DNA amplification fingerprinting of the Azolla-Anabaena symbiosis

The Azolla-Anabaena symbiosis has been used for centuries as a nitrogen biofertilizer in rice paddies. Genetic improvement of the symbiosis has been limited by the difficulty in identifying Azolla-Anabaena accessions and Anabaena azollae strains. The recently developed technique of DNA amplification fingerprinting (DAF) was applied to this problem. DAF uses single, short, oligonucleotide primers of arbitrary sequence to direct amplification of a characteristic set of DNA products by a thermostable DNA polymerase in a thermocycling reaction. The products are separated in polyacrylamide gels and detected by silver staining. DAF could easily distinguish and positively identify accessions of Azolla-Anabaena with DNA extracted from the intact symbioses. The contribution of prokaryotic Anabaena sequences to the fingerprint of the intact symbioses, however, ranged from 0 to 77%, depending on the primer sequence. Therefore, DNA extracted from the intact symbioses would not be suitable for Azolla taxonomy studies. The fingerprints of Anabaena strains isolated by sucrose gradient centrifugation from different species of Azolla could be easily distinguished, and DAF patterns were used to confirm the maternal pattern of transmission of Anabaena in a sexual hybrid. Template DNA extracted from roots was used to produce fingerprints for Azolla without interference from the microsymbiont. Comparison of the patterns from the parents and a hybrid gave strong evidence confirming sexual hybridization.     

PHYLOGENETIC PATTERNS AMONG NOSTOC CYANOBIONTS WITHIN BI‐ AND TRIPARTITE LICHENS OF THE GENUS PANNARIA1

Phylogenetic relationships between Nostoc cyanobionts in the lichen genus Pannaria were studied to evaluate their correlation to geography, habitat ecology, and other patterns previously reported. The 16S rRNA gene sequences of a total of 37 samples of 21 Pannaria species from seven countries from the Northern and Southern hemispheres were analyzed and compared with 69 free‐living and symbiotic cyanobacterial strains. The sequences from Pannaria were distributed throughout a branch of Nostoc sequences previously called “the Nephroma guild,” and within two subgroups from another branch, referred to as the “Peltigera guild,” although there was a gradual transition between the two major groups. There is a more diverse pattern of relationships between Nostoc sequences from bipartite versus tripartite lichen species in Pannaria, compared with other well‐studied genera, such as Nephroma and Peltigera. Cyanobionts from several tripartite Pannaria species from the Southern Hemisphere and corticolous bipartite species from both hemispheres were grouped together. Four sequences of Pannaria and Pseudocyphellaria cyanobionts from rocks in the Chilean Juan Fernández Islands were nested within corticolous cyanobionts, whereas the terricolous “Pannaria sphinctrina clade” was placed with other terricolous strains. The cluster patterns derived from phylogenetic analysis were partly reflecting lichen taxonomy, in two groups of lichen species, possibly indicating coevolution. The phylogram partly also reflected lichen ecology. Three Pannaria species have very different cyanobiont strains when they grow in different habitats.     

Schizymenia jonssonii sp. nov. (Nemastomatales,Rhodophyta): a relict or an introduction into the North Atlantic after the last glacial maximum?

North-Atlantic records of Schizymenia dubyi extend along the eastern shores of the North Atlantic from Morocco to southern Britain and Ireland, and the species is also recorded from Iceland. A study was undertaken to confirm the identity of the specimens from Iceland that were geographically separate from the main distribution of S. dubyi and in contrast to other species of the genus did not have gland cells. We analyzed rbcL and COI molecular sequence data from Icelandic specimens and compared the results with those for Schizymenia specimens available in GenBank. For both markers, Schizymenia was shown to be a monophyletic genus. The Icelandic specimens were clearly genetically distinct from S. dubyi and formed a well-supported clade with Schizymenia species from the Northern Pacific. Based on these results, we have described a new species, Schizymenia jonssonii, which can be distinguished by molecular phylogeny, its lack of gland cells and by being strictly intertidal. Crustose tetrasporophytes with identical COI and rbcL sequences were found at the same locations as foliose plants. Schizymenia apoda is reported for the first time in the UK, its identity confirmed by rbcL sequence data. In light of these findings, it is likely that by further molecular analysis of the genus Schizymenia in the north-eastern Atlantic and the Mediterranean, a higher diversity of Schizymenia spp. will be discovered in this region.     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.     

