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Gene trees of holoparasitic plants usually show distinctly longer branch lengths than seen in photosynthetic closest relatives. Such substitution rate jumps have made it difficult to infer the absolute divergence times of parasites. An additional problem is that parasite clades often lack a fossil record. Using nuclear and mitochondrial DNA sequences of Apodanthaceae, a worldwide family of endoparasites living inside Fabaceae and Salicaceae, we compared several dating approaches: (i) an uncorrelated lognormal (UCLN) model calibrated with outgroup fossils, (ii) ages of host lineages as a maximal age in an UCLN model, (iii) user-assigned local clocks, and (iv) outgroup-fossil-calibrated random local clocks (RLC) with varying prior probabilities on the number of permitted rate changes (RLCu and RLCp models), a variable that has never been explored. The resulting dated phylogenies include all 10 species of the family, three in Australia, one in Iran, one in Africa, and the remainder in the Americas. All clock models infer a drastic rate jump between nonparasitic outgroups and Apodanthaceae, but since they distribute the rate heterogeneity differently, they result in much-different age estimates. Bayes factors using path and stepping-stone sampling indicated that the RLCp model fit poorly, while for matR, topologically unconstrained RLCu and UCLN models did not differ significantly and for 18S, the UCLN model was preferred. Under the equally well fitting models, the Apodanthaceae appear to be a relatively old clade, with a stem age falling between 65 and 81 my, the divergence of Apodanthes from Pilostyles between 36 and 57 my ago, and the crown age of the Australian clade 8–18 my ago. In our study system, host-age calibrations did not yield well-constrained results, but they may work better in other parasite clades. For small data sets where statistical convergence can be reached even with complex models, random local clocks should be explored as an alternative to the exclusive reliance on UCLN clocks.

The economically most important group of bees is the “corbiculates”, or pollen basket bees, some 890 species of honeybees (Apis), bumblebees (Bombus), stingless bees (Meliponini), and orchid bees (Euglossini). Molecular studies have indicated that the corbiculates are closest to the New World genera Centris, with 230 species, and Epicharis, with 35, albeit without resolving the precise relationships. Instead of concave baskets, these bees have hairy hind legs on which they transport pollen mixed with floral oil, collected with setae on the anterior and middle legs. We sampled two-thirds of all Epicharis, a third of all Centris, and representatives of the four lineages of corbiculates for four nuclear gene regions, obtaining a well-supported phylogeny that has the corbiculate bees nested inside the Centris/Epicharis clade. Fossil-calibrated molecular clocks, combined with a biogeographic reconstruction incorporating insights from the fossil record, indicate that the corbiculate clade arose in the New World and diverged from Centris 84 (72–95) mya. The ancestral state preceding corbiculae thus was a hairy hind leg, perhaps adapted for oil transport as in Epicharis and Centris bees. Its replacement by glabrous, concave baskets represents a key innovation, allowing efficient transport of plant resins and large pollen/nectar loads and freeing the corbiculate clade from dependence on oil-offering flowers. The transformation could have involved a novel function of Ubx, the gene known to change hairy into smooth pollen baskets in Apis and Bombus.

Absolute times from calibrated DNA phylogenies can be used to infer lineage diversification, the origin of new ecological niches, or the role of long distance dispersal in shaping current distribution patterns. Molecular-clock dating of non-vascular plants, however, has lagged behind flowering plant and animal dating. Here, we review dating studies that have focused on bryophytes with several goals in mind, (i) to facilitate cross-validation by comparing rates and times obtained so far; (ii) to summarize rates that have yielded plausible results and that could be used in future studies; and (iii) to calibrate a specieslevel phylogeny for Nothoceros, a model for plastid genome evolution in hornworts. Including the present work, there have been 18 molecular clock studies of liverworts, mosses, or hornworts, the majority with fossil calibrations, a few with geological calibrations or dated with previously published plastid substitution rates. Over half the studies cross-validated inferred divergence times by using alternative calibration approaches. Plastid substitution rates inferred for ‘‘bryophytes’’ are in line with those found in angiosperm studies, implying that bryophyte clock models can be calibrated either with published substitution rates or with fossils, with the two approaches testing and cross-validating each other. Our phylogeny of Nothoceros is based on 44 accessions representing all suspected species and a matrix of six markers of nuclear, plastid, and mitochondrial DNA. The results show that Nothoceros comprises 10 species, nine in the Americas and one in New Zealand (N. giganteus), with the divergence between the New Zealand species and its Chilean sister species dated to the Miocene and therefore due to long-distance dispersal. Based on the new tree, we formally transfer two species of Megaceros into Nothoceros, resulting in the new combinations N. minarum (Nees) J.C. Villarreal and N. schizophyllus (Gottsche ex Steph.) J.C. Villarreal, and we also newly synonymize eight names described in Megaceros.

