Phylogeny of the cycads based on multiple single-copy nuclear genes: congruence of concatenated parsimony, likelihood and species tree inference methods.

BACKGROUND AND AIMS: Despite a recent new classification, a stable phylogeny for the cycads has been elusive, particularly regarding resolution of Bowenia, Stangeria and Dioon. In this study, five single-copy nuclear genes (SCNGs) are applied to the phylogeny of the order Cycadales. The specific aim is to evaluate several gene tree-species tree reconciliation approaches for developing an accurate phylogeny of the order, to contrast them with concatenated parsimony analysis and to resolve the erstwhile problematic phylogenetic position of these three genera. METHODS: DNA sequences of five SCNGs were obtained for 20 cycad species representing all ten genera of Cycadales. These were analysed with parsimony, maximum likelihood (ML) and three Bayesian methods of gene tree-species tree reconciliation, using Cycas as the outgroup. A calibrated date estimation was developed with Bayesian methods, and biogeographic analysis was also conducted. KEY RESULTS: Concatenated parsimony, ML and three species tree inference methods resolve exactly the same tree topology with high support at most nodes. Dioon and Bowenia are the first and second branches of Cycadales after Cycas, respectively, followed by an encephalartoid clade (Macrozamia-Lepidozamia-Encephalartos), which is sister to a zamioid clade, of which Ceratozamia is the first branch, and in which Stangeria is sister to Microcycas and Zamia. CONCLUSIONS: A single, well-supported phylogenetic hypothesis of the generic relationships of the Cycadales is presented. However, massive extinction events inferred from the fossil record that eliminated broader ancestral distributions within Zamiaceae compromise accurate optimization of ancestral biogeographical areas for that hypothesis. While major lineages of Cycadales are ancient, crown ages of all modern genera are no older than 12 million years, supporting a recent hypothesis of mostly Miocene radiations. This phylogeny can contribute to an accurate infrafamilial classification of Zamiaceae.

Phylogenetic relationships among arecoid palms (Arecaceae: Arecoideae).

BACKGROUND AND AIMS: The Arecoideae is the largest and most diverse of the five subfamilies of palms (Arecaceae/Palmae), containing >50 % of the species in the family. Despite its importance, phylogenetic relationships among Arecoideae are poorly understood. Here the most densely sampled phylogenetic analysis of Arecoideae available to date is presented. The results are used to test the current classification of the subfamily and to identify priority areas for future research. METHODS: DNA sequence data for the low-copy nuclear genes PRK and RPB2 were collected from 190 palm species, covering 103 (96 %) genera of Arecoideae. The data were analysed using the parsimony ratchet, maximum likelihood, and both likelihood and parsimony bootstrapping. KEY RESULTS AND CONCLUSIONS: Despite the recovery of paralogues and pseudogenes in a small number of taxa, PRK and RPB2 were both highly informative, producing well-resolved phylogenetic trees with many nodes well supported by bootstrap analyses. Simultaneous analyses of the combined data sets provided additional resolution and support. Two areas of incongruence between PRK and RPB2 were strongly supported by the bootstrap relating to the placement of tribes Chamaedoreeae, Iriarteeae and Reinhardtieae; the causes of this incongruence remain uncertain. The current classification within Arecoideae was strongly supported by the present data. Of the 14 tribes and 14 sub-tribes in the classification, only five sub-tribes from tribe Areceae (Basseliniinae, Linospadicinae, Oncospermatinae, Rhopalostylidinae and Verschaffeltiinae) failed to receive support. Three major higher level clades were strongly supported: (1) the RRC clade (Roystoneeae, Reinhardtieae and Cocoseae), (2) the POS clade (Podococceae, Oranieae and Sclerospermeae) and (3) the core arecoid clade (Areceae, Euterpeae, Geonomateae, Leopoldinieae, Manicarieae and Pelagodoxeae). However, new data sources are required to elucidate ambiguities that remain in phylogenetic relationships among and within the major groups of Arecoideae, as well as within the Areceae, the largest tribe in the palm family.

Molecular phylogenetics of the palm subtribe Ptychospermatinae (Arecaceae).