The white-spotted longicorn beetle, <Emphasis Type="Italic">Anoplophora malasiaca</Emphasis> (Coleoptera: Cerambycidae), with a blueberry as host plant,utilizes host chemicals for male orientation

Volatile chemicals from Citrus and Salix host plants evoke orientation behavior in males of the species Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae). These chemicals are emitted from wounded branches. We hypothesized that when released, these chemicals may indicate the presence of an individual to other conspecifics. Insects that originate from different host plants may use different plant chemicals from their own host to communicate with conspecifics. To further explore this theory, we investigated this communication system in a population of A. malasiaca from a third host plant, blueberry (Vaccinium spp.). Males from a blueberry host (Vaccinium population) were attracted to the odor of wounded Vaccinium branches when released near a female model in the laboratory, as has been observed in males found on Citrus and Salix host plants. The Vaccinium branch extract that was attractive to the males was separated into six fractions, of which two were active. Three active compounds were subsequently identified: β-caryophyllene and sulfur from the hexane fraction, and (E)-phytol in the weakly polar fraction. The latter two active compounds of Vaccinium branches were different from those found in Citrus and Salix.     

POLYSACCHARIDES FROM THE ENVELOPES OF HETEROCYSTS AND SPORES OF THE BLUE-GREEN ALGAE ANABAENA VARIABILIS AND CYLINDROSPERMUM LICHENIFORME1

The polysaccharides from the envelopes of heterocysts of Cylindrospermum licheniforme Kütz., and of heterocysts and spores of Anabaena variabilis Kütz., like those from the differentiated cells of Anabaena cylindrica Lemm., have a 1,3-linked backbone consisting of glucosyl and mannosyl residues in a molar ratio of approximately 3:1. As is the case with A. cylindrica the polysaccharides from A. variabilis and from the heterocysts of C. licheniforme have terminal xylosyl and galactosyl residues as side branches. In addition, the polysaccharide from C. licheniforme resembles that from A. cylindrica in having terminal mannosyl residues as side branches (absent from A. variabilis). The polysaccharides from A. variabilis resemble that from A. cylindrica in having glucose-containing side branches (absent from the heterocyst polysaccharide from C. licheniforme), but in contrast to the polysaccharides from the other two species they also have terminal arabinosyl residues as side branches. All of the polysaccharides mentioned appear to be structurally related; we present tentative structures for those not previously investigated. In contrast, the envelope of spores of C. licheniforme contains only a largely 4-linked galactan. The bulk of this envelope is not polysaccharide in nature, and contains aromatic groups.     

Regulatory potential of nonautonomous mariner elements and subfamily crosstalk

Two naturally occurring nonautonomous mariner elements were tested in vivo for their ability to down-regulate excision of a target element in the presence of functional mariner transposase. The tested elements were the peach element isolated from Drosophila mauritiana which encodes a transposase that differs from the autonomous element Mos1 in four amino acid replacements, and the DTBZ1 element isolated from D. teissieri which encodes a truncated protein consisting of the first 132 residues at the amino end of the normally 345-residue transposase. We provide evidence that the protein from the peach element does interact to down-regulate wildtype transposase, indicating that at least some nonautonomous elements in natural populations that retain their open reading frame may play a regulatory role. In contrast, our tests reveal at most a weak interaction between transposase from the autonomous Mos1 element and the truncated protein from DTBZ1 and none between Mos1 transposase and that from the distantly related mariner-like element Himar1 identified in the horn fly Haematobia irritans. Hence, the extent of regulatory crosstalk between mariner-like elements may be limited to closely related ones. The evolutionary implications of these results are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus OF4 encoding an ABC-type transporter with similarity of sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter

A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the alkaliphile natCAB catalyzes Na⁺ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na⁺ extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na⁺/H⁺ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs. Received: August 30, 1998 / Accepted: November 13, 1998     

The speciation of coliform genera from above and below a sewer outfall and their susceptibilities to antimicrobial agents

The occurrence of coliforms in a small water course was shown to increase by a factor of thirty six below the outfall of a sewage treatment plant. Speciation of the bacteria from above and below the sewer outfall showed thatEscherichia coli andEnterobacter species predominated. Drug resistance levels were significant in microorganisms from both sampling sites and the occurrence of a significant number of multiple-resistant microorganisms, particularlyE. coli, is reported. BothE. coli andEnterobacter species from below the sewer outfall show a statistically significant increase in resistance to ampicillin andE. coli from below the outfall also shows a statistically significant increase in resistance to sulphamethoxazole as compared with isolates from above the outfall.     