Studies over the past ten years have shown that the crown groups of most conifer genera are only about 15-25 Ma old. The genus Picea (spruces, Pinaceae), with around 35 species, appears to be no exception. In addition, molecular studies of co-existing spruce species have demonstrated frequent introgression. Perhaps not surprisingly therefore previous phylogenetic studies of species relationships in Picea, based mostly on plastid sequences, suffered from poor statistical support. We therefore generated mitochondrial, nuclear, and further plastid DNA sequences from carefully sourced material, striking a balance between alignability with outgroups and phylogenetic signal content. Motif duplications in mitochondrial introns were treated as characters in a stochastic Dollo model; molecular clock models were calibrated with fossils; and ancestral ranges were inferred under maximum likelihood. In agreement with previous findings, Picea diverged from its sister clade 180 million years ago (Ma), and the most recent common ancestor of today's spruces dates to 28 Ma. Different from previous analyses though, we find a large Asian clade, an American clade, and a Eurasian clade. Two expansions occurred from Asia to North America and several between Asia and Europe. Chinese P. brachytyla, American P. engelmannii, and Norway spruce, P. abies, are not monophyletic, and North America has ten, not eight species. Divergence times imply that Pleistocene refugia are unlikely to be the full explanation for the relationships between the European species and their East Asian relatives. Thus, northern Norway spruce may be part of an Asian species complex that diverged from the southern Norway spruce lineage in the Upper Miocene, some 6 Ma, which can explain the deep genetic gap noted in phylogeographic studies of Norway spruce. The large effective population sizes of spruces, and incomplete lineage sorting during speciation, mean that the interspecific relationships within each of the geographic clades require further studies, especially based on genomic information and population genetic data.

Papaya (Carica papaya) is a crop of great economic importance, and the species was among the first plants to have its genome sequenced. However, there has never been a complete species-level phylogeny for the Caricaceae, and the crop's closest relatives are therefore unknown. We investigated the evolution of the Caricaceae based on sequences from all species and genera, the monospecific Carica, African Cylicomorpha with two species, South American Jacaratia and Vasconcellea with together c. 28 species, and Mexican/Guatemalan Jarilla and Horovitzia with four species. Most Caricaceae are trees or shrubs; the species of Jarilla, however, are herbaceous. We generated a matrix of 4711 nuclear and plastid DNA characters and used maximum likelihood (ML) and Bayesian analysis to infer species relationships, rooting trees on the Moringaceae. Divergence times were estimated under relaxed and strict molecular clocks, using different subsets of the data. Ancestral area reconstruction relied on a ML approach. The deepest split in the Caricaceae occurred during the Late Eocene, when the ancestor of the Neotropical clade arrived from Africa. In South America, major diversification events coincide with the Miocene northern Andean uplift and the initial phase of the tectonic collision between South America and Panama resulting in the Panamanian land bridge. Carica papaya is sister to Jarilla/Horovitzia, and all three diverged from South American Caricaceae in the Oligocene, 27 (22-33) Ma ago, coincident with the early stages of the formation of the Panamanian Isthmus. The discovery that C. papaya is closest to a clade of herbaceous or thin-stemmed species has implications for plant breeders who have so far tried to cross papaya only with woody highland papayas (Vasconcellea).

