Plastid phylogenomics and cytonuclear discordance in Rubioideae, Rubiaceae.

In this study of evolutionary relationships in the subfamily Rubioideae (Rubiaceae), we take advantage of the off-target proportion of reads generated via previous target capture sequencing projects based on nuclear genomic data to build a plastome phylogeny and investigate cytonuclear discordance. The assembly of off-target reads resulted in a comprehensive plastome dataset and robust inference of phylogenetic relationships, where most intratribal and intertribal relationships are resolved with strong support. While the phylogenetic results were mostly in agreement with previous studies based on plastome data, novel relationships in the plastid perspective were also detected. For example, our analyses of plastome data provide strong support for the SCOUT clade and its sister relationship to the remaining members of the subfamily, which differs from previous results based on plastid data but agrees with recent results based on nuclear genomic data. However, several instances of highly supported cytonuclear discordance were identified across the Rubioideae phylogeny. Coalescent simulation analysis indicates that while ILS could, by itself, explain the majority of the discordant relationships, plastome introgression may be the better explanation in some cases. Our study further indicates that plastomes across the Rubioideae are, with few exceptions, highly conserved and mainly conform to the structure, gene content, and gene order present in the majority of the flowering plants.

Phylogenomics and topological conflicts in the tribe Anthospermeae (Rubiaceae).

Genome skimming (shallow whole-genome sequencing) offers time- and cost-efficient production of large amounts of DNA data that can be used to address unsolved evolutionary questions. Here we address phylogenetic relationships and topological incongruence in the tribe Anthospermeae (Rubiaceae), using phylogenomic data from the mitochondrion, the nuclear ribosomal cistron, and the plastome. All three genomic compartments resolve relationships in the Anthospermeae; the tribe is monophyletic and consists of three major subclades. Carpacoce Sond. is sister to the remaining clade, which comprises an African subclade and a Pacific subclade. Most results, from all three genomic compartments, are statistically well supported; however, not fully consistent. Intergenomic topological incongruence is most notable in the Pacific subclade but present also in the African subclade. Hybridization and introgression followed by organelle capture may explain these conflicts but other processes, such as incomplete lineage sorting (ILS), can yield similar patterns and cannot be ruled out based on the results. Whereas the null hypothesis of congruence among all sequenced loci in the individual genomes could not be rejected for nuclear and mitochondrial data, it was rejected for plastid data. Phylogenetic analyses of three subsets of plastid loci identified using the hierarchical likelihood ratio test demonstrated statistically supported intragenomic topological incongruence. Given that plastid genes are thought to be fully linked, this result is surprising and may suggest modeling or sampling error. However, biological processes such as biparental inheritance and inter-plastome recombination have been reported and may be responsible for the observed intragenomic incongruence. Mitochondrial insertions into the plastome are rarely documented in angiosperms. Our results indicate that a mitochondrial insertion event in the plastid trnS GGA - rps4 IGS region occurred in the common ancestor of the Pacific clade of Anthospermeae. Exclusion/inclusion of this locus in phylogenetic analyses had a strong impact on topological results in the Pacific clade.

No phylogenomic support for a Cenozoic origin of the "living fossil" Isoetes.

PREMISE: The isoetalean lineage has a rich fossil record that extends to the Devonian, but the age of the living clade is unclear. Recent results indicate that it is young, from the Cenozoic, whereas earlier work based on less data from a denser taxon sampling yielded Mesozoic median ages. METHODS: We analyzed node ages in Isoetes using two genomic data sets (plastome and nuclear ribosomal cistron), three clock models implemented in MrBayes (ILN, WN, and TK02 models), and a conservative approach to calibration. RESULTS: While topological results were consistently resolved in Isoetes estimated crown group ages range from the latest Paleozoic (mid-Permian) to the Mesozoic depending on data type and clock model. The oldest estimates were retrieved using the autocorrelated TK02 clock model. An (early) Cenozoic age was only obtained under one specific condition (plastome data analyzed with the uncorrelated ILN clock model). That same plastome data set also yielded the oldest (mid-Permian) age estimate when analyzed with the autocorrelated TK02 clock model. Adding the highly divergent, recently established sister species Isoetes wormaldii to the data set approximately doubled the average median node depth to the Isoetes crown group. CONCLUSIONS: There is no consistent support for a Cenozoic origin of the living clade Isoetes. We obtained seemingly well-founded, yet strongly deviating results depending on data type and clock model. The single most important future improvement is probably to add calibration points, which requires an improved understanding of the isoetalean fossil record or alternative bases for calibration.

