BackgroundDespite considerable progress in our understanding of land plant phylogeny, several nodes in the green tree of life remain poorly resolved. Furthermore, the bulk of currently available data come from only a subset of major land plant clades. Here we examine early land plant evolution using complete plastome sequences including two previously unexamined and phylogenetically critical lineages. To better understand the evolution of land plants and their plastomes, we examined aligned nucleotide sequences, indels, gene and nucleotide composition, inversions, and gene order at the boundaries of the inverted repeats.ResultsWe present the plastome sequences of Equisetum arvense, a horsetail, and of Isoetes flaccida, a heterosporous lycophyte. Phylogenetic analysis of aligned nucleotides from 49 plastome genes from 43 taxa supported monophyly for the following clades: embryophytes (land plants), lycophytes, monilophytes (leptosporangiate ferns + Angiopteris evecta + Psilotum nudum + Equisetum arvense), and seed plants. Resolution among the four monilophyte lineages remained moderate, although nucleotide analyses suggested that P. nudum and E. arvense form a clade sister to A. evecta + leptosporangiate ferns. Results from phylogenetic analyses of nucleotides were consistent with the distribution of plastome gene rearrangements and with analysis of sequence gaps resulting from insertions and deletions (indels). We found one new indel and an inversion of a block of genes that unites the monilophytes.ConclusionsMonophyly of monilophytes has been disputed on the basis of morphological and fossil evidence. In the context of a broad sampling of land plant data we find several new pieces of evidence for monilophyte monophyly. Results from this study demonstrate resolution among the four monilophytes lineages, albeit with moderate support; we posit a clade consisting of Equisetaceae and Psilotaceae that is sister to the "true ferns," including Marattiaceae.

The conjugating green algae represent a lineage of charophyte green algae known for their structural diversity and unusual mode of sexual reproduction, conjugation. These algae are ubiquitous in freshwater environments, where they are often important primary producers, but few studies have investigated evolutionary relationships in a molecular systematic context. A 109-taxon data set consisting of three gene fragments (two from the chloroplast and one from the mitochondrial genome) was used to estimate the phylogeny of the genera of the conjugating green algae. Maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) were used to estimate relationships from the 4,047 alignable nucleotides. This study confirmed the polyphyly of the Zygnemataceae and Mesotaeniaceae with respect to one another. The Peniaceae were determined to be paraphyletic, and two genera traditionally classified among the Zygnematales appear to belong to the lineage that gave rise to the Desmidiales. Six genera, Euastrum, Cosmarium, Cylindrocystis, Mesotaenium, Spondylosium, and Staurodesmus, were polyphyletic in this analysis. These findings have important implications for the evolution of structural characteristics in the group and will require some taxonomic changes. More work will be required to delineate lineages of Zygnematales in particular and to identify structural synapomorphies for some of the newly identified clades.

Natural populations of Zygnema were collected from 80 stream sites across California, and eight species were identified and characterized morphologically. Generic and infrageneric concepts of Zygnema and Zygogonium were tested with cox3 and rbcL gene sequence analysis. Strains of Zygnema were positioned in a single monophyletic clade sister to Zygogonium tunetanum Gauth.-Lièvre. In both the rbcL and cox3 phylogenies, strains of Zygnema formed two major clades. The first clade contained species that have zygospores with a blue-colored mesospore or akinetes with a colorless mesospore. The second clade contained species that have a yellow or brown mesospore. The existing taxonomic concepts for Zygnema classification are not consistent with our molecular phylogeny and do not correspond to natural groups. We propose that mesospore color may be useful in the infrageneric classification of Zygnema. Newly described Zygnema aplanosporum sp. nov. and Zygnema californicum sp. nov. have zygospores with a blue mesospore formed in the conjugation tube and separated by a cellulosic sporangial wall. Z. aplanosporum also possessed a combination of vegetative and reproductive features characteristic of Zygogonium, such as presence of short branches, rhizoidal outgrowths, thickened vegetative cell walls, purple-colored cell content, small compressed-globular chloroplasts as well as predominant asexual reproduction. Z. aplanosporum and Z. californicum were deeply embedded in a larger clade of Zygnema both in rbcL and cox3 analyses. Based on our observations, there are no features or combination of features that separate Zygnema and Zygogonium. Therefore, we conclude that Zygogonium is probably a synonym of Zygnema.

Chromosome I from the yeast Saccharomyces cerevisiae contains a DNA molecule of -231 kbp and is the smallest naturally occurring functional eukaryotic nuclear chromosome so far characterized. The nucleotide sequence of this chromosome has been determined as part of an international collaboration to sequence the entire yeast genome. The yeast Saccharomyces cerevisiae has been the focus of intensive study as a model eukaryote. As part of this effort, an international program is under way to determine the nucleotide sequence of the 16 chromosomes that constitute its 13.5-Mbp nuclear genome. This endeavor will provide both a complete eukaryotic gene set and a reference set of experimentally amenable genes for comparison with those of other organisms. Currently, four yeast chromosomes have been sequenced (1-4); all have a high gene density, and a majority of the genes found are newly sequenced and of unknown function. Chromosome I is the smallest S. cerevisiae chromosome. It contains a DNA molecule that is only 231 kbp, making it the smallest known fully functional nuclear chromosome. This chromosome has been studied intensively, and mutants are available for a large number of its genes (5-7). Here we report the nucleotide sequence of chromosome I and describe several unusual features of its gene organization and chromosome structure as well as many newly discovered genes.** MATERIALS AND METHODS DNA Sources. Four sources of chromosome I DNA, all from S288C-derived yeast strains, were used to generate the tem-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.plates for DNA sequencing. These were the library of Riles et aL (8), a cosmid from the collection of Dujon (9), chromosome walking (10), and PCR amplified fragments of genomic DNA. DNA fragments, except those generated by PCR which were used directly, were subcloned into the Bluescript KS(+) plasmid from Stratagene prior to sequencing. All DNA sequencing was performed using double-stranded DNA templates.DNA Sequencing. Two methods were used for sequencing DNA templates: manual sequencing and machine-based sequencing with an Applied Biosystems sequencing machine (model 373A). Our manual sequencing used unidirectional nested deletions and was carried out as described (11, 12). For machine-based sequencing, three sets of templates were used: unidirectional nested deletions, PCR amplified chromosomal DNA, and, for the region spanning YAL062 to CDC24, cosmid DNA was shotgun cloned into Bluescript KS(+). In summary, the procedure for the Applied Biosystems machine (model 373A) used dye-labeled dideoxynucleotide terminators and a cycle sequencing kit (Prism Ready reaction dye terminator kit; Perkin-Elmer) and the protocol provided by the supplier. This method allowed us to process all four sequencing reactions in a single reaction tube. The cycle amplification reactions were performed with a Perkin-Elmer ...

