Chloroplast DNA restriction site variation in theVernonieae (Asteraceae), an initial appraisal of the relationship of New and Old World taxa and the monophyly ofVernonia

Chloroplast DNA restriction site variation was examined for 35 taxa in theVernonieae and four outgroup tribes, using 17 restriction enzymes mapped for ca. 900 restriction sites per species; 139 mutations were found to be phylogenetically informative. Phylogenetic trees were constructed using Wagner and weighted parsimony, and evaluated by bootstrap and decay analyses. Relationships of Old and New World taxa indicate complex geographical relationships; there was no clear geographic separation by hemisphere. The relationships between Old and New World Vernonias found here support prior morphological analyses. The sister group to all New and most Old World taxa was composed of a small group of Old World species including yellow-flowered, trinervate-leaved species previously postulated to be basal in the tribe. The majority of both New and Old World taxa are derived from a lineage beginning with the monotypic genusStokesia, an endemic of the southeastern United States. The genusVernonia was also found to be paraphyletic within both the New and Old World. Available data do not support either the separation ofVernonia or the tribeVernonieae into geographically distinct lineages. The pattern of relationships within theVernonieae for taxa from North America, Asia, Africa, Central and South America is most similar to that of several other groups of both plants and animals with a boreotropical origin, rather than an origin in Gondwanaland. Such a pattern of distribution suggests more ancient vicariant events than are routinely postulated for theAsteraceae.     

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg²⁺-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase -dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergenta availability and cation sensitivity on the apparent distribution of PAPH in neutrophil sub-cellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzyme was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.     

CHLOROPLAST DNA EVIDENCE FOR THE INTERRELATIONSHIPS OF TOMATOES,POTATOES, AND PEPINOS (SOLANACEAE)

We used chloroplast DNA restriction site analysis to test hypotheses of relationships of Solarium subgenus Potatoe (including potatoes and pepinos), two other Solanum, Cyphomandra (the tree tomatoes), and Lycopersicon (the tomatoes). Capsicum and Datura were used as outgroups. The results support two main clades among the taxa we studied: 1) Solanum subgenus Potatoe and Lycopersicon; and 2) other Solanum and Cyphomandra. Within the first clade, the following groups were supported: a) sect. Basarthrum and sect. Anarrhichomenum; b) sect. Etuberosum; c) sect. Petota; d) sect. Juglandifolium, including subsect. Lycopersicoides; and e) the genus Lycopersicon. These results, in combination with an analysis of morphological data, advocate the controversial, but previously suggested, treatment of Lycopersicon as congeneric with Solanum in subgenus Potatoe. Thus, the cultivated tomato will be recognized as Solanum lycopersicum L. Solanum chmielewskii and Solanum lycopersicum var. cerasiforme are proposed as new combinations; Solanum neorickii is proposed as a new name for Lycopersicon parviflorum. Our data also suggest that Cyphomandra should be included within Solanum.     

Chloroplast DNA variation and phylogeny of theRanunculaceae

Restriction site mapping of chloroplast DNA from 31 species representing 26 genera of theRanunculaceae was performed using eleven restriction endonucleases. The chloroplast genome varies in length from approximately 152 to 160 kb. Length variants are frequent in theRanunculaceae and range from usually less than 300 bp to rarely 1.5 kb. The inverted repeat is extended into the large single copy (LSC) region by 4–4.5 kb inAnemone, Clematis, Clematopsis, Hepatica, Knowltonia, andPulsatilla. Several inversions are present in the LSC-region of the cpDNA in all these genera and inAdonis. The frequency of restriction site mutations varies within the chloroplast genome in theRanunculaceae between 4 and 32 mutations per kilobase, and is lowest in the inverted repeat and the regions containing the ATPase-genes and the genespsaA, psaB, psbA, rpoB, andrbcL. A total of 547 phylogenetically informative restriction sites was utilized in cladistic analyses of the family using Wagner, Dollo, and weighted parsimony. These three parsimony analyses result in different tree topologies. Four, six, and one equally most parsimonious trees were obtained with Wagner, Dollo, and weighted parsimony, respectively. The amount of support for the monophyletic groups was evaluated using bootstrapping and decay analysis. All three parsimony methods suggest thatHydrastis is the sister group to the remainder of theRanunculaceae, and that theAnemone-Clematis group, which shares several derived cpDNA rearrangements, is monophyletic. Only a few of the traditional groups in theRanunculaceae are supported by cpDNA restriction side data. Only Dollo parsimony provides support for the hypothesis thatThalictroideae andRanunculoideae are monophyletic.     