Target capture data resolve recalcitrant relationships in the coffee family (Rubioideae, Rubiaceae).

Subfamily Rubioideae is the largest of the main lineages in the coffee family (Rubiaceae), with over 8,000 species and 29 tribes. Phylogenetic relationships among tribes and other major clades within this group of plants are still only partly resolved despite considerable efforts. While previous studies have mainly utilized data from the organellar genomes and nuclear ribosomal DNA, we here use a large number of low-copy nuclear genes obtained via a target capture approach to infer phylogenetic relationships within Rubioideae. We included 101 Rubioideae species representing all but two (the monogeneric tribes Foonchewieae and Aitchinsonieae) of the currently recognized tribes, and all but one non-monogeneric tribe were represented by more than one genus. Using data from the 353 genes targeted with the universal Angiosperms353 probe set we investigated the impact of data type, analytical approach, and potential paralogs on phylogenetic reconstruction. We inferred a robust phylogenetic hypothesis of Rubioideae with the vast majority (or all) nodes being highly supported across all analyses and datasets and few incongruences between the inferred topologies. The results were similar to those of previous studies but novel relationships were also identified. We found that supercontigs [coding sequence (CDS) + non-coding sequence] clearly outperformed CDS data in levels of support and gene tree congruence. The full datasets (353 genes) outperformed the datasets with potentially paralogous genes removed (186 genes) in levels of support but increased gene tree incongruence slightly. The pattern of gene tree conflict at short internal branches were often consistent with high levels of incomplete lineage sorting (ILS) due to rapid speciation in the group. While concatenation- and coalescence-based trees mainly agreed, the observed phylogenetic discordance between the two approaches may be best explained by their differences in accounting for ILS. The use of target capture data greatly improved our confidence and understanding of the Rubioideae phylogeny, highlighted by the increased support for previously uncertain relationships and the increased possibility to explore sources of underlying phylogenetic discordance.

Phylogeny of the Madagascar-centred tribe Danaideae (Rubiaceae) as a precursor to taxonomic revision: insights into its generic and species limits, affinities and distribution.

BACKGROUND AND AIMS: The tribe Danaideae (Rubiaceae) is almost exclusively endemic to the Western Indian Ocean Region (WIOR), and encompasses the genera Danais, Payera and Schismatoclada that occur in humid, sub-humid and mountain and mountain bio climate zones. Much of the species diversity is endemic to restricted, remote and/or mountainous areas of Madagascar and recent field work on the island indicates substantial unknown diversity of the Danaideae. Furthermore, the monophyly of the Malagasy genera Payera and Schismatoclada has been questioned in previous work, species delimitations and phylogenetic relationships within the genera are poorly understood, and the distribution and evolution of gross morphological features have not been assessed. METHODS: We conducted morphological investigations, and produced robust phylogenies of Danaideae based on nuclear and plastid sequence data from 193 terminals. Ample plant material has been newly collected in the WIOR for the purpose of the present study, including potentially new species unknown to science. We performed Bayesian non-clock and relaxed-clock analyses employing three alternative clock models of a dataset with a dense sample of taxa from the entire geographical ranges of Danaideae. Based on the results, we discuss species diversity and distribution, relationships, and morphology in Danaideae. KEY RESULTS: Our results demonstrate the monophyly of Danaideae, its three genera and 42 species. Nine species are resolved as non-monophyletic. Many geographically distinct but morphologically heterogeneous lineages were identified, and morphological features traditionally considered diagnostic of subgroups of the genera, used for example in species identification keys, are not clade-specific. CONCLUSIONS: Our results demonstrate that Madagascar contains ample previously undocumented morphological and species diversity of Danaideae. Our novel approach to molecular phylogenetic analyses as a precursor to taxonomic revisions provides numerous benefits for the latter. There are tentative indications of parallel northward diversification in Payera and Schismatoclada on Madagascar, and of geographical phylogenetic clustering despite the anemochorous condition of Danaideae.

Phylogeny of Merlin's grass (Isoetaceae): revealing an "Amborella syndrome" and the importance of geographic distribution for understanding current and historical diversity.