Hallucinogenic or toxic species of Brunfelsia (Solanaceae: Petunieae) are important in native cultures throughout South America, and the genus also contains several horticulturally important species. An earlier morphological revision of the c. 50 species recognized three main groups, one consisting of the 23 Antillean species, another of southern South American and Andean species, and a third of species from the Amazon Basin and Guiana Shield. Based on plastid and nuclear DNA sequences from up to 65 accessions representing 80% of the species, we generated a phylogeny and a calibrated chronogram for Brunfelsia to infer clade expansion and shifts in pollinators and fruit types. Brunfelsia flowers offer nectar, and attract lepidoptera, hummingbirds, or bees; the fruits are dry or fleshy. Our results imply that Brunfelsia is 16-21 Myr old and entered the Antilles from South America early during its history, with subsequent expansion along the island arc. The ancestor of the Antillean clade was hawk-moth-pollinated and had fleshy capsules, perhaps facilitating dispersal by birds. The only shift to hummingbird pollination occurred on Cuba, which also harbors the largest single radiation, with 11 species (10 included in our study) that apparently arose over the past 4 Myr. Jamaica, Hispaniola, and Puerto Rico each sustained smaller radiations. The data also reveal at least one new species.

Alocasia comprises over 113 species of rainforest understorey plants in Southeast Asia, the Malesian region, and Australia. Several species, including giant taro, Alocasia macrorrhizos, and Chinese taro, Alocasia cucullata, are important food plants or ornamentals. We investigated the biogeography of this genus using plastid and nuclear DNA sequences (5200 nucleotides) from 78 accessions representing 71 species, plus 25 species representing 16 genera of the Pistia clade to which Alocasia belongs. Divergence times were inferred under strict and relaxed clock models, and ancestral areas with Bayesian and maximum likelihood approaches. Alocasia is monophyletic and sister to Colocasia gigantea from the SE Asian main- land, whereas the type species of Colocasia groups with Steudnera and Remusatia, requiring taxonomic realignments. Nuclear and plastid trees show topological conflict, with the nuclear tree reflecting morphological similarities, the plastid tree species' geographic proximity, suggesting chloroplast capture. The ancestor of Alocasia diverged from its mainland sister group c. 24 million years ago, and Borneo then played a central role in the expansion of Alocasia: 11–13 of 18–19 inferred dispersal events originated on Borneo. The Philippines were reached from Borneo 4–5 times in the Late Miocene and Early Pliocene, and the Asian mainland 6–7 times in the Pliocene. Domesticated giant taro originated on the Philippines, Chinese taro on the Asian mainland.

The tribe Delphinieae (Ranunculaceae) comprises two species-rich genera, Aconitum and Delphinium, the latter including Consolida and Aconitella. The 650–700 species are distributed in Eurasia and North America; three species occur on tropical African mountains. Maximum likelihood analyses of 2088 aligned nucleotides of plastid and nuclear sequences obtained from up to 185 species of Delphinieae from throughout the geographic range (plus relevant outgroups) show that three short-lived (facultative annual or biennial) Mediterranean species belonging to Delphinium subgenus Staphisagria are the sister clade to all other Delphinieae, implying that Staphisagria needs to be raised to genus status if Delphinium and Aconitum are to become mutually monophyletic. Molecular clock dating suggests an origin of the sampled Delphinieae in the Early Oligocene (c. 32.3 Ma) and expansion to North America of Aconitum and Delphinium around 3.3 and 2.9 Ma ago, respectively; the East African Mts. were reached by long-distance dispersal some 2.4 Ma ago, coincident with the major uplift of the East African Rift system. The ancestral growth form of the Delphinieae could not be reconstructed, but Late Miocene bursts in diversification rates in the Himalayan and southwestern Chinese clades of Aconitum and Delphinium appear to be associated with transitions from short-lived to long-lived life histories.

