Although horizontal gene transfer is well documented in microbial genomes, no case has been reported in higher plants. We discovered horizontal transfer of the mitochondrial nad1 intron 2 and adjacent exons b and c from an asterid to Gnetum (Gnetales, gymnosperms). Gnetum has two copies of intron 2, a group II intron, that differ in their exons, nucleotide composition, domain lengths, and structural characteristics. One of the copies, limited to an Asian clade of Gnetum, is almost identical to the homologous locus in angiosperms, and partial sequences of its exons b and c show characteristic substitutions unique to angiosperms. Analyses of 70 seed plant nad1 exons b and c and intron 2 sequences, including representatives of all angiosperm clades, support that this copy originated from a euasterid and was horizontally transferred to Gnetum. Molecular clock dating, using calibrations provided by gnetalean macrofossils, suggests an age of 5 to 2 million years for the Asian clade that received the horizontal transfer.H orizontal gene transfer is the basis for the genetic engineering of commercially important crops, and natural horizontal gene transfers across kingdoms have been documented between Agrobacterium and Nicotiana (1), Wolbachia and its insect host Callosobruchus (2), and bacteria and land plants (3, 4). Among eukaryotic lineages, however, very few natural horizontal transfers have been reported, and none of them involve transfers across groups of seed plants. As part of an investigation of the phylogeny of Gnetum (Gnetales, gymnosperms), we studied the distribution of a group II intron in the mitochondrial (mt) nad1 gene, which encodes subunit 1 of the respiratory chain NADH dehydrogenase. The nad1 gene consists of five exons that are cisor trans-located and separated by 7-294 kb . The intervening introns are cis-or trans-spliced accordingly (9, 10). The second intron of the nad1 gene, located between exons b and c, is a group II intron (ref. 11 and Fig. 1 A). Group II introns are self-splicing RNAs that are typical components of contemporary organellar genomes in plants, algae, fungi, protists, and eubacteria (10,(12)(13)(14). They are characterized by a uniform structure of six major domains radiating from a central wheel (Fig. 1B), with domains I and V most crucial for the introns' enzymatic activity (12, 14) and ribozymic function (11,12). Because of these characteristics and the presence of reverse transcriptase ORFs, they are believed to be the ancestors of spliceosomal introns and non-long-terminal repeat retroelements (13, 15). Unlike group I introns, at least one of which appears to have been traded within flowering plants (16), group II introns in plants have been thought to be strictly vertically inherited (17)(18)(19), and the only known horizontal transfer of a group II intron in eukaryotes occurred in haptophytes, marine unicellular flagellates (20).During work on the phylogeny of Gnetum, we developed specific primers to amplify the second intron in the nad1 gene, plus flanking exons, from n...

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.

Abstract.-Siparunaceae comprise Glossocalyx with one species in West Africa and Siparuna with 65 species in the neotropics; all have unisexual owers, and 15 species are monoecious, 50 dioecious. Parsimony and maximum likelihood analyses of combined nuclear ribosomal ITS and chloroplast trnL-trnF intergenic spacer sequences yielded almost identical topologies, which were used to trace the evolution of the two sexual systems. The African species, which is dioecious, was sister to all neotropical species, and the monoecious species formed a grade basal to a large dioecious Andean clade. Dioecy evolved a second time within the monoecious grade. Geographical mapping of 6,496 herbarium collections from all species sorted by sexual system showed that monoecy is con ned to low-lying areas (altitude < 700 m) in the Amazon basin and southern Central America. The only morphological trait with a strong phylogenetic signal is leaf margin shape (entire or toothed), although this character also correlates with altitude, probably re ecting selection on leaf shapes by temperature and rainfall regimes. The data do not reject the molecular clock, and branch lengths suggest that the shift to dioecy in the lowlands occurred many million years after the shift to dioecy in the ancestor of the Andean clade. [Altitudinal distribution of sexual systems; dioecy; molecular clock; monoecy; sexual system evolution.] About 6% (14,620 of 240,000) of the species of owering plants are dioecious, and about 7% (959 of 13,500) of the genera are completely or partly dioecious (Renner and Ricklefs, 1995). Comparative evidence suggests that dioecy has evolved many times from perfect-owered (monoclinous) or monoecious (with diclinous owers on each plant) ancestors (Lewis, 1942;Charlesworth and Charlesworth, 1978;Lloyd, 1980). Here we study a small tropical family with diclinous owers and monoecious or dioecious species, using a phylogeny to infer the frequency of shifts between these sexual systems. We also address the possible relation between an ecological factor (altitude) and switches in sexual system and discuss differences between some major evolutionary pathways to dioecy. Two key terms, paradioecy and gynodioecy, need to be de ned at the outset. Paradioecy is the inconstant presence of male or female owers in the males and females of dioecious species, with the inconstancies being of similar magnitude in both sexes (Lloyd, 1972a). Gynodioecy is the regular coexistence of perfect-owered and pistillate individuals (

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