BACKGROUND: Merlin's grass (Isoetes, Isoetaceae, Lycopsida), is the extant remnant of the isoetalean wood-producing lycopsids that originated during the Paleozoic, possibly in aquatic or boggy habitats. Modern day species are aquatic, semi-aquatic or terrestrial and occur almost worldwide. They display little morphological variation; the lobed corm has helically arranged leaves with internal air channels and basal sporangia. Genetic variation has also proven limited, which has hampered phylogenetic inference. We investigate evolutionary relationships in Isoetes, using molecular data and an extended sample of species compared to previous work, adding species that have never before been included in a phylogenetic study. RESULTS: Our results reveal an unexpected discovery of an "Amborella syndrome" in Isoetaceae: a single poorly known species is sister to the remaining family. The species, Isoetes wormaldii, is a rare endemic to the Eastern Cape of South Africa. Its leaves are flattened with a rounded point, which sharply contrasts with the awl-shaped leaves of most other species of Isoetes. The remaining species of Isoetes are resolved in five major clades, also indicated in previous work. While the phylogeny shows geographic structure, the patterns are complex. For example, tropical-southern African species occur in at least five clades, and Indian, Australian and Mediterranean species in at least three clades each. CONCLUSION: The evolutionary and biogeographical history of Isoetes is not easily explained, and may conceivably include ample extinction and a mixture of ancient and more recent processes. Previously shown difficulties with node age estimation increase the problem. The here demonstrated sister-relationship between the phylogenetically, morphologically and genetically distinct Isoetes wormaldii and the remaining family appears to bridge the morphological gap between Isoetes and its extinct relatives, although further studies are needed. Moreover, it shortens the branch length to its living sister genus Selaginella, and may enhance node age estimation in future studies. Isoetes wormaldii is critically endangered, known only from one (to a few) minor populations. Immediate actions need to be taken if we want to prevent this unique species from going extinct.

Conflicting results from mitochondrial genomic data challenge current views of Rubiaceae phylogeny.

PREMISE OF THE STUDY: Reconstruction of plant phylogeny has heavily relied on single-gene or multigene plastid data. New sequencing methods have led to an increasing number of studies based on data from the entire plastid, but the mitochondrion has rarely been used to infer plant phylogeny because of an assumed information poverty and demonstrated lateral transfer of mitochondrial gene regions between distantly related species. METHODS: We explored phylogenetic information from the plant mitochondrion using 57 representatives of the species-rich coffee family as study system and assessed consistency with previous results based (mostly) on plastid data. KEY RESULTS: We showed that the mitochondrial genome can provide structured and statistically significant information on plant phylogeny. While most of our results are consistent with those based on plastid data, some surprising and statistically significant conflicts emerge, and our study demonstrates with striking clarity that the phylogeny of Rubiaceae is far from resolved. CONCLUSIONS: It appears unlikely that conflicts between results retrieved from the different genomic compartments would be restricted to Rubiaceae. Rather, they are probably a general phenomenon and an important factor behind longstanding "difficult" phylogenetic questions. The biological processes responsible for the conflicting results detected here are unclear, but some conflicts are likely caused by hybridization events that occurred tens of millions of years ago. Whether such ancient events can be reconstructed based on molecular data from extant plants remains to be seen, but future studies of the nuclear genome may provide a way forward.

Historical biogeography and phylogeny of the pantropical Psychotrieae alliance (Rubiaceae), with particular emphasis on the Western Indian Ocean Region.

PREMISE OF THE STUDY: The Western Indian Ocean Region (WIOR) is a biodiversity hotspot providing an ideal setting for exploring the origins of insular biodiversity and dynamics of island colonization. We aimed to investigate the origins of the WIOR Psychotrieae alliance (Rubiaceae) with typically small, probably mainly bird-dispersed drupes, and the timing and direction or sequence of its colonization events in the region. METHODS: We used the program BEAST to estimate divergence times and Lagrange for biogeographic reconstruction. KEY RESULTS: The alliance has reached the WIOR at least 14 times via dispersals from Africa along with Asia and the Pacific mostly during the last 10 My, with at least one back-colonization to Africa. We inferred the earliest dispersal to Madagascar from the Pacific or Asia in the Miocene and numerous out-of-Madagascar dispersals to the nearby archipelagos but no dispersal out of those archipelagos. Gynochthodes with multiple fruits reached Madagascar twice from the Pacific possibly via ocean drifting. Psychotria with dry fruits (schizocarps) colonized Madagascar from the Pacific or Asia before reaching the Comoros from Madagascar possibly via wind dispersal. CONCLUSIONS: This study reinforces the pivotal role of dispersal in shaping the WIOR biodiversity and as the critical initiating step in the generation of endemic biodiversity on its islands. The WIOR alliance shows strong Asian and Pacific affinities despite the proximity of the region to Africa. Madagascar has served as a stepping-stone for subsequent dispersal to the rest of the region. The Afro-Malagasy-Seychelles genus Craterispermum and the Malagasy Puffia may represent relictual lineages.

