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A point mutation changes an individual nucleotide position, introducing heritable sequence variation. Replication errors and spontaneous chemical damage are possible origins; mutation does not occur because a future change would be useful. Natural selection changes the frequencies of variants through their effects on reproduction, while genetic drift causes random frequency changes, especially in small populations. A sequence difference fixed between lineages is a substitution; many new mutations disappear and never become substitutions. Under a simple neutral diploid model, $2N_e\mu$ new neutral variants per generation times fixation probability $1/(2N_e)$ gives substitution rate $\mu$, motivating a molecular clock for suitable neutral sites.

To infer a phylogenetic tree, compare homologous, preferably orthologous sequences and align corresponding sites. A nucleotide substitution model converts observed sequence differences into estimated evolutionary branch lengths, allowing for multiple changes at one site. For example, in the equal-frequency, equal-rate Jukes-Cantor model, a site changes to any alternative with rate $\mu /3$, where $\mu$ is its total substitution rate. The probability of matching its initial state after elapsed time $t$ is $1/4+(3/4)e^{-4\mu t/3}$. Thus for an evolutionary separation with expected substitutions per site $d$, the observed mismatch fraction is $p=(3/4)(1-e^{-4d/3})$ andFor small $p$, $d\simeq p$; near saturation $p=3/4$, large evolutionary distances become difficult to distinguish. The correction does not rescue fully saturated data. Real analyses may allow unequal nucleotide frequencies, transition biases and variation of rates between sites.

Tree topology and branch lengths can be estimated using maximum likelihood estimation or Bayesian phylogenetic inference, with support assessed from resampling or posterior uncertainty. A gene tree need not equal the species tree, because incomplete lineage sorting, genetic recombination and horizontal gene transfer can produce different histories. Rooting with an appropriate outgroup establishes ancestral direction; pairwise differences alone do not supply a root.

Branch lengths measured in substitutions per site are not yet calendar times. Under a strict molecular clock with rate $\mu$ per site per year, a branch of duration $t$ has length $\mu t$; two contemporaneously sampled descendants separated at time $t$ have total path length $d=2\mu t$, hence $\boxed{t=d/(2\mu )}$. Calibration comes from independently dated constraints such as fossil calibration, geological information or sufficiently informative sampling dates. A dated common ancestor is not automatically identical in age to the earliest observed fossil. A relaxed molecular clock permits branch-specific rates while imposing a statistical model on them.

Without an external temporal calibration or an independently known rate, multiplying all times by a constant and dividing all rates by that constant leaves expected sequence divergence unchanged. Absolute dates are therefore unidentifiable from sequence divergence alone. A useful time-calibrated phylogenetic tree reports uncertainty from sequence sampling, substitution models, rate variation, topology and calibration bounds, rather than displaying an unjustified exact date for each ancestor.

Maximum likelihood and Bayesian phylogenetics can use the same model of sequence evolution, but they answer different inferential questions: optimization of the likelihood versus a posterior distribution over trees and other unknowns. Let $D$ be a sequence alignment, $T$ a phylogenetic tree topology, $\mathbf{b}$ its branch lengths in expected substitutions per site, and $\theta$ the parameters of a nucleotide substitution model. Both methods can useThe likelihood function sums over unobserved ancestral states, rather than choosing one ancestral sequence and treating it as observed. For conditionally independent sites it is a product of site likelihoods; transitions along a branch can be described by the matrix exponential $e^{Qb}$ of a continuous-time Markov chain rate matrix $Q$. The phylogenetic pruning algorithm computes the ancestral sums efficiently.^[ref]

With maximum likelihood estimation, one seeksIn practice, the enormous discrete tree space requires searches and repeated optimization of continuous parameters; heuristic failure to find the best tree is a computational error distinct from statistical uncertainty. A tree's optimized likelihood is not the probability that it is correct. bootstrap analyses resample alignment columns and repeat inference to assess support under the resampling procedure; their frequencies are not automatically posterior probabilities. Independence assumptions matter, because linked sites and heterogeneous loci can make simple column resampling an imperfect representation of replication.