PREMISE OF THE STUDY: We examined the phylogeny and intergeneric relationships among the 12 genera of the palm subtribe Ptychospermatinae. While many of these taxa are familiar, cultivated ornamental palms in warm areas of the world, the monophyly of the subtribe and its component genera required testing. We also examined the biogeographic relationships of this lineage, which has a significant radiation east of Wallace's Line. METHODS: Phylogenetic analyses were based on maximum parsimony and Bayesian analyses of nucleotide sequences of two low-copy nuclear genes: intron 4 of phosphoribulokinase and intron 23 of RNA polymerase II. Biogeographical reconstructions were explored using S-DIVA. KEY RESULTS: The two-gene, combined analysis yielded a monophyletic subtribe with six major clades. The biogeographical analysis suggests that the subtribe originated in New Guinea. CONCLUSIONS: The phylogenetic hypotheses support the monophyly of the subtribe. The genera Drymophloeus, Ponapea, and Veitchia, as presently circumscribed, are not monophyletic. The resurrection and expanded circumscription of the genus Ponapea are supported. A newly discovered species of Adonidia is confirmed as sister species to Adonidia merrillii. Our phylogenetic hypothesis suggests that the Ptychospermatinae diverged into six major clades with repeated radiations into Australia and the western Pacific. The presence of Adonidia to the west of Wallace's Line is likely to be the result of long-distance dispersal. The following new combinations are made to restore monophyly to Veitchia and Ponapea: Veitchia pachyclada, V. subisticha, V. lepidota, and Ponapea hentyi.

Sweet drinks are made of this: conservation genetics of an endemic palm species from the Dominican Republic.

Pseudophoenix ekmanii is a threatened palm species endemic to the Dominican Republic. Sap from trees is extracted to make a local drink; once they are tapped the individual usually dies. Plants are also illegally harvested for the nursery trade and destroyed by poachers hunting the endemic and threatened Hispaniolan parrot. We used 7 DNA microsatellite markers to assist land managers in developing conservation strategies for this palm. We sampled 4 populations along the known distribution range of this species (3 populations from the mainland and 1 from the small island of Isla Beata), for a total sample of n = 104. We found strong evidence for genetic drift, inbreeding, and moderate gene flow (i.e., all populations had at least 4 loci that were not in Hardy-Weinberg equilibrium, at least 9 loci pairs were in linkage disequilibrium, the pairwise F(ST) values ranged from 0.069 to 0.266, and had positive F(IS) values). Data supported an isolation-by-distance model, and cluster analyses based on genetic distances resolved 2 groups that match a north-south split. The population from Isla Beata had the lowest levels of genetic diversity and was the only one in which we found pairs of individuals with identical shared multilocus genotypes.

Phylogenetic analyses of nucleotide sequences confirm a unique plant intercontinental disjunction between tropical Africa, the Caribbean, and the Hawaiian Islands.

Phylogenetic analyses of nucleotide sequences of the internal transcribed spacers and 5.8 regions of the nuclear ribosomal DNA and of the trnH-psbA spacer of the chloroplast genome confirm that the three taxa of the Jacquemontia ovalifolia (Choicy) Hallier f. complex (Convolvulaceae) form a monophyletic group. Levels of nucleotide divergence and morphological differentiation among these taxa support the view that each should be recognized as distinct species. These three species display unique intercontinental disjunction, with one species endemic to Hawaii (Jacquemontia sandwicensis A. Gray.), another restricted to eastern Mexico and the Antilles [Jacquemontia obcordata (Millspaugh) House], and the third confined to East and West Africa (J. ovalifolia). The Caribbean and Hawaiian species are sister taxa and are another example of a biogeographical link between the Caribbean Basin and Polynesia. We provide a brief conservation review of the three taxa based on our collective field work and investigations; it is apparent that J. obcordata is highly threatened and declining in the Caribbean.

An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies.