The chloroplast genome of Ephedra foeminea (Ephedraceae, Gnetales), an entomophilous gymnosperm endemic to the Mediterranean area.

This study presents the chloroplast genome of Ephedra foeminea, an entomophilous gymnosperm, sister to the remaining (wind-pollinated) species of Ephedra (Ephedraceae, Gnetales). Based on the reference-guided assembly, the length of the chloroplast genome was estimated to be 109 584 bp, comprising a large single copy region of 60 027 bp, a small single copy 8079 bp, and inverted repeat regions of 20 739 bp. In total, 118 genes were detected, including 73 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The gene density is 1.076 (genes/kb) and the GC content is 36.7%. The genomic sequence of the entomophilous, Mediterranean species E. foeminea, differs from that of the anemophilous, Asian species E. equisetina by 1018 point mutations and 1334 indels. The detected variation is useful for future development of new plastid markers for phylogenetic purposes. Our phylogenetic analysis based on 55 protein-coding chloroplast genes resolve Ephedra as monophyletic and sister to a Gnetum-Welwitschia clade. The Gnetales are sister to Cupressophytes.

Correction: A Revised Time Tree of the Asterids: Establishing a Temporal Framework For Evolutionary Studies of the Coffee Family (Rubiaceae).

[This corrects the article DOI: 10.1371/journal.pone.0126690.].

A revised time tree of the asterids: establishing a temporal framework for evolutionary studies of the coffee family (rubiaceae).

Divergence time analyses in the coffee family (Rubiaceae) have all relied on the same Gentianales crown group age estimate, reported by an earlier analysis of the asterids, for defining the upper age bound of the root node in their analyses. However, not only did the asterid analysis suffer from several analytical shortcomings, but the estimate itself has been used in highly inconsistent ways in these Rubiaceae analyses. Based on the original data, we here reanalyze the divergence times of the asterids using relaxed-clock models and 14 fossil-based minimum age constraints. We also expand the data set to include an additional 67 taxa from Rubiaceae sampled across all three subfamilies recognized in the family. Three analyses are conducted: a separate analysis of the asterids, which completely mirrors the original asterid analysis in terms of taxon sample and data; a separate analysis of the Gentianales, where the result from the first analysis is used for defining a secondary root calibration point; and a combined analysis where all taxa are analyzed simultaneously. Results are presented in the form of a time-calibrated phylogeny, and age estimates for asterid groups, Gentianales, and major groups of Rubiaceae are compared and discussed in relation to previously published estimates. Our updated age estimates for major groups of Rubiaceae provide a significant step forward towards the long term goal of establishing a robust temporal framework for the divergence of this biologically diverse and fascinating group of plants.

Phylogeny and generic limits in the sister tribes Psychotrieae and Palicoureeae (Rubiaceae): Evolution of schizocarps in Psychotria and origins of bacterial leaf nodules of the Malagasy species.

• Premise of the study: The pantropical, species-rich Psychotrieae and Palicoureeae are sister tribes of mostly drupe-bearing and nonbacterial leaf-nodulating species with problematic generic limits. This problem is more complicated in Psychotrieae due to the paraphyly of the genus Psychotria, the lack of diagnostic characters for some major lineages, and the poor sampling from some biodiversity hotspots. Schizocarps and bacterial leaf nodules have been used for recognizing formal groups in Psychotrieae, but their evolution and taxonomic value have not been studied using a robust phylogeny of the tribe.• Methods: We analyzed 287 samples from the entire ranges of the tribes, with particular emphasis on the Western Indian Ocean region, with the Bayesian Markov chain Monte Carlo method.• Key results: All allied Psychotria genera investigated are nested within a paraphyletic Psychotria. Schizocarps evolved independently two times within Psychotria, and one reversal back to the drupaceous condition is inferred. The Malagasy leaf-nodulated Psychotrieae (except Apomuria bullata) and the Comorian non-leaf-nodulated Psychotria conocarpa are nested within the (African) leaf-nodulated clade. Within Palicoureeae, Chassalia is paraphyletic with respect to Geophila sensu stricto, and the Malagasy Geophila gerrardii and the African Hymenocoleus are closely related.• Conclusions: A widely circumscribed Psychotria encompassing the entire Psychotrieae is supported. Within Psychotria, two separate origins of schizocarps from drupes, one reversal back to the drupaceous condition, and two independent origins of the Malagasy leaf-nodulated species are inferred. A new genus Puffia is described to accommodate Geophila gerrardii, and a narrow circumscription of Chassalia is adopted. Thirty-two new combinations, two lectotypifications, and 25 new names are presented.

