Nuclear ITS Region Research Articles

Friends or Relatives? Phylogenetics and Species Delimitation in the Controversial European Orchid Genus Ophrys

Highly variable, yet possibly convergent, morphology and lack of sequence variation have severely hindered production of a robust phylogenetic framework for the genus Ophrys. The aim of this study is to produce this framework as a basis for more rigorous species delimitation and conservation recommendations. Nuclear and plastid DNA sequencing and amplified fragment length polymorphism (AFLP) were performed on 85 accessions of Ophrys, spanning the full range of species aggregates currently recognized. Data were analysed using a combination of parsimony and Bayesian tree-building techniques and by principal co-ordinates analysis. Complementary phylogenetic analyses and ordinations using nuclear, plastid and AFLP datasets identify ten genetically distinct groups (six robust) within the genus that may in turn be grouped into three sections (treated as subgenera by some authors). Additionally, genetic evidence is provided for a close relationship between the O. tenthredinifera, O. bombyliflora and O. speculum groups. The combination of these analytical techniques provides new insights into Ophrys systematics, notably recognition of the novel O. umbilicata group. Heterogeneous copies of the nuclear ITS region show that some putative Ophrys species arose through hybridization rather than divergent speciation. The supposedly highly specific pseudocopulatory pollination syndrome of Ophrys is demonstrably 'leaky', suggesting that the genus has been substantially over-divided at the species level.

The Anopheles (Anopheles) maculipennis complex (Diptera: Culicidae) in Greece

Mosquitoes belonging to the Maculipennis Complex of Anopheles subgenus Anopheles were collected in 10 prefectures across Greece: Attiki, Evros, Florina, Fthiotida, Ioannina, Lakonia, Magnesia, Rodopi, Serres, and Xanthi. DNA was extracted from 276 specimens and sequences for the nuclear rDNA ITS2 region were obtained from 257 of these (93.1%). Four members of the An. maculipennis Meigen complex were identified: An. maculipennis, An. sacharovi Favre, An. melanoon Hackett, and An. messeae Falleroni. Species were identified based on correlation of their sequences with those available in GenBank. All four species have been incriminated as primary or secondary vectors of malaria. Species distributions in relation to potential threats of reintroduction of malaria in Greece are discussed. This study comprises the most complete study of the Maculipennis Complex in Greece to date, and contributes substantially to the current knowledge of the genetics and distributions of the four species.

Identification and characterization of a green-type mutant of Porphyra tenera Kjellman var. tamatsuensis Miura (Bangiales, Rhodophyta)

We identified two green-colored conchocelis strains of cultivated Porphyra, ‘Oba-green’ and HGT-6 (F1 strain of ‘Oba-green’), based on PCR-RFLP and sequence analyses of the nuclear ITS region and the plastid RuBisCO spacer region. These molecular analyses confirmed that the two strains were green-type mutants of the endangered species Porphyra tenera, although their blade color was not green. Under the same culture conditions, blade length increase was significantly higher in HGT-6 than in a wild strain HGT-1 of P. tenera, and the blade shape of HGT-6 was extremely elongate compared with that of HGT-1. From the growth characteristics and ITS-1 sequence data, HGT-6 was confirmed to be a green-type mutant of P. tenera var. tamatsuensis, a vigorously growing cultivar. Although photosynthetic pigment contents were lower in HGT-6 than in HGT-1, total content of four major free amino acids was higher and the blade thickness was almost the same in HGT-6 and HGT-1. These results suggest that the green-type mutant HGT-6 has potential as breeding material for further development of Porphyra breeding.

