | vauthors = Heath TA, Huelsenbeck JP, Stadler T | title = The fossilized birth-death process for coherent calibration of divergence-time estimates | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 29 | pages = E2957–E2966 | date = July 2014 | pmid = 25009181 | pmc = 4115571 | doi = 10.1073/pnas.1319091111 | name-list-style = amp | arxiv = 1310.2968 | doi-access = free | bibcode = 2014PNAS..111E2957H }}</ref> 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 (evolution)|lineage]]. This allows fossils to be placed on a branch above an extant organism, rather than being confined to the tips.<ref name=Gavryushkina06>{{cite journal | vauthors = Gavryushkina A, Heath TA, Ksepka DT, Stadler T, Welch D, Drummond AJ | title = Bayesian Total-Evidence Dating Reveals the Recent Crown Radiation of Penguins | journal = Systematic Biology | volume = 66 | issue = 1 | pages = 57–73 | date = January 2017 | pmid = 28173531 | pmc = 5410945 | doi = 10.1093/sysbio/syw060 | name-list-style = amp | arxiv = 1506.04797 }}</ref>

===Methods===
Bayesian methods can provide more appropriate estimates of divergence times, especially if large datasets—such as those yielded by [[phylogenomics]]—are employed.<ref name="Dos Reis2012">{{cite journal | vauthors = dos Reis M, Inoue J, Hasegawa M, Asher RJ, Donoghue PC, Yang Z | title = Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny | journal = Proceedings. Biological Sciences | volume = 279 | issue = 1742 | pages = 3491–3500 | date = September 2012 | pmid = 22628470 | pmc = 3396900 | doi = 10.1098/rspb.2012.0683 }}</ref>

==Non-constant rate of molecular clock==
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 (natural selection)|negative selection]] showed divergence rates of 0.7–0.8% per&nbsp;[[Myr]] in bacteria, mammals, invertebrates, and plants.<ref name=Ochman87>{{cite journal | vauthors = Ochman H, Wilson AC | title = Evolution in bacteria: evidence for a universal substitution rate in cellular genomes | journal = Journal of Molecular Evolution | volume = 26 | issue = 1–2 | pages = 74–86 | year = 1987 | pmid = 3125340 | doi = 10.1007/BF02111283 | s2cid = 8260277 | bibcode = 1987JMolE..26...74O }}</ref>  In the same study, genomic regions experiencing very high negative or purifying selection (encoding rRNA) were considerably slower (1% per 50&nbsp;Myr).