Inferring geographic range evolution of a pantropical tribe in the coffee family (Lasiantheae, Rubiaceae) in the face of topological uncertainty.

In this study we explore what historical biogeographic events are responsible for the wide and disjunct distribution of extant species in Lasiantheae, a pantropical group of trees and shrubs in the coffee family. Three of the genera in the group, Lasianthus, Saldinia, and Trichostachys, are found to be monophyletic, while there are indications that the fourth, Ronabea, is paraphyletic. We also address how the uncertainty in topology and divergence times affects the level of confidence in the biogeographic reconstruction. A data set consisting of chloroplast and nuclear ribosomal DNA data was analyzed using a Bayesian relaxed molecular clock approach to estimate phylogenetic relationships and divergence times, and the dispersal-extinction-cladogenesis (DEC) method to reconstruct geographic range evolution. Our results show that the Lasiantheae stem lineage originated in the neotropics, and the group expanded its range to the palaeotropics during the Eocene, either by continental migration through the boreotropics or by transatlantic long-distance dispersal. Two cases of Oligocene/Miocene over water-dispersal were also inferred, once from the paleotropics to the neotropics within Lasianthus, and once to Madagascar, concurrent with the origin of Saldinia. A lot of the diversification within Lasianthus took place during the Miocene and may have been influenced by climatic factors such as a period of markedly warm and moist climate in Asia and the aridification of the interior of the African continent. When biogeographic reconstructions were averaged over a random sample of 1000 dated phylogenies, the confidence in the biogeographic reconstruction decreased for most nodes, compared to when a single topology was used. A good understanding of phylogenetic relationships is necessary to understand the biogeographic history of a group, bit since the phylogeny is rarely completely known it is important to include phylogenetic uncertainty in biogeographic analysis. For nodes where the resolution is uncertain, the use of a single "best" topology as a basis for biogeographic analysis will result in inflated confidence in a biogeographic reconstruction which may be just one of several possible reconstructions.

Molecular dating and biogeography of fig-pollinating wasps.

Figs and fig-pollinating wasps are obligate mutualists that have coevolved for over 60 million years. But when and where did pollinating fig wasps (Agaonidae) originate? Some studies suggest that agaonids arose in the Late Cretaceous and the current distribution of fig-wasp faunas can be explained by the break-up of the Gondwanan landmass. However, recent molecular-dating studies suggest divergence time estimates that are inconsistent with the Gondwanan vicariance hypothesis and imply that long distance oceanic dispersal could have been an important process for explaining the current distribution of both figs and fig wasps. Here, we use a combination of phylogenetic and biogeographical data to infer the age, the major period of diversification, and the geographic origin of pollinating fig wasps. Age estimates ranged widely depending on the molecular-dating method used and even when using the same method but with slightly different constraints, making it difficult to assess with certainty a Gondwanan origin of agaonids. The reconstruction of ancestral areas suggests that the most recent common ancestor of all extant fig-pollinating wasps was most likely Asian, although a southern Gondwana origin cannot be rejected. Our analysis also suggests that dispersal has played a more important role in the development of the fig-wasp biota than previously assumed.

Occurrence and evolutionary origins of polyploids in the clubmoss genus Diphasiastrum (Lycopodiaceae).