Phylogeny of the Cyphomandra clade of the genus Solanum (Solanaceae) based on ITS sequence data

About 13 major clades can be recognized within the genus Solanum (Solanaceae) based on chloroplast DNA sequence data. One of these is the Cyphomandra clade, which includes about 50 neotropical species. These have traditionally been placed into two or three sections: S. section Pachyphylla (formerly recognized as the genus Cyphomandra), S. section Cyphomandropsis, and S. section Glaucophyllum (monotypic and sometimes placed in S. section Cyphomandropsis). Phylogenetic relationships among 61 accessions of 35 species of the Cyphomandra clade are investigated using sequence data from the nuclear ITS region analyzed by parsimony and Bayesian inference. The Cyphomandra clade forms a monophyletic group, but the ITS data are equivocal as to the monophyly of sections Pachyphylla and Cyphomandropsis. Four well‐supported groups of species can be recognized within the Cyphomandra clade; these conform in part to species groups proposed on the basis of morphology. The distribution of self‐incompatible and self‐compatible breeding systems is mapped onto the ITS cladogram, and patterns of evolution of enlarged anther connectives, osmophores, and volatile composition are discussed in light of hypothesized phylogenetic relationships.

Cladosiphon umezakiisp. nov. (Ectocarpales, Phaeophyceae) from Japan

SUMMARYThe new speciesCladosiphon umezakiiAjisaka (Ectocarpales, Phaeophyceae) is described from Japan based on morphology and DNA sequences. The species resemblesCladosiphon okamuranusTokida in its gross morphology; somewhat slimy, cylindrical, multiaxial and sympodial erect thallus, arising from a small disc‐shaped holdfast, and branching once to twice. However,C. umezakiihas considerably longer assimilatory filaments (up to 840 μm long, composed of up to 90 cells) than any known taxa of the genus. The species is a winter to spring annual, growing on lower intertidal to subtidal rocks of more or less exposed sites on the north‐eastern coast of Kyushu and on both the Pacific and the Sea of Japan coasts of Honshu. Specimens from the Sea of Japan coast had both unilocular and plurilocular zoidangia, whereas those from Kyushu and from the Pacific had only unilocular zoidangia. Unilocular zoidangia were formed on the basal part of assimilatory filaments, and plurilocular ones were transformed from the distal part of assimilatory filaments. DNA sequences of the Rubisco‐spacer (rbc‐spacer) region and the nuclear rDNA ITS region (ITS1, 5.8S and ITS2) supported the distinctness of the species.

Generic level phylogeny of Thalictroideae (Ranunculaceae) — implications for the taxonomic status of Paropyrum and petal evolution

The subfamily Thalictroideae, which is clearly defined by its chromosome characters, contains nine or ten genera. Here we use sequences of chloroplast rbcL and matK genes as well as non‐coding trnL‐F and nuclear ITS regions to reconstruct the generic level phylogeny of this subfamily. Our data recognize three major groups within Thalictroideae: (1) Thalictrum, Leptopyrum, Paropyrum, and Paraquilegia; (2) Urophysa, Semiaquilegia, and Aquilegia; and (3) Dichocarpum, Enemion, and Isopyrum. Isopyrum anemonoides, the sole species of Paropyrum, and Paraquilegia form a clade sister to Leptopyrum; together they make up the first group. Isopyrum manshuricum and I. thalictroides form another clade within the third group. Isopyrum anemonoides is sister to the monophyletic Paraquilegia. Our data suggest that Isopyrum is polyphyletic and support the segregate genus Paropyrum. Reconstruction of petal evolution suggests a secondary loss of petals in Enemion and Thalictrum, in contrast with the view that apetalous taxa are the most primitive. Petals with longer stalks in Dichocarpum are shown to be derivative, refuting the viewpoint that this type is a primitive condition. The flat petals in Paraquilegia may be derived from the tubular‐type petals in Paropyrum and Leptopyrum, and spurred petals are thought to have originated from a single ancestor.