The bitter gourd genus Momordica comprises 47 species in Africa and 12 in Asia and Australia. All have unisexual flowers, and of the African species, 24 are dioecious, 23 monoecious, while all Asian species are dioecious. Maximum likelihood analyses of 6257 aligned nucleotides of plastid, mitochondrial and nuclear DNA obtained for 122 accessions of Momordica and seven outgroups show that Momordica is monophyletic and consists of 11 well-supported clades. Monoecy evolved from dioecy seven times independently, always in Africa and mostly in savanna species with low population densities. Leaky dioecy, with occasional fruit-producing males, occurs in two African species and might be the first step in an evolutionary transition towards monoecy. Dated biogeographic analyses suggest that Momordica originated in tropical Africa and that the Asian species are the result of one long-distance dispersal event about 19 million years ago. The pantropical vegetable Momordica charantia is of African, not Asian origin as had previously been suggested.

The bee tribe Ctenoplectrini, with two genera, comprises nine species in tropical Africa and ten in Asia and Australia. Most of them collect floral oil, pollen, and nectar from Cucurbitaceae, but three species are thought to be cleptoparasites. The unusual morphology of Ctenoplectrini has made it difficult to infer their closest relatives, in turn preventing an understanding of these bees' geographic and temporal origin. We used two mitochondrial and two nuclear markers (4741 nucleotides) generated for most of the species to test the monophyly of the tribe, its relationships to other Apidae, and its biogeographic history. Ctenoplectrini are strongly supported as monophyletic and closest to the Long-horned bees, Eucerini. The presumably cleptoparasitic species form a clade (Ctenoplectrina) that is sister to the remaining species (Ctenoplectra), confirming the independent evolution of cleptoparasitism in this tribe. Tree topology and molecular dating together suggest that Ctenoplectrini originated in Africa in the Early Eocene and that Ctenoplectra dispersed twice from Africa to Asia, sometime in the Late Eocene, 30-40 my ago, from where one species reached the Australian continent via Indonesia and New Guinea in the mid-Miocene, c. 13 my ago. Dry and cool mid-Miocene climates also coincide with the divergence between Ctenoplectra bequaerti from West Africa and Ctenoplectra terminalis from East and South Africa, perhaps related to fragmentation of the equatorial African rainforest belt.

Based on nuclear and chloroplast sequences we resolve species relationships in Calycanthaceae and develop a biogeographic hypothesis that explains their intercontinental disjunctions and intra-continental diversification in eastern Asia. Fossil-calibrated penalized likelihood and Bayesian divergence time estimates indicate that the Northern Hemisphere Calycanthus and Chimonanthus diverged from each other in the mid-Miocene, while the Australian Idiospermum had already diverged by the Upper Cretaceous and likely represents a remnant of a former Gondwanan distribution of Calycanthaceae that included South America, as indicated by the occurrence of Cretaceous Calycanthaceae fossils in Brazil. Relationships within Calycanthus were difficult to resolve, but a shared 155-bp deletion in the trnL-F intergenic spacer unites the two North American species, which were also sisters in a cpDNA restriction site study. Their ancestor apparently crossed the Bering land bridge in the Miocene. The six species of Chimonanthus, by contrast, diverged from each other as recently as 1-2my ago, and a DIVA analysis with four areas of endemism recognized within China suggests three vicariance and two dispersal events within Chimonanthus, with initial vicariance having occurred between eastern and southwestern or central China. Further divergence then appears to have involved eastern and south-central China, and southwestern and central China.

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25–35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7–13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.

