Former diversity of Ephedra (Gnetales): evidence from Early Cretaceous seeds from Portugal and North America

BACKGROUND AND AIMS: The extant species of the seed plant group Gnetales (Ephedra, Gnetum and Welwitschia) have been considered a remnant of a much greater, now extinct, diversity due to the pronounced differences in form and ecology among the genera. Until recently, this hypothesis has not been supported by evidence from the fossil record. This paper adds to the expanding information on Gnetales from the Early Cretaceous and describes coalified seeds from Barremian-Albian localities in Portugal and USA. METHODS: The fossils were extracted from sediment samples by sieving in water. Adhering mineral matrix was removed by chemical treatment. Seeds were investigated using light and scanning electron microscopy. Morphology and anatomy of the seeds were documented and compared with those of extant species. KEY RESULTS: The fossils share characters with extant Ephedra, for example papillae on the inner surface of the seed envelope and in situ polyplicate pollen grains that shed the exine during germination. They differ from extant Ephedra seeds in morphological and anatomical details as well as in their smaller size. Two new species of Ephedra are described together with one species assigned to a new genus of Gnetales. Other Ephedra-like seeds, for which pollen and critical morphological details are currently unknown, are also present in the samples. CONCLUSIONS: These Cretaceous seeds document that key reproductive characters and pollen germination processes have remained unchanged within Ephedra for about 120 million years or more. There is sufficient variety in details of morphology to suggest that a diversity of Ephedra and Ephedra-like species were present in the Early Cretaceous flora. Their presence in Portugal and eastern North America indicates that they were widespread on the Laurasian continent. The fossil seeds are similar to seeds of Erdtmanithecales and this supports the previously suggested relationship between Erdtmanithecales and Gnetales.     

Estimation of the age of extant Ephedra using chloroplast rbcL sequence data

The distinctive gymnosperm genus Ephedra is sometimes considered to have originated over 200 million years (Myr) ago on the basis of "ephedroid" fossil pollen. In this article we estimate the age of extant Ephedra using chloroplast rbcL gene sequences. Relative rate tests fail to reject the null hypothesis of equal rates of nucleotide substitution of the rbcL sequences among three landmark lineages (Gnetales, Pinaceae, and Ginkgo). The most divergent sequences we have found in Ephedra differ by only 7 bp for an 1,110 bp region of rbcL sequence, whereas the differences among genera range from 92 to 107 bp in Gnetales and from 35 to 92 bp in Pinaceae. Using three landmark events, the age of extant Ephedra is estimated to be approximately 8-32 Myr. Our result is consistent with the current distribution of many Ephedra species in geologically recent habitats and points out difficulties in the identification of older ephedroid pollen fossils with the modern genus Ephedra.     

Failure of DNA barcoding in discriminating Calligonum species

We tested the effectiveness of four DNA barcoding markers (rbcL, matK, ITS and trnLF region) for land plants in identifying Calligonum species. High quality sequences were obtained for rbcL, matK and trnLF with the universal primers whereas ITS sequences were of poor quality. RbcL and matK were highly conservative and failed in species discrimination. When rbcL, matK and trnLF were combined, the species resolution was up to 6.25%. Low sequence variation resulted in poorly resolved tree topologies. Among the sixteen sampled species, only three were recovered as a monophyletic group. Our results show that although DNA barcoding is an important tool for species identification, it fails in discriminating Calligonum species. Further research will be needed to develop markers capable to discriminate species in this taxonomy complicated and recently diverged genus.     