Two polyploid taxa are commonly recognized in the genus Diphasiastrum, D. wightianum from Asia and D. zanclophyllum from South Africa and Madagascar. Here we present results from Feulgen DNA image densitometry analyses providing the first evidence for the polyploid origin of D. zanclophyllum. Also reported for the first time are data confirming that D. multispicatum and D. veitchii, representing the putative parent lineages of D. wightianum, are diploids. Phylogenetic analyses of the nuclear regions RPB2, LEAFY and LAMB4 reveal that putative tetraploid accessions are of allopolyploid origin. Diphasiastrum zanclophyllum shows close relationship to the North American taxon D. digitatum on the maternal side, but the paternal relationship is less clear. Two accessions from Asia, both inferred to be polyploid, have D. veitchii as the maternal parent, whereas the paternal paralogs show relationships to D. multispicatum and D. tristachyum, respectively. None of these parental combinations have previously been hypothesized.

Reconstructing the history of Campanulaceae with a Bayesian approach to molecular dating and dispersal-vicariance analyses.

We reconstruct here the spatial and temporal evolution of the Campanula alliance in order to better understand its evolutionary history. To increase phylogenetic resolution among major groups (Wahlenbergieae-Campanuleae), new sequences from the rbcL region were added to the trnL-F dataset obtained in a previous study. These phylogenies were used to infer ancestral areas and divergence times in Campanula and related genera using a Bayesian approach to molecular dating and dispersal-vicariance analyses that takes into account phylogenetic uncertainty. The new phylogenetic analysis confirms Platycodoneae as the sister group of Wahlenbergieae-Campanuleae, the two last ones inter-graded into a well-supported clade. Biogeographic and dating analyses suggest that Western Asia and the Eastern Mediterranean have played a major role as centers of migration and diversification within the Campanula alliance, probably in relation to the intense orogenic activity that took place in this region during the Late Neogene, and that could have promoted isolation and allopatric speciation within lineages. Diversification rates within several Campanula lineages would have increased at the end of the Miocene, coinciding with the Messinian Stage. Strong selective pressures from climate changes and the expansion of mountainous regions during this period are suggested to explain the adaptation to drought, cold or disturbed environments observed in many Campanula species. Several independent long-distance dispersal events to North America are inferred within the Rapunculus clade, which seem to be related to high ploidy levels.

Incongruence between primary sequence data and the distribution of a mitochondrial atp1 group II intron among ferns and horsetails.

Using DNA sequence data from multiple genes (often from more than one genome compartment) to reconstruct phylogenetic relationships has become routine. Augmenting this approach with genomic structural characters (e.g., intron gain and loss, changes in gene order) as these data become available from comparative studies already has provided critical insight into some long-standing questions about the evolution of land plants. Here we report on the presence of a group II intron located in the mitochondrial atp1 gene of leptosporangiate and marattioid ferns. Primary sequence data for the atp1 gene are newly reported for 27 taxa, and results are presented from maximum likelihood-based phylogenetic analyses using Bayesian inference for 34 land plants in three data sets: (1) single-gene mitochondrial atp1 (exon+intron sequences); (2) five combined genes (mitochondrial atp1 [exon only]; plastid rbcL, atpB, rps4; nuclear SSU rDNA); and (3) same five combined genes plus morphology. All our phylogenetic analyses corroborate results from previous fern studies that used plastid and nuclear sequence data: the monophyly of euphyllophytes, as well as of monilophytes; whisk ferns (Psilotidae) sister to ophioglossoid ferns (Ophioglossidae); horsetails (Equisetopsida) sister to marattioid ferns (Marattiidae), which together are sister to the monophyletic leptosporangiate ferns. In contrast to the results from the primary sequence data, the genomic structural data (atp1 intron distribution pattern) would seem to suggest that leptosporangiate and marattioid ferns are monophyletic, and together they are the sister group to horsetails--a topology that is rarely reconstructed using primary sequence data.

Molecular evidence on plant divergence times.

Estimation of divergence times from sequence data has become increasingly feasible in recent years. Conflicts between fossil evidence and molecular dates have sparked the development of new methods for inferring divergence times, further encouraging these efforts. In this paper, available methods for estimating divergence times are reviewed, especially those geared toward handling the widespread variation in rates of molecular evolution observed among lineages. The assumptions, strengths, and weaknesses of local clock, Bayesian, and rate smoothing methods are described. The rapidly growing literature applying these methods to key divergence times in plant evolutionary history is also reviewed. These include the crown group ages of green plants, land plants, seed plants, angiosperms, and major subclades of angiosperms. Finally, attempts to infer divergence times are described in the context of two very different temporal settings: recent adaptive radiations and much more ancient biogeographic patterns.