In Bayesian phylogenetic inference,The prior distribution supplies weights on topologies, branch lengths and model parameters. Integrating over nuisance parameters givesThus a very narrow likelihood peak can lose posterior support to a broader region with a slightly lower maximum. maximum a posteriori estimation is another optimization and does not replace this integration. Markov chain Monte Carlo provides dependent samples approximating the posterior distribution; a clade's posterior probability is the posterior mass of trees containing that clade. The early likelihood-based Bayesian formulation explicitly distinguished these probabilities from bootstrap support.^[ref]

Neither approach is protected against systematic error. Poor sequence alignment, incorrect orthology assignments, compositionally heterogeneous sequences, and an inadequate substitution process can support a wrong tree with high apparent confidence. A gene tree can differ from a species tree because of incomplete lineage sorting or introgression; extra sequence from that gene does not automatically fix the mismatch. Model comparison, biological examination of the data and sensitivity to locus sampling are needed in both approaches. In Bayesian analyses, agreement among short chains is not proof of convergence: independent runs, adequate mixing between topologies and an appropriate effective sample size of a Markov chain should be checked. A credible interval and a confidence interval also have different interpretations even if their numerical endpoints happen to resemble one another.

Time inference adds a particularly strong role for assumptions. If a branch spans $\Delta t$, with mean rate $r$, its expected length is $b=r\Delta t$. Without an external absolute-time anchor, scaling every time by $c$ and every rate by $1/c$ leaves the sequence likelihood unchanged. Even a very long alignment can identify branch lengths accurately without identifying an absolute calendar scale. For example, a lineage length $b=0.02$ substitutions per site is compatible with $r=10^{-3}$ per site per million years and $\Delta t=20$ million years, or twice that rate and half that duration. This is an identifiability issue, not merely a shortage of sampled bases.

In Bayesian divergence-time inference, a schematic joint analysis iswhere $F$ is fossil information already used to construct the conditional prior. An alternative generative analysis includes a fossil-sampling likelihood explicitly; the same fossil evidence must not be counted twice. A strict molecular clock equates rates across branches; a relaxed molecular clock allows variation, whose distribution and correlations also require specification. A narrow rate prior favors particular absolute ages, and a strong rate-smoothing assumption can redistribute inferred time among branches. A birth-death process prior on the tree encodes assumptions about lineage origination, extinction and sampling; it is not a neutral choice just because it is mathematically convenient.

fossil calibration must separate a fossil's geological age from the age of the lineage split it constrains. A confidently placed crown fossil gives a minimum age for the relevant crown, whereas a stem fossil does not automatically set that crown minimum. Uncertain placement and the incompleteness of the record complicate upper bounds. hard calibration bounds assign zero probability outside the permitted range; enough sequence cannot overcome a zero prior. soft calibration bounds allow small tail probabilities, exposing rather than eliminating the possibility of conflict. Multiple-calibration analyses demonstrate that changing the treatment of bounds changes inferred times and uncertainty.^[ref]

Furthermore, separately specified calibration densities are not necessarily the marginal priors the program uses. The rule that an ancestor must be older than its descendant truncates their joint possibilities. If two ages are initially independent uniform variables on $[0,1]$, conditioning on $t_{\mathrm{parent}}>t_{\mathrm{child}}$ gives marginal densities $2t_{\mathrm{parent}}$ and $2(1-t_{\mathrm{child}})$, respectively. Neither remains uniform. Combining tree-process priors, calibration densities and ordering constraints can therefore introduce substantial information unintentionally.^[ref]

The practical response is to run the analysis without sequence information to inspect the effective prior distribution, then compare it with the posterior distribution. Repeat under defensible calibration tails, rate models, tree priors and fossil placements; report which ages change and whether the data sharpen relative ages more than absolute ages. Examine prior predictive checks and posterior predictive checks, and keep numerical mixing diagnostics separate from biological model adequacy. Priors can legitimately supply the time information missing from sequences, but narrow posterior ages are persuasive only when that information and its sensitivity are explicit.