Phylogeny of seed plants based on evidence from eight genes

Relationships among the five groups of extant seed plants (cycads, Ginkgo, conifers, Gnetales, and angiosperms) remain uncertain. To explore relationships among groups of extant seed plants further and to attempt to explain the conflict among molecular data sets, we assembled a data set of four plastid (cpDNA) genes (rbcL, atpB, psaA, and psbB), three mitochondrial (mtDNA) genes (mtSSU, coxI, and atpA), and one nuclear gene (18S rDNA) for 19 exemplars representing the five groups of living seed plants. Analyses of the combined eight-gene data set (15?772 base pairs/taxon) with maximum parsimony (MP), maximum likelihood (ML), and Bayesian approaches reveal a gymnosperm clade that is sister to angiosperms. Within the gymnosperms, a conifer clade includes Gnetales as sister to Pinaceae. Cycads and Ginkgo are either successive sisters to this conifer clade (including Gnetales) or a clade that is sister to conifers and Gnetales. All analyses of the mtDNA partition and ML analyses of the nuclear partition yield very similar topologies. However, MP analyses of the combined cpDNA genes place Gnetales as sister to all other seed plants with strong bootstrap support, whereas ML and Bayesian analyses of the cpDNA data set place Gnetales as sister to Pinaceae. Maximum parsimony and ML analyses of first and second codon positions of the cpDNA partiation also place Gnetales as sister to Pinaceae. In contrast, MP analyses of third codon positions place Gnetales as sister to other seed plants, although ML analyses of third codon positions place Gnetales with Pinaceae. Thus, most of the discrepancies in seed plant topologies involve third codon positions of cpDNA genes. The likelihood ratio (LR) and Shimodaira-Hasegasa (SH) tests were applied to the cpDNA data. The preferred topology based on the LR test is that Gnetales are sister to Pseudotsuga. The SH test based on first and second codon and all three codon positions indicated that there is no significant difference between the best topology (Gnetales sister to Pseudotsuga) and Gnetales sister to a conifer clade. However, there is a significant difference between the best topology and topologies in which Gnetales are sister to the rest of the seed plants or Gnetales sister to angiosperms.     

Timing transantarctic disjunctions in the Atherospermataceae (Laurales): evidence from coding and noncoding chloroplast sequences

Previous studies of the small Southern Hemisphere family Atherospermataceae have drawn contradictory conclusions regarding the number of transantarctic disjunctions and role of transoceanic dispersal in its evolution. Clarification of intergeneric relationships is critical to resolving (1) whether the two Chilean species, Laurelia sempervirens and Laureliopsis philippiana, are related to different Austral-Pacific species, implying two transantarctic disjunctions as suggested by morphology; (2) where the group is likely to have originated; and (3) whether observed disjunctions reflect the breakup of Gondwana. We analyzed chloroplast DNA sequences from six regions (the rbcL gene, the rpl16 intron, and the trnL-trnF, trnT-trnL, psbA-trnH, and atpB-rbcL spacer regions; for all six regions, 4,372 bp) for all genera and most species of Atherospermataceae, using parsimony and maximum likelihood (ML). The family's sister group, the Chilean endemic Gomortega nitida (Gomortegaceae), was used to root the tree. Parsimony and ML yielded identical single best trees that contain three well-supported clades (> or = 75% bootstrap): Daphnandra and Doryphora from south-eastern Australia; Atherosperma and Nemuaron from Australia-Tasmania and New Caledonia, respectively; and Laurelia novac-zelandiac and Laureliopsis philippiana from New Zealand and Chile, respectively. The second Chilean species, Laurelia sempervirens, is sister to this last clade. Likelihood ratio testing did not reject the molecular clock assumption for the rbcL data, which can therefore be used for divergence time estimates. The atherosperm fossil record, which goes back to the Upper Cretaceous, includes pollen, wood, and leaf fossils from Europe, Africa, South America, Antarctica, New Zealand, and Tasmania. Calibration of rbcL substitution rates with the fossils suggests an initial diversification of the family at 100-140 million years ago (MYA), probably in West Gondwana, early entry into Antarctica, and long-distance dispersal to New Zealand and New Caledonia at 50-30 MYA by the ancestors of L. novae-zelandiae and Nemuaron.     