Diversity of the filamentous green algae in the genus Spirogyra (Zygnematophyceae) was investigated from more than 1,200 stream samples from California. We identified 12 species of Spirogyra not previously known for California (CA), including two species new to science, Spirogyra californica sp. nov. and Spirogyra juliana sp. nov. Environmental preferences of the Californian species are discussed in the light of their restricted distribution to stream habitats with contrasting nutrient levels. We also investigated the systematic relationships of Spirogyra species from several continents using the chloroplast-encoded genes ribulose-1,5-bisphosphate carboxylase/hydrogenase large subunit (rbcL) and the beta subunit of the ATP synthase (atpB). Californian species were positioned in most major clades of Spirogyra. The phylogeny of Spirogyra and its taxonomic implications are discussed, such as the benefits of combining structural and molecular data for more accurate and consistent species identification. Considerable infraspecific genetic variation of globally distributed Spirogyra species was observed across continental scales. This finding suggests that structurally similar species from distant regions may be genetically dissimilar and that Spirogyra may contain a large number of cryptic species. Correlating the morphological and genetic variation within the genus will be a major challenge for future researchers.

Reflectance, transmittance and absorptance spectral of "normal" and six types of nutrient-deficient (N, P, K, S, Mg, and Ca) maize (Zea mays L.) leaves were analyzed at 30 selected wavelengths from 500~2600 nm. The analysis of variance showed significant differences in reflectance, transmittance and absorptance in the visible wavelengths among leaf numbers 3, 4, and 5, among the seven treatments, and among the interactions of leaf number and treatments. In the infrared wavelengths only treatments produced significant differences.The chlorophyll content of leaves was reduced in all nutrientdeficient treatment s. Percent moisture 'was increased in S-, Mg-, and N-deficiencies. Positive correlations were obtained (r = 0.7) between moisture content and percent absorption at both 1450 and 1930 nm. Polynomial regression analysis of leaf thickness and leaf moisture content showed that these two variables were significantly and directly related (R = 0.894). Leaves from the P-and Ca-deficient plants absorbed less energy in the near infrared than the normal plants; S-, Mg-, K-, and N-deficient leaves absorbed more than the normal.Leaf thermo grams were prepared on normal and S-and Ndeficient leaves. Both S-and N-deficient leaves had higher temperatures than normal maize leaves.

The ultrastructure of mesophyll chloroplasts in full-nutrient and mineral-deficient maize (Zea mays) leaves was examined by electron microscopy after glutaraldehyde-osmium tetroxide fixation. Nitrogen, calcium, magnesium, phosphorus, potassium, and sulfur deficiencies were induced by growing the plants in nutrient culture. Distinctive chloroplast types were observed with each deficiency. Chloroplasts from nitrogendeficient plants were reduced in size and had prominent osmiophilic globules and large grana stacks. Magnesium deficiency was characterized by the accumulation of osmiophilic globules and the progressive disruption of the chloroplast membranes. In calcium deficiency, the chloroplast envelope was often ruptured. Chloroplasts from potassium-or phosphorus-deficient plants possessed an extensive system of stroma lamellae. Sulfur deficiency resulted in a pronounced decrease of stroma lamellae, an increase in grana stacking, and the frequent occurrence of long projections extending from the body of the chloroplast. These morphological changes were correlated with functional alterations in the chloroplasts as measured by photosystem I and II activities. In chloroplasts of the nitrogen-and sulfur-deficient plants an increase in grana stacking was associated with an increase in photosystem II activity.Only a limited number of studies on deficiencies of the major mineral nutrients in higher plants have been concerned with the effects of the deficiency at the ultrastructural level and in particular on the fine structure of the chloroplast (8-10, 14-17). These studies have utilized a variety of plant species, a mixture of macro-and micronutrient deficiencies, and primarily potassium permanganate fixation.The present study is a report of the ultrastructural changes observed in chloroplasts in glutaraldehyde-osmium tetroxide-

Using standard operant conditioning techniques, a killer whale, Orcinus orca Linnaeus, was trained to respond to pure-tone auditory signals by pushing a response manipulandum. An audiogram was obtained for frequencies between 500 Hz and 31 kHz. Greatest sensitivity to the signal was observed at 15 kHz at a level of −70±5 dB re 1 dyn/cm2. The observed upper limit of hearing was 32 kHz. At no time during training or testing did the animal respond to an undistorted signal above 32 kHz. Frequencies below 500 Hz were not tested, owing to high ambient tank noise levels.