PHYLOGENETIC RELATIONSHIPS IN SUBTRIBE ASCLEPIADINAE (APOCYNACEAE: ASCLEPIADOIDEAE)1

A first approximation of evolutionary relationships within the Asclepiadinae (Apocynaceae: Asclepiadoideae) is inferred from two DNA data sets, from the nuclear ITS region, and from plastid trnT-L and trnL-F spacers and trnL intron. Both the subtribe as a whole and the phylogenetic radiation of largely herbaceous genera around Asclepias are monophyletic and well supported. While resolution within the Asclepias complex is limited, the lineages identified demonstrate that current generic delimitation is unsatisfactory. Asclepias should be either restricted to New World members of the subtribe or expanded to encompass the entire radiation. In the African species, it is shown that, although the corona is taxonomically important for recognizing species, it can be misleading as an indicator of phylogenetic relationship. Vegetative similarities often prove a more reliable guide, but more detailed studies are needed to refine these lineages and to detect morphological characters to aid in their practical recognition.

Relationships of cucumbers and melons unraveled: molecular phylogenetics ofCucumisand related genera (Benincaseae, Cucurbitaceae)

Cucumis (Cucurbitaceae) comprises 33 species of annuals and perennials with a major native center of diversity in tropical and southern Africa. The genus includes some economically important and widely grown vegetables such as cucumbers and melons. Monophyly of the genus has been disputed in previous studies, but with only limited sampling. Relationships within Cucumis are thus poorly understood; moreover, the validity of the closely related genera has not been thoroughly tested. The present study was undertaken to test the monophyly of Cucumis and several closely related genera, to test sectional circumscriptions within Cucumis, and to understand the biogeographical history of the genus. We sequenced the nuclear ITS and plastid trnS-trnG regions for 40 ingroup and three outgroup taxa, representing all recognized subgenera and sections. Parsimony, maximum likelihood, and Bayesian analyses found Cucumella, Oreosyce, Mukia, Myrmecosicyos, and Dicaelospermum nested within Cucumis. The clades recovered within the Cucumis complex in some instances represent the first phylogenetically derived hypothesis of relationships, whereas others correspond to previous subgeneric and sectional classifications. At least four introductions from Africa to Asia, as well as one reintroduction to Africa, are suggested within the Cucumis complex. Cucumis sativus (cucumber) is strongly supported as sister to the eastern Asian C. hystrix, whereas C. melo (melon) is strongly supported as sister to C. sagittatus in southern Africa.

Development of DNA markers for hybrid identification in Leucadendron (proteaceae)

A rapid and reliable method to accurately identify hybrids at an early age is essential to the success of Leucadendron breeding programs because identification based on morphology can be difficult or impossible when the seedlings are young. DNA based PCR-RFLP and random amplified microsatellite polymorphism (RAMP) markers were developed for this purpose. Unexpected non-parental fragments appeared during the PCR-RFLP analysis of the nuclear ITS region of L. uliginosum 05 × L. procerum 04 hybrids. Mixing DNA from both parents in a single PCR also produced the non-parental fragment, suggesting that PCR recombination had introduced a novel restriction site into the products from the hybrids. Sequencing of individual amplified ITS products from the hybrids confirmed this conclusion. To avoid this complication, RAMP markers were developed for accurate hybrid identification in Leucadendron. RAMP analysis generated a considerable number of polymorphic products, and showed more discrimination in identifying Leucadendron hybrids than did PCR-RFLP.

PHYLOGENETIC RELATIONSHIPS IN THE TRIBES ANCHONIEAE, CHORISPOREAE, EUCLIDIEAE, AND HESPERIDEAE (BRASSICACEAE) BASED ON NUCLEAR RIBOSOMAL ITS DNA SEQUENCES