Diadromus pulchellus in North America: field release against leek moth and new characters to distinguish it from Diadromus subtilicornis,a native diamondback moth parasitoid

We report successful overwintering of Diadromus pulchellus in North America (Ontario) following introduction of this species from Europe to control the leek moth, Acrolepiopsis assectella, a recently established alien species. Field rearing revealed that the native Diadromus subtilicornis emerged only from diamondback moth, Plutella xylostella, whereas D. pulchellus was reared almost exclusively from leek moth. The single D. pulchellus reared from diamondback moth was anticipated because host range studies found this species could develop on both leek moth and diamondback moth in the laboratory, although, it had not been previously reported from diamondback moth in the field in Europe. DNA barcoding of specimens of both Diadromus spp. confirmed their species status and novel morphological characters are presented to distinguish D. pulchellus from D. subtilicornis. In addition, DNA from specimens of D. subtilicornis from Europe clustered with DNA from specimens across Canada, confirming that it is a single Holarctic species. Finally, a new host association for D. subtilicornis is recorded from the dame's rocket moth: Pseudoplutella porrectella.     

Purification and Characterization of an Endo-Polygalacturonase from a Mutant of Saccharomyces cerevisiae

An extracellular endo-polygalacturonase (PGase) produced by a mutant of Saccharomyces cerevisiae was isolated. The enzyme was regarded, immunologically, as a PGase belonging to the Kluyveromyces marxianus group. The enzyme had properties similar to the PGase from K. marxianus in heat and pH stability, and N-terminal amino acid sequence. However, the enzyme showed different properties in optimum pH and temperature, molecular weight, and reactivity in antiserum against PGase from K. marxianus, indicating that the enzyme has a different molecular structure from the PGase from K. marxianus.     

Genetic differentiation of continental and island populations of Bombus terrestris (Hymenoptera: Apidae) in Europe

Ten microsatellite loci and a partial sequence of the COII mitochondrial gene were used to investigate genetic differentiation in B. terrestris, a bumble bee of interest for its high-value crop pollination. The analysis included eight populations from the European continent, five from Mediterranean islands (six subspecies altogether) and one from Tenerife (initially described as a colour form of B. terrestris but recently considered as a separate species, B. canariensis). Eight of the 10 microsatellite loci displayed high levels of polymorphism in most populations. In B. terrestris populations, the total number of alleles detected per polymorphic locus ranged from 3 to 16, with observed allelic diversity from 3.8 ± 0.5 to 6.5 ± 1.4 and average calculated heterozygosities from 0.41 ± 0.09 to 0.65 ± 0.07. B. canariensis showed a significantly lower average calculated heterozygosity (0.12 ± 0.08) and observed allelic diversity (1.5 ± 0.04) as compared to both continental and island populations of B. terrestris. No significant differentiation was found among populations of B. terrestris from the European continent. In contrast, island populations were all significantly and most of them strongly differentiated from continental populations. B. terrestris mitochondrial DNA is characterized by a low nucleotide diversity: 0.18%± 0.07%, 0.20%± 0.04% and 0.27%± 0.04% for the continental populations, the island populations and all populations together, respectively. The only haplotype found in the Tenerife population differs by a single nucleotide substitution from the most common continental haplotype of B. terrestris. This situation, identical to that of Tyrrhenian islands populations and quite different from that of B. lucorum (15 substitutions between terrestris and lucorum mtDNA) casts doubts on the species status of B. canariensis. The large genetic distance between the Tenerife and B. terrestris populations estimated from microsatellite data result, most probably, from a severe bottleneck in the Canary island population. Microsatellite and mitochondrial DNA data call for the protection of the island populations of B. terrestris against importation of bumble bees of foreign origin which are used as crop pollinators.     