Comparison between cellulose and amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases for enantiomeric separation of 17 amidotetralins

Direct enantiomeric separations of 17 chiral amidotetralins by means of high performance liquid chromatography were performed on stationary phases composed of tris(3,5-dimethylphenylcarbamate) derivatives of cellulose and amylose, coated on silica gel. The enantiomers of 15 out of 17 amidotetralins were resolved with a resolution of more than 1.5 by at least one of the chiral stationary phases. The stationary phases showed complementary results with regard to the separation of the amidotetralins, that is, pairs that did not separate on the cellulose-type column were well separated on the amylose-type column, and vice versa. There was no significant correlation between the chromatographic properties of the chiral stationary phases. © 1993 Wiley-Liss, Inc.     

EVOLUTIONARY IMPLICATIONS OF INTRASPECIFIC CHLOROPLAST DNA VARIATION IN DWARF DANDELIONS (KRIGIA; ASTERACEAE)

Intraspecific chloroplast DNA polymorphisms were examined for 51 populations of seven species in the genus Krigia. A total of 1,100 restriction sites was surveyed and 46 of these were variable at the intraspecific level. Twenty-two of the variable sites were found within K. virginica, giving this species one of the highest levels of intraspecific chloroplast DNA divergence of any examined species. In contrast, no restriction site variation was detected within K. dandelion, K. wrightii, and K. occidentalis. Five polymorphisms were identified from the 16 populations of the K. cespitosa-gracilis complex, but no mutations distinguished the K. cespitosa and K. gracilis types. Krigia montana and K. biflora showed 11 and eight restriction site polymorphisms, respectively. The chloroplast genome of the hexaploid K. montana was derived from the diploid K. biflora rather than the tetraploid K. montana. High levels of polymorphism were found in species having different ploidy levels, such as K. virginica, K. biflora, and K. montana. Furthermore, most mutations found in these three species were recorded from the tetraploid lineages. As a result, evolutionary rates between different ploidy levels differ significantly. The chloroplast DNA restriction site data suggest that all surveyed populations of the autotetraploid K. virginica originated from a common ancestor. Our results also indicate that certain regions of the chloroplast genome have changed more rapidly than others and have the potential to resolve evolutionary questions at the population level.     

RESTRICTION FRAGMENT ANALYSIS OF CHLOROPLAST DNA AND THE SYSTEMATICS OF VIGUIERA AND RELATED GENERA (ASTERACEAE: HELIANTHEAE)

Phylogenetic relationships among 31 species representing variously Helianthus, Helianthopsis, Heliomeris, Simsia, Viguiera, and Tithonia were assessed by chloroplast DNA restriction site mapping. A total of 147 mutations including 92 informative ones was detected using 16 restriction enzymes, with an estimated sequence divergence within the group of 1.4%. Parsimony analysis produced a single most parsimonious tree with a length of 161 steps and a consistency index of 0.91. Statistically significant clades, as assessed by the bootstrap method, correspond to a number of taxa recognized previously, including Helianthus (3 species), Helianthopsis (5 species), and several groups within Viguiera, including sect. Maculatae (4 species), the Baja California group (6 species), sect. Paradosa (2 species) and V. dentata (3 samples). However, species of Viguiera collectively form a highly paraphyletic group relative to species of other genera. Helianthus and Helianthopsis were separated into different clades, supporting their recent segregation. Placement of H. porteri in Helianthus rather than Heliomeris was confirmed; the single sample of the latter genus was most similar to the Baja California group of Viguiera. An expected relationship between Simsia (2 species) and one member of Viguiera ser. Grammatoglossae was confirmed (although not with two other putatively related members of Viguiera) and an unexpected relationship between Simsia and Tithonia was suggested. The presence of Mexican taxa as the more basal groups in the tree points toward a possible Mexican origin for Viguiera and related genera. A molecular clock hypothesis is rejected in many pairwise comparisons involving woody taxa with herbaceous ones, although it could not be rejected in most pairwise comparisons involving taxa of similar habit.     

Single embryo transfer in IVF to prevent multiple pregnancies.

As the success rates of IVF clinics improve, one of the adverse consequences is the increased incidence of twins, due largely to the number of embryos transferred. Even if the number of embryos transferred is restricted to two, the twinning rate can exceed 40% of the pregnancies. An obvious way to reduce this high twin rate would be to transfer only one embryo. This would require that cryopreservation of the supernumerary embryos be efficacious enough so that the chance of achieving an ongoing pregnancy is not diminished by transferring a single embryo in the stimulated cycle. Previous studies utilising embryos on day 2 and 3 of development have shown that the pregnancy rates can be acceptable (about 40%) and that the cumulative rate can be up to 60%. Most of these studies, however, do not include a comparison with the cumulative pregnancy rate with two embryos transferred in the stimulated cycle. Therefore, the efficacy has not been proven. We present clinical data from the past few years to illustrate the increase in success rates and the concomitant increase in twinning rates. The increased success in the cryopreservation program has enabled us to trial a single embryo transfer program and compare the results to the transfer of two embryos. The results strongly suggest that the transfer of a single embryo is the better clinical option.