In addition to such variation in rate with genomic position, since the early 1990s variation among taxa has proven fertile ground for research too,<ref name=Douzery03>{{cite journal | vauthors = Douzery EJ, Delsuc F, Stanhope MJ, Huchon D | title = Local molecular clocks in three nuclear genes: divergence times for rodents and other mammals and incompatibility among fossil calibrations | journal = Journal of Molecular Evolution | volume = 57 | issue = Suppl 1 | pages = S201–S213 | year = 2003 | pmid = 15008417 | doi = 10.1007/s00239-003-0028-x | s2cid = 23887665 | citeseerx = 10.1.1.535.897 | bibcode = 2003JMolE..57S.201D }}</ref> even over comparatively short periods of evolutionary time (for example [[mockingbird]]s<ref name=Hunt01>{{cite journal | vauthors = Hunt JS, Bermingham E, Ricklefs RE |year=2001 |title=Molecular systematics and biogeography of Antillean thrashers, tremblers, and mockingbirds (Aves: Mimidae) |journal=[[Auk (journal)|Auk]] |volume=118 |issue=1 |pages=35–55|doi=10.1642/0004-8038(2001)118[0035:MSABOA]2.0.CO;2 |s2cid=51797284 |issn=0004-8038|doi-access=free }}</ref>). [[Procellariiformes|Tube-nosed seabirds]] have molecular clocks that on average run at half speed of many other birds,<ref name=Rheindt05>{{cite journal |author1=Rheindt, F. E.  |author2=Austin, J.  |name-list-style=amp |year=2005 |title=Major analytical and conceptual shortcomings in a recent taxonomic revision of the Procellariiformes – A reply to Penhallurick and Wink (2004) |journal=[[Emu (journal)|Emu]] |volume=105 |issue=2 |pages=181–186|url=http://www.publish.csiro.au/?act=view_file&file_id=MU04039.pdf |doi=10.1071/MU04039|bibcode=2005EmuAO.105..181R |s2cid=20390465 }}</ref> 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.<ref name=Avise92>{{cite journal | vauthors = Avise JC, Bowen BW, Lamb T, Meylan AB, Bermingham E | title = Mitochondrial DNA evolution at a turtle's pace: evidence for low genetic variability and reduced microevolutionary rate in the Testudines | journal = Molecular Biology and Evolution | volume = 9 | issue = 3 | pages = 457–473 | date = May 1992 | pmid = 1584014 | doi = 10.1093/oxfordjournals.molbev.a040735 | doi-access = free }}</ref> 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.<ref name=Ayala99>{{cite journal | vauthors = Ayala FJ | title = Molecular clock mirages | journal = BioEssays | volume = 21 | issue = 1 | pages = 71–75 | date = January 1999 | pmid = 10070256 | doi = 10.1002/(SICI)1521-1878(199901)21:1<71::AID-BIES9>3.0.CO;2-B | url = http://www3.interscience.wiley.com/cgi-bin/abstract/60000186/ABSTRACT?CRETRY=1&SRETRY=0 | url-status = dead | archive-url = https://archive.today/20121216135641/http://www3.interscience.wiley.com/cgi-bin/abstract/60000186/ABSTRACT?CRETRY=1&SRETRY=0 | archive-date = 16 December 2012 | url-access = subscription }}</ref><ref name=Schwartz06>{{cite journal |author1=Schwartz, J. H.  |author2=Maresca, B.  |name-list-style=amp |year=2006 |title=Do Molecular Clocks Run at All? A Critique of Molecular Systematics |journal=Biological Theory |volume=1 |pages=357–371|doi=10.1162/biot.2006.1.4.357 |issue=4|citeseerx=10.1.1.534.4502  |s2cid=28166727}}
*{{cite press release |date=February 12, 2007 |title=No Missing Link? Evolutionary Changes Occur Suddenly, Professor Says |website=[[ScienceDaily]] |url=https://www.sciencedaily.com/releases/2007/02/070210170623.htm}}</ref><ref name=Pascual2019>{{cite journal | vauthors = Pascual-García A, Arenas M, Bastolla U | title = The Molecular Clock in the Evolution of Protein Structures | journal = Systematic Biology | volume = 68 | issue = 6 | pages = 987–1002 | date = November 2019 | pmid = 31111152 | doi = 10.1093/sysbio/syz022 |doi-access= | name-list-style = amp | hdl = 20.500.11850/373053 | hdl-access = free }}</ref> According to Ayala's 1999 study, five factors combine to limit the application of molecular clock models:

* Changing generation times (If the rate of new mutations depends at least partly on the number of generations rather than the number of years)
* Population size ([[Genetic drift]] is stronger in small populations, and so more mutations are effectively neutral)
* Species-specific differences (due to differing metabolism, ecology, evolutionary history,&nbsp;...)
* Change in function of the protein studied (can be avoided in closely related species by utilizing [[non-coding DNA]] sequences or emphasizing [[silent mutation]]s)
* Changes in the intensity of natural selection.

[[File:Molecular evolution bamboos.svg|thumb|300px|left|alt=Phylogram showing three groups, one of which has strikingly longer branches than the two others|Woody bamboos (tribes [[Arundinarieae]] and [[Bambuseae]]) have long generation times and lower mutation rates, as expressed by short branches in the [[phylogenetic tree]], than the fast-evolving herbaceous bamboos ([[Olyreae]]).]]

Molecular clock users have developed workaround solutions using a number of statistical approaches including [[maximum likelihood]] techniques and later [[Bayesian statistics|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'''<ref name=Drummond06>{{cite journal | vauthors = Drummond AJ, Ho SY, Phillips MJ, Rambaut A | title = Relaxed phylogenetics and dating with confidence | journal = PLOS Biology | volume = 4 | issue = 5 | pages = e88 | date = May 2006 | pmid = 16683862 | pmc = 1395354 | doi = 10.1371/journal.pbio.0040088 | doi-access = free }}</ref> because they represent an intermediate position between the 'strict' molecular clock hypothesis and [[Joseph Felsenstein]]'s many-rates model<ref name=Felsenstein01>{{cite journal | vauthors = Felsenstein J | title = Taking variation of evolutionary rates between sites into account in inferring phylogenies | journal = Journal of Molecular Evolution | volume = 53 | issue = 4–5 | pages = 447–455 | year = 2001 | pmid = 11675604 | doi = 10.1007/s002390010234 | s2cid = 9791493 | bibcode = 2001JMolE..53..447F }}</ref> and are made possible through [[Markov chain Monte Carlo|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 (genetic)|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 function|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.<ref>Marshall, D. C., et al. 2016. Inflation of molecular clock rates and dates: molecular phylogenetics, biogeography, and diversification of a global cicada radiation from Australasia (Hemiptera: Cicadidae: Cicadettini). [https://academic.oup.com/sysbio/article/65/1/16/2461540/Inflation-of-Molecular-Clock-Rates-and-Dates Systematic Biology 65(1):16–34].</ref>