Indirect immunofluorescence procedures reported thus far are not effective at localizing two antigens in the same preparation when both primary antibodies are raised in the same species. In this case, the secondary antibodies can crossreact with both primary antibodies. We report here a protocol in which mouse monoclonal antibodies (MAb) specific for actin and myosin were used sequentially to stain the same frozen section of guinea pig skeletal muscle. The myosin-specific mAb was applied first and was localized with a rabbit anti-mouse IgG-rhodamine secondary antibody. The sections were then "blocked" with a non-binding mouse MAb and unconjugated goat anti-mouse IgG F(ab) fragments. The actin-specific mAb was then applied and localized with a rabbit anti-mouse IgG-fluorescein secondary antibody. Laser scanning confocal microscopy and image analysis demonstrated that the I-bands, the A-bands, and the H-bands of each sarcomere were clearly identifiable by this approach. This protocol is not limited to use with mouse MAb but can be easily modified to permit indirect immunolocalization of two antigens in the same sample using any pair of same-species primary antibodies.

Acetylenic Vinyllithiums: Consecutive Cycloisomerization-[4 + 2] Cycloaddition Reactions.

Acetylenic vinyllithiums (2), which were generated from the corresponding acetylenic vinyl bromides (3) by low-temperature lithium-bromine exchange, cyclize on warming to give, following quench with water, isomerically pure conjugated bis-exocyclic 1,3-dienes (1) in good to excellent yield. Both five-membered and six-membered outer-ring dienes may be prepared: 5-exo closure of an acetylenic vinyllithium, which proceeds with total stereocontrol via syn-addition to give the E-isomer of a five-membered outer-ring diene, tolerates aryl-, silyl-, or alkyl-substituents at the distal acetylenic carbon; the corresponding 6-exo process is less facile and seems to be confined to substrates bearing an anion-stabilizing substituent, such as phenyl or trimethylsilyl, at the terminal acetylenic carbon. The highly reactive bis-exocyclic 1,3-dienes serve as precursors to polycyclic materials through subsequent Diels-Alder reaction with a wide variety of dienophiles. The consecutive exchange-cyclization-cycloaddition methodology, which can be conducted in one pot without isolation of intermediates, provides an efficient, operationally simple, and diastereoselective route to diverse polycyclic ring systems.

Evolution of lamina anatomy in the palm family (Arecaceae).

The unique properties of tree building in Arecaceae strongly constrain their architectural lability. Potentially compensating for this limitation, the extensive diversification of leaf anatomical structure within palms involves many characters whose alternate states may confer disparate mechanical or physiological capabilities. In the context of a recent global palm phylogeny, we analyzed the evolution of 10 such lamina anatomical characters and leaf morphology of 161 genera, conducting parsimony and maximum likelihood ancestral state reconstructions, as well as tests of correlated evolution. Lamina morphology evolves independently from anatomy. Although many characters do optimize as synapomorphic for major clades, anatomical evolution is highly homoplasious. Nevertheless, it is not random: analyses indicate the recurrent evolution of different cohorts of correlated character states. Notable are two surface layer (epidermis and hypodermis) types: (1) a parallel-laminated type of rectangular epidermal cells with sinuous anticlinal walls, with fibers present in the hypodermis and (2) a cross-laminated type of hexagonal cells in both layers. Correlated with the cross-laminated type is a remarkable decrease in the volume fraction of fibers, accompanied by changes in the architecture and sheath cell type of the transverse veins. We discuss these and other major patterns of anatomical evolution in relation to their biomechanical and ecophysiological significance.

Immunolocalization of the ecto-ATPase and ecto-apyrase in chicken gizzard and stomach. Purification and N-terminal sequence of the stomach ecto-apyrase.