Monophagy in a polyphagous grasshopper,Schistocerca shoshone

The feeding behavior of different populations of the grasshopper,Schistocerca shoshone, was investigated in the southwestern United States. Insects from three riparian populations, with a broad spectrum of plants available to them, tended to eat plants roughly in relation to their availability except that broad-leaved herbaceous plants were avoided. Insects from a desert population in a plantation ofSimmondsia fed exclusively on that plant, as did those from another population in the Tucson mountains, despite the availability of a range of other plants. Insects from a third desert population, near Portal, fed mainly onProsopis, the dominant woody plant. In detailed behavioral experiments in the laboratory, insects from Tucson mountains readily acceptedSimmondsia, and less readily acceptedProsopis. Three other common woody plants from the habitat were generally rejected without feeding. Insects from Portal acceptedProsopis andSimmondsia with approximately equal readiness. Breeding experiments suggested that the differences between the plantation insects and those from Portal was genetic and not induced by experience. The insects from both populations were potentially polyphagous and ate a wide range of plants in the laboratory if given no alternative.     

Discriminating plants using the DNA barcode rbcLb: an appraisal based on a large data set

The ideal DNA barcode for plants remains to be discovered, and the candidate barcode rbcL has been met with considerable skepticism since its proposal. In fact, the variability within this gene has never been fully explored across all plant groups from algae to flowering plants, and its performance as a barcode has not been adequately tested. By analysing all of the rbcL sequences currently available in GenBank, we attempted to determine how well a region of rbcL performs as a barcode in species discrimination. We found that the rbcLb region was more variable than the frequently used rbcLa region. Both universal and plant group‐specific primers were designed to amplify rbcLb, and the performance of rbcLa and rbcLb was tested in several ways. Using blast , both regions successfully identified all families and nearly all genera; however, the successful species identification rates varied significantly among plant groups, ranging from 24.58% to 85.50% for rbcLa and from 36.67% to 90.89% for rbcLb. Successful species discrimination ranged from 5.19% to 96.33% for rbcLa and from 22.09% to 98.43% for rbcLb in species‐rich families, and from 0 to 88.73% for rbcLa and from 2.04% to 100% for rbcLb in species‐rich genera. Both regions performed better for lower plants than for higher plants, although rbcLb performed significantly better than rbcLa overall, particularly for angiosperms. Considering the applicability across plants, easy and unambiguous alignment, high primer universality, high sequence quality and high species discrimination power for lower plants, we suggest rbcLb as a universal plant barcode.     

STUDIES IN SOME FOLIOSE RED ALGAE OF THE PACIFIC COAST. III. DUMONTIACEAE,WEEKSIACEAE, KALLYMENIACEAE1

The female reproductive structures and their development, and the vegetative structure are studied in 17 species of red algae in the Cryptonemiales (Rhodophyceae). Three genera, Weeksia, Constantinea, and the type species of Leptocladia, are removed from the Dumontiaceae to a newly created family, the Weeksiaceae, because of differing postfertilization events leading to the development of the gonimoblast from a cell of the carpogonial branch. Three genera of Dumontiaceae are studied: Pikea, including P. californica, the type species, and Pikea robusta a newly described species; Dilsea californica, and a newly described species of Neodilsea, a genus heretofore known only from the northwestern Pacific. Two transfers are made from the genus Leptocladia, 1 to Farlowia, as F. conferta, and 1 to Rhodophyllis (Gigartinales) as R. peruviana. Three species in the Kallymeniaceae are redescribed: Kallymenia pacifica, a rare and nearly unknown species from southern California and adjacent Pacific Mexico; K. norrisii from central California; and K. oblongifructa from Washington, Oregon, and northern California.     

Molecular data reveal cryptic lineages within the northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina edwardsii complex (Mollusca: Gastropoda: Muricidae)