At very short time scales, many differences between samples do not represent [[Fixation (population genetics)|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 [[Fixation (population genetics)|fixed]]
leads to a potentially dramatic inflation of the apparent rate of the molecular clock at very short timescales.<ref name=":1">{{cite journal | vauthors = Ho SY, Phillips MJ, Cooper A, Drummond AJ | title = Time dependency of molecular rate estimates and systematic overestimation of recent divergence times | journal = Molecular Biology and Evolution | volume = 22 | issue = 7 | pages = 1561–1568 | date = July 2005 | pmid = 15814826 | doi = 10.1093/molbev/msi145 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Peterson GI, Masel J | title = Quantitative prediction of molecular clock and ka/ks at short timescales | journal = Molecular Biology and Evolution | volume = 26 | issue = 11 | pages = 2595–2603 | date = November 2009 | pmid = 19661199 | pmc = 2912466 | doi = 10.1093/molbev/msp175 }}</ref>

==Uses==
The molecular clock technique is an important tool in [[molecular systematics]], [[macroevolution]], and [[phylogenetic comparative methods]]. Estimation of the dates of [[phylogeny|phylogenetic]] events, including those not documented by [[fossils]], such as the divergences between living [[taxon|taxa]] has allowed the study of macroevolutionary processes in organisms that had limited fossil records. Phylogenetic comparative methods rely heavily on calibrated phylogenies.

== See also ==
* [[Charles Darwin]]
* [[Gene orders]]
* [[Human mitochondrial molecular clock]]
* [[Mitochondrial Eve]] and [[Y-chromosomal Adam]]
* [[Models of DNA evolution]]
* [[Molecular evolution]]
* [[Neutral theory of molecular evolution]]
* [[Glottochronology]]

== References ==
<!-- ZoolScripta35:531 molecular evolution speeds can differ markedly after few million years already -->
{{Reflist|2}}

== Further reading ==
* {{Cite book | editor = Ho, S.Y.W. | year = 2020 | title = The Molecular Evolutionary Clock: Theory and Practice | publisher = Springer, Cham | doi=10.1007/978-3-030-60181-2 | isbn = 978-3-030-60180-5 | s2cid = 231672167 }}
* {{cite journal | vauthors = Kumar S | title = Molecular clocks: four decades of evolution | journal = Nature Reviews. Genetics | volume = 6 | issue = 8 | pages = 654–662 | date = August 2005 | pmid = 16136655 | doi = 10.1038/nrg1659 | s2cid = 14261833 }}
* {{cite journal | vauthors = Morgan GJ | title = Emile Zuckerkandl, Linus Pauling, and the molecular evolutionary clock, 1959-1965 | journal = Journal of the History of Biology | volume = 31 | issue = 2 | pages = 155–178 | year = 1998 | pmid = 11620303 | doi = 10.1023/A:1004394418084 | s2cid = 5660841 }}
* {{Cite book | vauthors = Zuckerkandl E, Pauling LB | year = 1965 | title = Evolving Genes and Proteins | chapter = Evolutionary divergence and convergence in proteins | veditors = Bryson V, Vogel HJ | pages = 97–166 | publisher = Academic Press, New York|author2-link=Linus Pauling |author-link=Emile Zuckerkandl }}

== External links ==
* [https://web.archive.org/web/20100525101859/http://awcmee.massey.ac.nz/aw.htm Allan Wilson and the molecular clock]
* [https://web.archive.org/web/20090213150149/http://rtis.com/nat/user/elsberry/evobio/evc/argresp/sequence.html Molecular clock explanation of the molecular equidistance phenomenon]
* [https://web.archive.org/web/20061107013958/http://www.fossilrecord.net/dateaclade/index.html Date-a-Clade service for the molecular tree of life]

{{Chronology}}

{{DEFAULTSORT:Molecular Clock}}
[[Category:Evolutionary biology concepts]]
[[Category:Molecular evolution]]
[[Category:Molecular genetics]]
[[Category:Phylogenetics]]