Sequence data from the nuclear ITS region of 118 species (152 accessions) were used to test the monophyletic status and interrelationships of four related tribes in the Brassicaceae: Anchonieae, Chorisporeae, Euclidieae, and Hesperideae. Both maximum parsimony and maximum likelihood analyses support the recognition of the tribes Hesperideae (unigeneric, Hesperis L.) and Chorisporeae (3 genera, including Chorispora R. Br. ex DC., Diptychocarpus Trautv., and Parrya R. Br.), whereas the Anchonieae and the Euclidieae each separate into two distinct and distant clades (designated here as Anchonieae I and II and Euclidieae I and II). The data also support the exclusion of eight genera from the latter four tribes, with appropriate tribal assignment given in parentheses: Aubrieta Adans. (Arabideae), Blennodia R. Br. (Camelineae), Erysimum L. (Camelineae), Goldbachia DC. (unresolved), Hesperidanthus (B. L. Rob.) Rydb. (Schizopetaleae), Notothlaspi Hook. f. (unresolved), Pseudocamelina (Boiss.) N. Busch (Thlaspideae), and Zuvanda (Dvorák) Askerova (unresolved). The genus Malcolmia R. Br. (ca. 35 species) was paraphyletic and divided into three tribal clades, providing support for the separate genera Malcolmia, Strigosella Boiss., and Zuvanda. The genera Anchonium DC., Desideria Pamp., Eremobium Boiss., Erysimum, Iskandera N. Busch, Matthiola R. Br., Morettia DC., Neotorularia Hedge & J. Léonard, Rhammatophyllum O. E. Schulz, Sisymbriopsis Botsch. & Tzvelev, Sterigmostemum M. Bieb., and Tetracme Bunge each fell within a single tribal clade but were not monophyletic.

Phylogenetic relationships in Alisma (Alismataceae) based on RAPDs, and sequence data from ITS and trnL

The phylogeny of Alisma (Alismataceae), a genus of approximately nine species of aquatic plants mainly distributed in the Northern Hemisphere, was reconstructed with parsimony analysis on RAPD data and sequences of the nuclear ITS and chloroplast trnL regions, and with MDS on RAPD data. Separate analyses were performed on each data set. Butomus umbellatus was used as outgroup in the analysis of sequence data, and Luronium and Baldellia in the analyses of RAPD data. Among diploid Alisma, two major groups were found: (i) the gramineum group consisting of A. gramineum and A. wahlenbergii, and (ii) the plantago-aquatica group with A. plantago-aquatica, A. orientale, A. subcordatum, A. juzepczukii and A. ``bottnicum''. Taxa within the groups were poorly separated. The Baltic endemics A. wahlenbergii, A. juzepczukii and A. ``bottnicum'' have probably originated relatively recently from local populations of A. gramineum (i.e. A. wahlenbergii) and A. plantago-aquatica (i.e. A. juzepczukii and A. ``bottnicum''). The exact origin of the polyploid taxa, i.e. A. lanceolatum, A. triviale, A. canaliculatum and A. rariflorum, is still unclear.

여수해역에서 채집한 보름달 둥근 물해파리의 핵과 미토콘드리아 DNA 염기서열을 이용한 유연 관계 분석

This study presents the molecular phylogenetic analysis of Korean Aurelia aurita Linnaeus collected from Yeosu in the southern waters of Korea using nuclear ITS1 region and mitochondrial COI gene sequences. The use of oligonucleotide primers F5 (forward) and R5 (reverse) targeted to ITS1 and LCO1490 (forward) and HCO2198 (reverse) targeted to COI amplified 267 bp and 643 bp fragments, respectively. The shortest genetic distance towards the ITS1 region is estimated at 0.023 when comparing Korean A. aurita to Aurelia sp. collected from California, USA. In particular, Korean and American/Swedish A. aurita were located far away in terms of genetic distance, ranging from 0.393 to 0.395. On the other hand, the genetic distance between Korean and English/Turkish/Swedish/ American A. aurita regarding the mitochondrial DNA COI gene ranged from 0.201 to 0.205. However, a sister-ship with Korean and American A. aurita showed an extremely high bootstrap value (100%). The predicted secondary RNA structure of the mitochondrial DNA COI gene showed many different folding structures with a similar energy between Korean and American A. aurita. These results suggest that ITS1 and the mitochondrial DNA COI gene could be used as genetic markers for identification of the biogeographic populations.