A fossil-calibrated relaxed clock for Ephedra indicates an Oligocene age for the divergence of Asian and New World clades and Miocene dispersal into South America

Ephedra comprises approximately 50 species, which are roughly equally distributed between the Old and New World deserts, but not in the intervening regions （amphitropical range）. Great heterogeneity in the substitution rates of Gnetales （Ephedra, Gnetum, and Welwitschia） has made it difficult to infer the ages of the major divergence events in Ephedra, such as the timing of the Beringian disjunction in the genus and the entry into South America. Here, we use data from as many Gnetales species and genes as available from GenBank and from a recent study to investigate the timing of the major divergence events. Because of the tradeoff between the amount of missing data and taxon/gene sampling, we reduced the initial matrix of 265 accessions and 12 loci to 95 accessions and 10 loci, and further to 42 species （and 7736 aligned nucleotides） to achieve stationary distributions in the Bayesian molecular clock runs. Results from a relaxed clock with an uncorrelated rates model and fossil-based calibration reveal that New World species are monophyletic and diverged from their mostly Asian sister clade some 30 mya, fitting with many other Beringian disjunctions. The split between the single North American and the single South American clade occurred approximately 25 mya, well before the closure of the Panamanian Isthmus. Overall, the biogeographic history of Ephedra appears dominated by long-distance dispersal, but finer-scale studies are needed to test this hypothesis.     

Dated Phylogenies of the Sister Genera Macaranga and Mallotus (Euphorbiaceae): Congruence in Historical Biogeographic Patterns?

Molecular phylogenies and estimates of divergence times within the sister genera Macaranga and Mallotus were estimated using Bayesian relaxed clock analyses of two generic data sets, one per genus. Both data sets were based on different molecular markers and largely different samples. Per genus three calibration points were utilised. The basal calibration point (crown node of all taxa used) was taken from literature and used for both taxa. The other three calibrations were based on fossils of which two were used per genus. We compared patterns of dispersal and diversification in Macaranga and Mallotus using ancestral area reconstruction in RASP (S-DIVA option) and contrasted our results with biogeographical and geological records to assess accuracy of inferred age estimates. A check of the fossil calibration point showed that the Japanese fossil, used for dating the divergence of Mallotus, probably had to be attached to a lower node, the stem node of all pioneer species, but even then the divergence time was still younger than the estimated age of the fossil. The African (only used in the Macaranga data set) and New Zealand fossils (used for both genera) seemed reliably placed. Our results are in line with existing geological data and the presence of stepping stones that provided dispersal pathways from Borneo to New Guinea-Australia, from Borneo to mainland Asia and additionally at least once to Africa and Madagascar via land and back to India via Indian Ocean island chains. The two genera show congruence in dispersal patterns, which corroborate divergence time estimates, although the overall mode and tempo of dispersal and diversification differ significantly as shown by distribution patterns of extant species.     

Evolutionary rates analysis of Leguminosae implicates a rapid diversification of lineages during the tertiary

Tertiary macrofossils of the flowering plant family Leguminosae (legumes) were used as time constraints to estimate ages of the earliest branching clades identified in separate plastid matK and rbcL gene phylogenies. Penalized likelihood rate smoothing was performed on sets of Bayesian likelihood trees generated with the AIC-selected GTR+ Gamma +I substitution model. Unequivocal legume fossils dating from the Recent continuously back to about 56 million years ago were used to fix the family stem clade at 60 million years (Ma), and at 1-Ma intervals back to 70 Ma. Specific fossils that showed distinctive combinations of apomorphic traits were used to constrain the minimum age of 12 specific internal nodes. These constraints were placed on stem rather than respective crown clades in order to bias for younger age estimates. Regardless, the mean age of the legume crown clade differs by only 1.0 to 2.5 Ma from the fixed age of the legume stem clade. Additionally, the oldest caesalpinioid, mimosoid, and papilionoid crown clades show approximately the same age range of 39 to 59 Ma. These findings all point to a rapid family-wide diversification, and predict few if any legume fossils prior to the Cenozoic. The range of the matK substitution rate, 2.1-24.6 x 10(-10) substitutions per site per year, is higher than that of rbcL, 1.6- 8.6 x 10(-10), and is accompanied by more uniform rate variation among codon positions. The matK and rbcL substitution rates are highly correlated across the legume family. For example, both loci have the slowest substitution rates among the mimosoids and the fastest rates among the millettioid legumes. This explains why groups such as the millettioids are amenable to species-level phylogenetic analysis with these loci, whereas other legume groups are not.     