We have examined the in vivo localization of extracellular ecto-ATPase and ecto-apyrase (ATPDase) in adult chicken gizzard and stomach by immunofluorescence and laser scanning confocal microscopy. In chicken gizzard, the ecto-ATPase was distributed in discrete clusters restricted to the sarcolemma of the smooth muscle cells. Anti-ecto-apyrase antibody detected a single 80-kDa band (putative apyrase) in Western blots of both chicken gizzard membrane extracts and partially purified anion exchange fractions, but the antibody did not detect ecto-apyrase in immunolabeled gizzard cryosections. In adult chicken stomach, the ecto-apyrase was observed at the apical membrane of the glandular oxyntico-peptic cells as described in previous immunoperoxidase studies (Stout, J. G., R. S. Strobel, and T. L. Kirley (1995) Biochem. Mol. Biol. Int. 36, 529-535). However, ecto-ATPase was clustered in the sarcolemma of the organized layer of circular smooth muscle and in smooth muscle cells of the septa surrounding the glandular tissue, but not in the glandular cells containing the ecto-apyrase. The findings indicate compartmentalization of the two related extracellular nucleotide hydrolyzing enzymes and suggest differential functions that are specialized for different regions of the chicken stomach. We also partially purified the ecto-apyrase of chicken stomach, an 80-kDa membrane glycoprotein. Chicken stomach membranes were solubilized in digitonin, glycoproteins were separated from solubilized proteins by lectin chromatography, and nucleotide-binding glycoproteins were selected by immobilized Cibacron blue chromatography. Further purification by size exclusion and anion exchange chromatography yielded purification of 94-fold. The ATPase specific activity of the purified stomach ecto-apyrase was 75,000 micromol of Pi/mg of protein/h, and the purified preparation consisted of a major band (55% of total protein) at 80 kDa. The purified enzyme could be deglycosylated with peptide N-glycosidase-F to a core molecular mass of 54 kDa. The N-terminal sequence of the 80-kDa stomach ecto-apyrase band (which reacted with anti-ecto-ATPDase antibodies) was determined to be: MEYKGKVVAGLLTATWV. Immunological cross-reactivity data indicate that the stomach 80-kDa protein isolated is an ecto-apyrase and is related to both the chicken liver and oviduct ecto-ATPDase enzymes characterized earlier, as well as to the human lymphoid cell activation antigen, CD39.

Cross-linking induces homodimer formation and inhibits enzymatic activity of chicken stomach ecto-apyrase.

We have investigated the effect of cross-linking on the enzymatic activity and oligomer formation of the chicken stomach ecto-apyrase. Cross-linking with the hydrophobic, lysine-specific dithiobis(succinimidylpropionate) (DSP) caused equal inhibition of ATPase and ADPase activity in both the membrane-bound and detergent-solubilized ecto-apyrase. The inhibitory effect of cross-linking was reversed upon the addition of the reductant dithiothreitol. Western blots of aliquots of the cross-linked samples show decreased amounts of the monomeric 80 kDa ecto-apyrase and the appearance of a 160 kDa dimer under conditions inducing enzyme inhibition. Therefore, the chicken stomach ecto-apyrase, like the chicken gizzard ecto-ATPase, is likely a homodimer in vivo. Unlike the related gizzard ecto-ATPase, however, the native stomach ecto-apyrase is not stimulated, but rather inhibited by cross-linking, presumably due to different quaternary structural stability of the two enzymes.

Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase.

A human brain E-type ATPase (HB6 ecto-apyrase) was subjected to site-directed mutagenesis to assess the functional significance of two highly conserved tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly expressed ecto-apyrase completely devoid of nucleotidase activity. Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular distribution clearly different from that of the wild-type enzyme, with the majority of the immunoreactivity concentrated in the interior of the cell. Unlike the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron Blue and was sensitive to proteolytic digestion by chymotrypsin. These results suggest alteration of the tertiary structure, causing the enzyme to be improperly folded and retained within the cell. In contrast, mutation of tryptophan 459 to alanine resulted in an ecto-apyrase with enhanced NTPase activity, but diminished NDPase activity. Immunolocalization of this active mutant ecto-apyrase revealed a cellular pattern similar to that of the wild-type enzyme, distributed along the cell periphery and in cell processes. Coupling this active W459A mutation to a previously described mutation (D219E) resulted in an enzyme which preferentially hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In addition, the double mutant is substantially less sensitive to inhibition by azide, a more potent inhibitor of ecto-apyrases than ecto-ATPases. Thus, mutation of only two amino acids of an E-type ATPase essentially converts an ecto-apyrase to an ecto-NTPase.