Short communication. A new host and phenotypic variation of Phytophthora hedraiandra in Spain

The oomycete Phytophthora hedraiandra De Cock & Man in't Veld is first reported in Spain on the ornamental plant Rhododendron catawbiense Michx., as well as on leaves of Viburnum tinus L. in nurseries. The identification of these isolates was carried out by examining their morphological and cultural features, and by comparing the sequence of the nuclear rDNA ITS region and the mitochondrial cox1 gene with those published in GenBank database. The phenotypes of the isolates fitted the species description, but a higher intraspecific variation was noticed in respect to their sporangial size, colony pattern and radial growth rates. Due to the similarities between P. cactorum (Lebert et Cohn) and P. hedraiandra, the taxonomy and host ranges of the P. cactorum/P. hedraiandra complex in ornamental nurseries and natural ecosystems need reviewing.

Spatiotemporal analysis of population genetic structure in Geomonhystera disjuncta (Nematoda, Monhysteridae) reveals high levels of molecular diversity

Species identification in the phylum Nematoda is complicated due to the paucity of easily obtainable diagnostic morphological features. Furthermore, the cosmopolitan distribution of several species despite low dispersal abilities makes cryptic diversity potentially substantial within this phylum. We conducted a population genetic survey in the marine nematode Geomonhystera disjuncta in Belgium and The Netherlands in two seasons. The mitochondrial cytochrome oxidase c subunit 1 (COI) gene was screened with the single-strand conformation polymorphism method in 759 individuals. The 43 haplotypes were grouped into five lineages, with low divergences within (<3%) and high divergences between lineages (>14%). Analysis of the nuclear ITS region yielded concordant tree topologies, indicating the presence of five cryptic taxa within G. disjuncta. Analysis of Molecular Variance (AMOVA) illustrated a significant structuring in all lineages and temporal fluctuations in haplotype frequencies within and between locations. Metapopulation dynamics and/or priority effects best explained this structuring. Finally, our data indicate that the COI gene may be useful for DNA barcoding purposes.

Phylogeny and morphological evolution of tribe Menispermeae (Menispermaceae) inferred from chloroplast and nuclear sequences

Abstract The Menispermaceae family contains ca. 72 genera with 450 species that are almost entirely tropical. Its phylogeny at the tribal level has never been examined using molecular data. Here we used DNA sequences of the chloroplast matK gene and trnL - F regions, and the nuclear ITS region to study the delimitation and position of the tribe Menispermeae within the family and its subtribal monophyletic groups. Family-wide phylogenetic analyses of the chloroplast data produced two strongly supported clades. The first clade contains two subclades: Coscinieae including Arcangelisia and Anamirta , and Tinosporeae sensu lato including Fibraureae, supported by morphological characters, such as traits of the cotyledon, stylar scar and embryo. The second clade consists of the tribes Menispermeae sensu DC. and Tiliacoreae Miers. All our analyses surprisingly recognized that tribe Menispermeae is not monophyletic unless tribe Tiliacoreae is included, suggesting that characters of cotyledon and stylar scar are very important for the infrafamilial classification, and that endosperm presence vs. absence was over-emphasized in traditionally tribal division of the family. Our topologies indicate a secondary loss of endosperm. The monophyly of two subtribes of the tribe Menispermeae, Stephaniinae and Cissampelinae, is supported by the cpDNA and ITS data, as well as by morphological characters, including aperture types and shapes, and colpal membrane features of pollen grains, and sepal number of male flowers. The Cocculinae was recognized as a paraphyletic group containing the remaining genera of the tribe Menispermeae.