We used a molecular phylogenetic approach to investigate species delimitations and diversification in the mussel drills of the Ocinebrina edwardsii complex by means of a combination of nuclear (internal transcribed spacer 2, ITS2) and mitochondrial [cytochrome oxidase subunit I (COI) and 16S] sequences. Our sample included 243 specimens ascribed to seven currently accepted species from 51 sites. Five of the samples were from either the type locality of a nominal species or a close nearby locality (O. edwardsii from Corsica, O. carmelae and O. piantonii from the Kerkennah Islands, O. hispidula from the Gulf of Gabès and O. leukos from the Canary Islands), one from the inferred original locality (O. ingloria from Venice Lagoon), and specimens assigned in the recent literature to O. nicolai. We used a combination of distance‐ and tree‐based species delimitation methods to identify Molecular Operational Taxonomic Units (MOTUs) to compare with the a priori species identifications. The consensus tree obtained by BEAST on the COI alignment allows the recognition of several distinct clades supported by the three species delimitation methods employed. The eight‐MOTUs scenario, shared by the Automatic Barcode Gap Discovery (ABGD) and Generalized Mixed Yule‐Coalescent (GMYC) methods, comprises the following major clades: clade A contains the south Tunisian species Ocinebrina piantonii Cecalupo, Buzzurro & Mariani from which the sympatric taxon O. carmelae Cecalupo, Buzzurro & Mariani (new synonym) cannot be separated; clades B and C bring together all populations from the Aegean Sea and some from the Ionian Sea, respectively; clade D groups, on the one hand, the south Tunisian samples morphologically assigned to O. hispidula Pallary and, on the other, Atlantic and Alboran Sea samples (including the Canarian taxon O. leukos Houart); clade E includes a sample from the type locality of O. edwardsii and several samples from the Tyrrhenian Sea; clades F and G correspond to a few samples from the Venice Lagoon and the Tyrrhenian Sea, respectively; clade H groups the bulk of samples from the Adriatic Sea, including samples from the Venice Lagoon morphologically identified as Ocinebrina ingloria (Crosse), and some from the Ionian Sea. No final conclusions could be reached to reconcile the currently recognized morphological taxa with the clades suggested by the COI data. The geographical structure proposed by the mitochondrial markers is similar to that found in other marine invertebrates and partially corresponds to the species defined by shell characters. We propose here a framework for the revision of the Ocinebrina edwardsii species complex, suggesting a geographical pattern for the diversification of this group in the studied area. © 2013 The Linnean Society of London     

Isolation of polymorphic microsatellite loci in Hemerocallis fulva and Hemerocallis citrina (Hemerocallidaceae)

Twenty microsatellite loci were isolated from a hybrid of two daylilies, Hemerocallis fulva and Hemerocallis citrina. We characterized individuals from two H. fulva populations and two H. citrina populations in Japan and observed three to 20 alleles per locus in H. fulva and one to 19 alleles per locus in H. citrina. Mean observed heterozygosity within populations ranged from 0.35 to 0.85 in H. fulva and from 0 to 0.95 in H. citrina. In about a half of the loci, the observed heterozygosity did not deviate from Hardy–Weinberg equilibrium. These loci are proved useful in studying gene flow and qualitative trait loci mapping using the two species.     

Quinolone-resistant mutations of the gyrA gene of Escherichia coli

Summary DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones. The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E. coli chromosome. It encoded a polypeptide of 875 amino acids with a molecular weight of about 97000. The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively. These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA.     

A new homeobox-leucine zipper gene from Arabidopsis thaliana

We have isolated a homeobox-containing gene from Arabidopsis thaliana using a degenerate oligonucleotide probe corresponding to the most conserved region of the homeodomain. This strategy has been used previously to isolate homeobox-containing genes from Caenorhabditis, and recently from A. thaliana. The Arabidopsis genes have an unusual structure in that they have a leucine zipper motif adjacent to the carboxy terminal region of the homeo domain, a feature not found in homeobox-containing genes isolated from animals. We report the isolation and primary structure of a new member of this Arabidopsis homeobox-leucine zipper gene family. This new member has the homeodomain and leucine-zipper motif similar to the two genes previously identified, but differs from these genes in the part corresponding to the carboxy terminus of the polypeptide, as well as in size and isoelectric point of the protein.     

Prasiola (Prasiolales,Trebouxiophyceae) in Japan: a survey of freshwater populations and new records of marine taxa

Recent collections from marine and freshwater locations have enabled the investigation of diversity of Prasiola in Japan. Sequence data from the rbc L and tuf A markers revealed the presence of three marine species and one freshwater species. Prasiola delicata was confirmed to occur on Daikokujima, Prasiola calophylla was found for the first time in Japan from Hokkaido, and a species within the P. meridionalis/linearis/stipitata complex was found on both Hokkaido and Daikokujima. Collections from a range of populations of freshwater Prasiola, identified here as P. japonica, were found to be conspecific and identical in rbc L and tuf A sequences to freshwater collections from Nepal, Korea, and China.