40种蕨类植物matK基因的系统分类及分子进化研究

采用“放松分子钟”模型、氨基酸位点正选择模型和分子内共进化网络估算方法,对蕨类植物Ⅱ型内含子成熟酶蛋白K(Maturase K,MATK)编码基因matK的进化趋势进行研究。结果显示:matK基因在蕨类植物系统学研究中具有一定的应用价值,与rbcL基因和psaA基因联合后能显著提升系统发育树的可信度;蕨类植物MATK蛋白中存在少数曾经历正选择的位点;MATK蛋白内部有多对氨基酸位点共同构成共进化网络。在被子植物兴起环境改变后,MATK蛋白部分位点发生适应性进化,通过位点间共进化网络协同作用方式提升蕨类植物对新光合环境的适应能力。     

Multiple Miocene Melastomataceae dispersal between Madagascar, Africa and India

Melastomataceae sensu stricto (excluding Memecylaceae) comprise some 3000 species in the neotropics, 1000 in Asia, 240 in Africa, and 230 in Madagascar. Previous family-wide morphological and DNA analyses have shown that the Madagascan species belong to at least three unrelated lineages, which were hypothesized to have arrived by trans-oceanic dispersal. An alternative hypothesis posits that the ancestors of Madagascan, as well as Indian, Melastomataceae arrived from Africa in the Late Cretaceous. This study tests these hypotheses in a Bayesian framework, using three combined sequence datasets analysed under a relaxed clock and simultaneously calibrated with fossils, some not previously used. The new fossil calibration comes from a re-dated possibly Middle or Upper Eocene Brazilian fossil of Melastomeae. Tectonic events were also tentatively used as constraints because of concerns that some of the family's fossils are difficult to assign to nodes in the phylogeny. Regardless of how the data were calibrated, the estimated divergence times of Madagascan and Indian lineages were too young for Cretaceous explanations to hold. This was true even of the oldest ages within the 95% credibility interval around each estimate. Madagascar's Melastomeae appear to have arrived from Africa during the Miocene. Medinilla, with some 70 species in Madagascar and two in Africa, too, arrived during the Miocene, but from Asia. Gravesia, with 100 species in Madagascar and four in east and west Africa, also appears to date to the Miocene, but its monophyly has not been tested. The study afforded an opportunity to compare divergence time estimates obtained earlier with strict clocks and single calibrations, with estimates based on relaxed clocks and different multiple calibrations and taxon sampling.     

The first mistletoes: origins of aerial parasitism in Santalales

Past molecular phylogenetic work has shown that aerial parasites have evolved five times independently in the sandalwood order (Santalales), but the absolute timing of these diversifications was not addressed. DNA sequences from nuclear SSU and LSU rDNA, and chloroplast rbcL, matK and trnL-F from 39 santalalean taxa were obtained. Separate and combined data partitions were analyzed with maximum parsimony and Bayesian inference. Time estimates were performed with Bayesian relaxed molecular clock and penalized likelihood methods using published fossil data. Both methods gave comparable divergence dates for the major clades. These data confirm five origins of aerial parasitism, first in Misodendraceae ca. 80 Mya and subsequently in Viscaceae (72 Mya), "Eremolepidaceae" (53 Mya), tribe Amphorogyneae in Santalaceae (46 Mya), and Loranthaceae (28 Mya). The rapid adaptive radiation and speciation in Loranthaceae coincides with the appearance of savanna biomes during the Oligocene. In all clades except Misodendraceae, it appears that aerial parasites evolved from ancestors that were polymorphic for either root or stem parasitism-a condition here termed amphiphagous. Convergences in morphological features associated with the mistletoe habit have occurred such as the squamate habit, seed attachment structures, unisexual flowers, and loss of chlorophyll.     

DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species

There is currently international interest in the application of DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of five candidate plant DNA barcoding regions [rbcL, matK, trnH-psbA, trnL-F and internal transcribed spacer (ITS)] in Eurasian yews. This group of species is taxonomically difficult because of a lack of clear-cut morphologically differences between species and hence represents a good test case for DNA barcoding. Forty-seven accessions were analysed, representing all taxa treated in current floristic works and covering most of the distribution range of Taxus in Eurasia. As single loci, trnL-F and ITS showed the highest species discriminatory power, each resolving 11 of 11 lineages (= barcode taxa). Species discrimination using matK, trnH-psbA and rbcL individually was lower, with matK resolving 8 of 10, trnH-psbA 7 of 11 and rbcL 5 of 11 successfully sequenced lineages. The proposed CBOL core barcode (rbcL + matK) resolved 8 of 11 lineages. Combining loci generally increased the robustness (measured by clade support) of the barcoding discrimination. Based on overall performance, trnL-F and ITS, separately or combined, are proposed as barcode for Eurasian Taxus. DNA barcoding discriminated recognized taxa of Eurasian Taxus, namely T. baccata, T. cuspidata, T. fuana and T. sumatrana, and identified seven lineages among the T. wallichiana group, some with distinct geographical distributions and morphologies, and potentially representing new species. Using the proposed DNA barcode, a technical system can be established to rapidly and reliably identify Taxus species in Eurasia for conservation protection and for monitoring illegal trade.     

The age of the angiosperms: a molecular timescale without a clock

The age of the angiosperms has long been of interest to botanists and evolutionary biologists. Many early efforts to date the age of the angiosperms and evolutionary divergences within the angiosperm clade using a molecular clock have yielded age estimates that are grossly inconsistent with the fossil record. We investigated the age of angiosperms using Bayesian relaxed clock (BRC) and penalized likelihood (PL) approaches. Both of these methods allow the incorporation of multiple fossil constraints into the optimization procedure. The BRC method allows a range of values for among-lineage rate of substitution, from a nearly clocklike behavior to a condition in which each branch is allowed an optimal substitution rate, and also accounts for variation in molecular evolution across multiple genes. A topology derived from an analysis of genes from all three plant genomes for 71 taxa was used as a backbone. The effects on age estimates of different genes, single-gene versus concatenated datasets, and the inclusion and assumptions of fossils as age constraints were examined. In addition, the influence of prior distributions on estimates of divergence times was also explored. These results indicate that widely divergent age estimates can result from the different methods (198-139 million years ago), different sources of data (275-122 million years ago), and the inclusion of temporal constraints to topologies. Most dates, however, are between 180-140 million years ago, suggesting a Middle Jurassic-Early Cretaceous origin of flowering plants, predating the oldest unequivocal fossil angiosperms by about 45-5 million years. Nonetheless, these dates are consistent with other recent studies that have used methods that relax the assumption of a strict molecular clock and also agree with the hypothesis that the angiosperms may be somewhat older than the fossil record indicates.     

Testing DNA barcoding in closely related groups of Lysimachia L. (Myrsinaceae)

It has been suggested that rbcL and matK are the core barcodes in plants, but they are not powerful enough to distinguish between closely related plant groups. Additional barcodes need to be evaluated to improve the level of discrimination between plant species. Because of their well-studied taxonomy and extreme diversity, we used Chinese Lysimachia (Myrsinaceae) species to test the performance of core barcodes (rbcL and matK) and two additional candidate barcodes (trnH-psbA and the nuclear ribosomal ITS); 97 accessions from four subgenus representing 34 putative Lysimachia species were included in this study. And many closely related species pairs in subgen. Lysimachia were covered to detect their discriminatory power. The inefficiency of rbcL and matK alone or combined in closely related plant groups was validated in this study. TrnH-psbA combined with rbcL + matK did not yet perform well in Lysimachia groups. In contrast, ITS, alone or combined with rbcL and/or matK, revealed high resolving ability in Lysimachia. We support ITS as a supplementary barcode on the basis of core barcode rbcL and matK. Besides, this study also illustrates several mistakes or underlying evolutionary events in Lysimachia detected by DNA barcoding.     

Occurrence of matK in a trnK group II intron in charophyte green algae and phylogeny of the Characeae

A group II intron containing the matK gene, which encodes a splicing-associated maturase, was found in the trnK (lysine tRNA) exon in the chloroplast genome of the six extant genera of green algae in the family Characeae, which among green algae are the sister group to embryophytes (land plants). The characean trnK intron (～2.5 kilobases [kb]) and matK ORF (～1.5 kb) are comparable in size to the intron and ORF of land plants, in which they are similarly found inserted in the trnK exon. Domain X, a sequence of conserved amino acid residues within matK, occurs in the Characeae. Phylogenetic analysis using maximum likelihood (GTR + I + gamma likelihood model) and parsimony (branch and bound search) yielded one tree with high bootstrap support for all branches. The matK tree was congruent with the rbcL tree for the same taxa. The number and proportion of informative sites was higher in matK (501, 31% of matK sequence) compared to rbcL (122, 10%). Characeae branch lengths were on average more than five times longer for matK compared to rbcL and provided better resolution within the Characeae. These findings along with recent genomic analyses demonstrate that the intron and matK invaded the chloroplast genome of green algae prior to the evolution of land plants.     