A phylogeny of the “evil tribe” (Vernonieae: Compositae) reveals Old/New World long distance dispersal: Support from separate and combined congruent datasets ( trnL-F, ndhF, ITS)

The Vernonieae is one of the major tribes of the largest family of flowering plants, the sunflower family (Compositae or Asteraceae), with ca. 25,000 species. While the family’s basal members (the Barnadesioideae) are found in South America, the tribe Vernonieae originated in the area of southern Africa/Madagascar. Its sister tribe, the Liabeae, is New World, however. This is the only such New/Old World sister tribe pairing anywhere in the family. The Vernonieae is now found on islands and continents worldwide and includes more than 1500 taxa. The Vernonieae has been called the “evil tribe” because overlapping character states make taxonomic delimitations difficult at all levels from the species to the subtribe for the majority of taxa. Juxtaposed with these difficult-to-separate entities are monotypic genera with highly distinctive morphologies and no obvious affinities to any other members of the tribe. The taxonomic frustration generated by these contrary circumstances has resulted in a lack of any phylogeny for the tribe until now. A combined approach using DNA sequence data from two chloroplast regions, the ndhF gene and the noncoding spacer trnL-F, and from the nuclear rDNA ITS region for 90 taxa from throughout the world was used to reconstruct the evolutionary history of the tribe. The data were analyzed separately and in combination using maximum parsimony (MP), minimum evolution neighbor-joining (NJ), and Bayesian analysis, the latter producing the best resolved and most strongly supported tree. In general, the phylogeny shows Old World taxa to be basal and New World taxa to be derived, but this is not always the case. Old and New World species are found together in two separate and only distantly related clades. This is best explained by long-distance dispersal with a minimum of two trans-oceanic exchanges. Meso/Central America has had an important role in ancient dispersals between the Old and New World and more recent movements from South to North America in the New World.

Taxonomic Revision of &lt;I&gt;Geranium&lt;/I&gt; Subsect. &lt;I&gt;Mediterranea&lt;/I&gt; (Geraniaceae)

Abstract Geranium subsect. Mediterranea (Geraniaceae) consists of ten species. The highest diversity of the group is located in the Caucasus and neighbouring areas of Turkey and Iran, with five endemic species. Other species reach western Europe and northwestern Africa. In contrast to the current literature, we consider G. montanum and G. ibericum subsp. jubatum to be synonyms of G. ibericum. A univariate morphometric study revealed some valuable quantitative characters useful for the identification of these species. Micromorphological features of pollen, stigmas, seeds, and mericarps were investigated by SEM. A new key is provided, as well as new and detailed descriptions. Geranium kurdicum is here illustrated for the first time. Eleven lectotypes are designated, and distribution maps are presented. Maximum parsimony and Bayesian analyses of chloroplastic trnL-trnF and ribosomal nuclear ITS regions suggest that sect. Mediterranea is monophyletic. Two clades are recovered, one including the annual species...

Suspended mummies in Aleiodes species (Hymenoptera: Braconidae: Rogadinae) with descriptions of six new species from western Uganda based largely on DNA sequence data

A group of species of the rogadine braconid genus Aleiodes are shown to produce a distinctive mummy, which is “J”‐shaped and is formed after the host larva, in all cases an ennomine geometrid moth, has dropped from a plant suspended in midair by a silk thread. The group includes one described species, A. buzurae He & Chen from China, and a species complex from tropical Africa (W. Uganda). All the African specimens reared from suspended mummies looked morphologically virtually indistinguishable, though there was considerable colour variation that segregated the specimens into five groups. Three gene fragments (nuclear 28S D2‐3 rDNA, the nuclear ITS2 region and part of the mitochondrial cytochrome oxidase 1 gene (CO1)) were sequenced to assess if these specimens represented a single variable species or a complex of morphologically cryptic species. Results show variation in all three gene fragments, with strong signal in the CO1 gene, parsimony analysis of which revealed six well supported groups corresponding to the colour variants, except that two specimens with nearly identical colour differed considerably in their CO1 sequences. Large, and difficult to align, variation was found in the ITS2 fragments, which by eye also supported the same six groupings. Limited variation was found in the 28S fragment, but one position supported monophyly of the two specimens belonging to one of the species circumscribed by the other genes. These groups are considered to correspond to separate species, which are described as new: A. barnardae Quicke & Shaw, A. basutai Quicke & Shaw, A. kanyawarensis Quicke & Shaw, A. kasenenei Quicke & Shaw, A. mubfsi Quicke & Shaw and A. trevelyanae Quicke & Shaw. The possible function of the specialised mummification behaviour is discussed and some observations on rates of hyperparasitism are presented.