The monocot family Aponogetonaceae (Alismatales) consists only of Aponogeton, with 57 species occurring in Africa, Madagascar, India and Sri Lanka, Southeast Asia and Australia. Earlier studies inferred a Madagascan or Australian origin for the genus. Aponogeton-like pollen is documented from the Late Cretaceous of Wyoming, the early mid-Eocene of Canada, and the late mid-Eocene of Greenland. We obtained nuclear and plastid DNA sequences for 42 species and generated a time-calibrated phylogeny, rooted on appropriate outgroups. Statistical biogeographic analyses were carried out with or without the fossils incorporated in the phylogeny. The recent-most common ancestor of living Aponogetonaceae appears to date to the mid-Eocene and to have lived in Madagascar or Africa (but not Australia). Three transoceanic dispersal events from Africa/Madagascar to Asia sometime during the Miocene could explain the observed species relationships. As inferred in earlier studies, an ancient Australian species is sister to all other Aponogetonaceae, while the remaining Australian species stem from an Asian ancestor that arrived about 5 million years ago. The family’s ancient Northern Hemisphere fossil record and deepest extant divergence between a single Australian species and an Africa/Madagascar clade are statistically well-supported and rank among the most unusual patters in the biogeography of flowering plants.

The Anisophylleaceae comprise 29–34 species of shrubs and trees occurring in lowland forests and swamps in tropical Africa, Asia, and South America. These species are placed in four genera with disjunct geographic distributions; Anisophyllea has 25–30 species in South America, Africa, and Malesia; Combretocarpus has one species in Sumatra and Borneo; Poga one species in equatorial Africa; and Polygonanthus two in the Amazon Basin. Here we use a phylogeny based on six nuclear and plastid loci sequenced for 15 species representing the four genera to infer their relationships and the relative and absolute ages of the range disjunctions. Combretocarpus is sister to the other three genera, and Polygonanthus then sister to Poga and Anisophyllea. Ansiophyllea, represented by 12 species from all three continents, is monophyletic. A relaxed Bayesian clock calibrated with the oldest fossils from a relevant outgroup, Tetramelaceae, suggests that the disjunctions between Combretocarpus, Poga, and Polygonanthus date back to the Cretaceous, Mid-, and Upper Eocene, whereas the intercontinental disjunctions within Anisophyllea appear to date back only some 22–23 million years and thus probably result from long-distance dispersal.

Cucurbitaceae contain c. 800 species in 130 genera and are among the economically most important families of plants. We inferred their phylogeny based on chloroplast DNA sequences from two genes, one intron, and two spacers (rbcL, matK, trnL, trnL-trnF, rpl20-rps12) obtained for 171 species in 123 genera. Molecular data weakly support the traditional subfamilies Cucurbitoideae (111 genera) and Nhandiroboideae (19 genera, 60 species), and recover most of the eleven tribes, but almost none of the subtribes. Indofevillea khasiana is sister to all other Cucurbitoideae, and the genera of Joliffieae plus a few Trichosantheae form a grade near the base of Cucurbitoideae. A newly discovered large clade consists of the ancestrally Asian genera Nothoalsomitra, Luffa, Gymnopetalum, Hodgsonia, Trichosanthes, and the New World tribe Sicyeae. Genera that are poly- or paraphyletic include Ampelosicyos, Cucumis, Ibervillea, Neoachmandra, Psiguria, Trichosanthes, and Xerosicyos. Flower characters, especially number of free styles, fusion of filaments and/or anthers, tendril type, and pollen size, exine, and aperture number correlate well with the chloroplast phylogeny, while petal and fruit characters as well as karyotype exhibit much evolutionary flexibility.

Hoya (Marsdenieae, Apocynaceae) includes at least 200 species distributed from India to the Pacific Islands. We here infer major species groups in the genus based on combined sequences from the chloroplast atpB-rbcL spacer, the trnL region, and nuclear ribosomal DNA ITS region for 42 taxa of Hoya and close relatives. To assess levels of ITS polymorphism, ITS sequences for a third of the accessions were obtained by cloning. Most ITS clones grouped by species, indicating that speciation in Hoya usually predates ITS duplication. One ITS sequence of H. carnosa, however, grouped with a sequence of the morphologically similar H. pubicalyx, pointing to recent hybridization or the persistence of paralogous copies through a speciation event. The topology resulting from the combined chloroplast and nuclear data recovers some morphology-based sections, such as Acanthostemma and Eriostemma, as well as a well-supported Australian/New Guinean clade. The combined data also suggest that morphological adaptations for ant-symbiosis evolved at least three times within Hoya.