Angiosperm divergence times: the effect of genes, codon positions, and time constraints

An understanding of the evolution of modern terrestrial ecosystems requires an understanding of the dynamics associated with angiosperm evolution, including the timing of their origin and diversification into their extraordinary present-day diversity. Molecular estimates of angiosperm age have varied widely, and many substantially predate the Early Cretaceous fossil appearance of the group. In this study, the effect of different genes, codon positions, and chronological constraints on node ages are examined on divergence time estimates across seed plants, with a special focus on angiosperms. Penalized likelihood was used to estimate divergence times on a phylogenetic hypothesis for seed plants derived from Bayesian analysis, with branch lengths estimated with maximum likelihood. The plastid genes atpB, psaA, psbB, and rbcL were used individually and in combination, using first and second, third, and the three codon positions, including and excluding age constraints on 20 nodes derived from a critical examination of the land-plant fossil record. The optimal level of rate smoothing according to each unconstrained and constrained dataset was obtained with penalized likelihood. Tests for a molecular clock revealed significantly unclocklike rates in all datasets. Addition of fossil constraints resulted in even greater departures from constancy. Consistently with significant deviations from a clock, estimated optimal smoothing values were low, but a strict correlation between rate heterogeneity and optimal smoothing value was not found. Age estimates for nodes across the phylogeny varied, sometimes substantially, with gene and codon position. Nevertheless, estimates based on the four concatenated genes are very similar to the mean of the four individual gene estimates. For any given node, unconstrained age estimates are more variable than constrained estimates and are frequently younger than well-substantiated fossil members of the clade. Constrained estimates of ages of clades are older than unconstrained estimates and oldest fossil representatives, sometimes substantially so. Angiosperm age estimates decreased as rate smoothing increased. Whereas the range of unconstrained angiosperm age estimates spans the fossil age of the clade, the range of constrained estimates is narrower (and older) than the earliest angiosperm fossils. Results unambiguously indicate the relevance of constraints in reducing the variability of ages derived from different partitions of the data and diminishing the effect of the smoothing parameter. Constrained optimizations of divergence times and substitution rates across the phylogeny suggest appreciably different evolutionary dynamics for angiosperms and for gymnosperms. Whereas the gymnosperm crown group originated shortly after the origin of seed plants, a long time elapsed before the origin of crown group angiosperms. Although absolute age estimates of angiosperms and angiosperm clades are older than their earliest fossils, the estimated pace of phylogenetic diversification largely agrees with the rapid appearance of angiosperm lineages in stratigraphic sequences.     

Is Oligomeris (Resedaceae) indigenous to North America? Molecular evidence for a natural colonization from the Old World

Oligomeris linifolia constitutes one of the few examples of intercontinental disjunctions at the species level between the arid regions of the Old World and SW North America. The status of the American populations has been obscure, with some authors considering the populations to be introduced, whereas others believe them to be native. To clarify these conflicting opinions, we performed phylogeographic analyses using nuclear ribosomal ITS and plastid trnL-F and rps16 sequences to infer the origin of the disjunct American populations. Two independent molecular clock approaches based on ITS and cpDNA sequences (rbcL, matK, trnL-F) were used to estimate a divergence time of O. linifolia. Low levels of sequence divergence and estimates of relatively recent splits of Oligomeris lineages disagree with the vicariance hypotheses traditionally suggested to account for New-Old World disjunctions. In addition, significant genetic differentiation of American populations does not indicate a recent anthropogenic introduction. Morphological uniformity and the sharing of haplotypes between disjunct populations, together with the molecular clock results, suggest that a long-distance dispersal event from the Old Word to SW North America may have taken place during the Quaternary, in spite of limited dispersal mechanisms in Oligomeris.     

