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This article reviews the most common methods used today for estimating divergence times and rates of molecular evolution. The methods are grouped into three main classes: (1) methods that use a molecular clock and one global rate of substitution, (2) methods that correct for rate heterogeneity, and (3) methods that try to incorporate rate heterogeneity. Additionally, links to the most important literature on molecular dating are given, including articles comparing the performance of different methods, papers that investigate problems related to taxon, gene and partition sampling, and literature discussing highly debated issues like calibration strategies and uncertainties, dating precision and the calculation of error estimates. KeywordsDivergence time estimation, molecular dating methods, rate heterogeneity, review. INTRODUCTIONThe use of DNA sequences to estimate the timing of evolutionary events is increasingly popular. The idea of dating evolutionary divergences using calibrated sequence differences was first proposed in 1965 by Zuckerkandl and Pauling (1965). The authors postulated that the amount of difference between the DNA molecules of two species is a function of the time since their evolutionary separation. This was shown by comparing protein sequences (hemoglobins) from different species and further comparing amino acid substitution rates with ages estimated from fossils. Based on this central idea, molecular dating has been used in countless studies as a method to investigate mechanisms and processes of evolution. For example, the timing of the eucaryotic evolution (Douzery et al ., 2004), the Early Cambrian origin of the main phyla of animals (Cambrian explosion; Wray et al ., 1996;Smith & Peterson, 2002;Aris-Brosou & Yang, 2003), the replacement of dinosaurs by modern birds and mammals in the late Tertiary (Madsen et al ., 2001), and the age of the last common ancestor of the main pandemic strain of human immunodeficiency virus (HIV; Korber et al. , 2000) have all been investigated using molecular dating. Also in plants, there are numerous studies where molecular dating methods have been used to investigate the timeframe of evolutionary events, e.g. for testing biogeographical hypotheses or to investigate the causes of recent radiations (for a more complete review see Sanderson et al ., 2004). For example, dating techniques have been applied on taxa from very different taxonomic levels, e.g. to infer the age of plastid-containing eucaryotes (Yoon et al ., 2004) The goal of this article is to give a short overview on the most commonly used molecular dating methods. To allow for easier comparisons, the different methods for estimating divergence times are also summarized in Tables 1-3. A. The ideal case scenario: a molecular clock and one global rate of substitutionIn the special case of a molecular clock, all branches of a phylogenetic tree evolve at the same, global substitution rate. The clock-like tree is ultrametric, which means that the total distance between the root and every tip is ...

Aim  The flowering plant family Proteaceae is putatively of Gondwanan age, with modern and fossil lineages found on all southern continents. Here we test whether the present distribution of Proteaceae can be explained by vicariance caused by the break‐up of Gondwana. Location  Africa, especially southern Africa, Australia, New Zealand, South America, New Caledonia, New Guinea, Southeast Asia, Sulawesi, Tasmania. Methods  We obtained chloroplast DNA sequence data from the rbcL gene, the rbcL‐atpB spacer, and the atpB gene from leaf samples of forty‐five genera collected from the field and from living collections. We analysed these data using Bayesian phylogenetic and molecular dating methods, with five carefully selected fossil calibration points to obtain age estimates for the nodes within the family. Results  Four of eight trans‐continental disjunctions of sister groups within our sample of the Proteaceae post‐date the break‐up of Gondwana. These involve independent lineages, two with an Africa‐Australia disjunction, one with an Africa–South America disjunction, and one with a New Zealand–Australasia disjunction. The date of the radiation of the bird‐pollinated Embothriinae corresponds approximately to the hypothesized date of origin of nectar‐feeding birds in Australia. Main conclusions  The findings suggest that disjunct distributions in Proteaceae result from both Gondwanan vicariance and transoceanic dispersal. Our results imply that ancestors of some taxa dispersed across oceans rather than rafting with Gondwanan fragments as previously thought. This finding agrees with other studies of Gondwanan plants in dating the divergence of Australian, New Zealand and New Caledonian taxa in the Eocene, consistent with the existence of a shared, ancestral Eocene flora but contrary to a vicariance scenario based on accepted geological knowledge.

Although recent methodological advances have allowed the incorporation of rate variation in molecular dating analyses, the calibration procedure, performed mainly through fossils, remains resistant to improvements. One source of uncertainty pertains to the assignment of fossils to specific nodes in a phylogeny, especially when alternative possibilities exist that can be equally justified on morphological grounds. Here we expand on a recently developed fossil cross-validation method to evaluate whether alternative nodal assignments of multiple fossils produce calibration sets that differ in their internal consistency. We use an enlarged Crypteroniaceae-centered phylogeny of Myrtales, six fossils, and 72 combinations of calibration points, termed calibration sets, to identify (i) the fossil assignments that produce the most internally consistent calibration sets and (ii) the mean ages, derived from these calibration sets, for the split of the Southeast Asian Crypteroniaceae from their West Gondwanan sister clade (node X). We found that a correlation exists between s values, devised to measure the consistency among the calibration points of a calibration set (Near and Sanderson, 2004), and nodal distances among calibration points. By ranking all sets according to the percent deviation of s from the regression line with nodal distance, we identified the sets with the highest level of corrected calibration-set consistency. These sets generated lower standard deviations associated with the ages of node X than sets characterized by lower corrected consistency. The three calibration sets with the highest corrected consistencies produced mean age estimates for node X of 79.70, 79.14, and 78.15 My. These timeframes are most compatible with the hypothesis that the Crypteroniaceae stem lineage dispersed from Africa to the Deccan plate as it drifted northward during the Late Cretaceous.

The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2,K  m = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of theLeCPK1 N terminus was found to be required for plasma membrane targeting.