Phylogenetic relationships of Clematis (Ranunculaceae) based on chloroplast and nuclear DNA sequences

Phylogenetic relationships within Clematis, including Naravelia, Archiclematis and Clematopsis, were analysed using nucleotide sequences of chloroplast DNA [(1) matK and trnK introns; (2) atpB–rbcL spacer; (3) rpoB–trnC spacer; (4) psbA–trnH–trnQ spacer; (5) rbcL–accD spacer] and the nuclear ITS regions. The phylogenetic trees resulting from these analyses suggested nine major clades. The genera Archiclematis, Naravelia and Clematopsis were found to be nested within the genus Clematis, and should be included within it. Within the genus Clematis, the traditional subgenus Viorna was found to be monophyletic. The remaining subgenera (Flammula, Clematis and Campanella) and sections Flammula, Clematis and Cheiropsis, however, were found to be paraphyletic or polyphyletic. Recircumscription of several groups in Clematis was suggested. Enormous morphological diversification and very few nucleotide substitutions within Clematis indicate the recent radiation of the genus. Clematidinae shares a gene order with Anemoninae between the starting point of large single copy region and trnQ of chloroplast DNA, although Hepatica, a genus in Anemoninae, shows a different order in the region near trnH.

Dating Dispersal and Radiation in the Gymnosperm Gnetum (Gnetales)—Clock Calibration When Outgroup Relationships Are Uncertain

Most implementations of molecular clocks require resolved topologies. However, one of the Bayesian relaxed clock approaches accepts input topologies that include polytomies. We explored the effects of resolved and polytomous input topologies in a rate-heterogeneous sequence data set for Gnetum, a member of the seed plant lineage Gnetales. Gnetum has 10 species in South America, 1 in tropical West Africa, and 20 to 25 in tropical Asia, and explanations for the ages of these disjunctions involve long-distance dispersal and/or the breakup of Gondwana. To resolve relationships within Gnetum, we sequenced most of its species for six loci from the chloroplast (rbcL, matK, and the trnT-trnF region), the nucleus (rITS/5.8S and the LEAFY gene second intron), and the mitochondrion (nad1 gene second intron). Because Gnetum has no fossil record, we relied on fossils from other Gnetales and from the seed plant lineages conifers, Ginkgo, cycads, and angiosperms to constrain a molecular clock and obtain absolute times for within-Gnetum divergence events. Relationships among Gnetales and the other seed plant lineages are still unresolved, and we therefore used differently resolved topologies, including one that contained a basal polytomy among gymnosperms. For a small set of Gnetales exemplars (n = 13) in which rbcL and matK satisfied the clock assumption, we also obtained time estimates from a strict clock, calibrated with one outgroup fossil. The changing hierarchical relationships among seed plants (and accordingly changing placements of distant fossils) resulted in small changes of within-Gnetum estimates because topologically closest constraints overrode more distant constraints. Regardless of the seed plant topology assumed, relaxed clock estimates suggest that the extant clades of Gnetum began diverging from each other during the Upper Oligocene. Strict clock estimates imply a mid-Miocene divergence. These estimates, together with the phylogeny for Gnetum from the six combined data sets, imply that the single African species of Gnetum is not a remnant of a once Gondwanan distribution. Miocene and Pliocene range expansions are inferred for the Asian subclades of Gnetum, which stem from an ancestor that arrived from Africa. These findings fit with seed dispersal by water in several species of Gnetum, morphological similarities among apparently young species, and incomplete concerted evolution in the nuclear ITS region.