A fossil‐calibrated relaxed clock for Ephedra indicates an Oligocene age for the divergence of Asian and New World clades and Miocene dispersal into South America

Abstract Ephedra comprises approximately 50 species, which are roughly equally distributed between the Old and New World deserts, but not in the intervening regions (amphitropical range). Great heterogeneity in the substitution rates of Gnetales (Ephedra, Gnetum, and Welwitschia) has made it difficult to infer the ages of the major divergence events in Ephedra, such as the timing of the Beringian disjunction in the genus and the entry into South America. Here, we use data from as many Gnetales species and genes as available from GenBank and from a recent study to investigate the timing of the major divergence events. Because of the tradeoff between the amount of missing data and taxon/gene sampling, we reduced the initial matrix of 265 accessions and 12 loci to 95 accessions and 10 loci, and further to 42 species (and 7736 aligned nucleotides) to achieve stationary distributions in the Bayesian molecular clock runs. Results from a relaxed clock with an uncorrelated rates model and fossil‐based calibration reveal that New World species are monophyletic and diverged from their mostly Asian sister clade some 30 mya, fitting with many other Beringian disjunctions. The split between the single North American and the single South American clade occurred approximately 25 mya, well before the closure of the Panamanian Isthmus. Overall, the biogeographic history of Ephedra appears dominated by long‐distance dispersal, but finer‐scale studies are needed to test this hypothesis.     

Phylogeny, biogeography, and molecular dating of cornelian cherries (Cornus, Cornaceae): tracking Tertiary plant migration

Data from four DNA regions (rbcL, matK, 26S rDNA, and ITS) as well as extant and fossil morphology were used to reconstruct the phylogeny and biogeographic history of an intercontinentally disjunct plant group, the cornelian cherries of Cornus (dogwoods). The study tests previous hypotheses on the relative roles of two Tertiary land bridges, the North Atlantic land bridge (NALB) and the Bering land bridge (BLB), in plant migration across continents. Three approaches, the Bayesian, nonparametric rate smoothing (NPRS), and penalized likelihood (PL) methods, were employed to estimate the times of geographic isolations of species. Dispersal and vicariance analysis (DIVA) was performed to infer the sequence and directionality of biogeographic pathways. Results of phylogenetic analyses suggest that among the six living species, C. sessilis from western North America represents the oldest lineage, followed by C. volkensii from Africa. The four Eurasian species form a clade consisting of two sister pairs, C. mas-C. officinalis and C. chinensis-C. eydeana. Results of DIVA and data from fossils and molecular dating indicate that the cornelian cherry subgroup arose in Europe as early as the Paleocene. Fossils confirm that the group was present in North America by the late Paleocene, consistent with the DIVA predictions that, by the end of the Eocene, it had diversified into several species and expanded its distribution to North America via the NALB and to Africa via the last direct connection between Eurasia and Africa prior to the Miocene, or via long-distance dispersal. The cornelian cherries in eastern Asia appear to be derived from two independent dispersal events from Europe. These events are inferred to have occurred during the Oligocene and Miocene. This study supports the hypothesis that the NALB served as an important land bridge connecting the North American and European floras, as well as connecting American and African floras via Europe during the early Tertiary.     

Relaxed molecular clocks for dating historical plant dispersal events

Age estimation from molecular sequences has emerged as a powerful tool for inferring when a plant lineage arrived in a particular area. Knowing the tenure of lineages within a region is key to understanding the evolution of traits, the evolution of biotic interactions, and the evolution of floras. New analytical methods model change in substitution rates along individual branches of a phylogenetic tree by combining molecular data with time constraints, usually from fossils. These "relaxed clock" approaches can be applied to several gene regions that need not all have the same substitution rates, and they can also incorporate multiple simultaneous fossil calibrations. Since 1995, at least 100 plant biogeographic studies have used molecular-clock dating, and about a fifth has used relaxed clocks. Many of these report evidence of long-distance dispersal. Meta-analyses of studies from the same geographic region can identify directional biases because of prevailing wind or water currents and the relative position and size of landmasses.