Predicted and verified distributions of <Emphasis Type="Italic">Ateles geoffroyi</Emphasis> and <Emphasis Type="Italic">Alouatta palliata</Emphasis> in Oaxaca,Mexico

Primate conservation requires a better knowledge of the distributions and statuses of populations in both large areas of habitat and in areas for which we currently have no information. We focused on spider monkeys (Ateles geoffroyi) and howler monkeys (Alouatta palliata) in the state of Oaxaca, Mexico. This Mexican state has protected large tracts of forest, and has historical records for both primates, although very little is known about them. To update our knowledge of the distributions of these primates and identify potential areas in which they are present, we modeled their geographic distributions by characterizing their ecological niches using the genetic algorithm for rule-set production (GARP), performed interviews and carried out field surveys. The predicted distributions, surveys and interviews indicate that the distributions of these primates are restricted to northeastern Oaxaca. The results suggest that spider monkeys occupy a wider area and elevational range than howler monkeys. Throughout that range there is a wide variety of suitable habitats for these primates. Most of the sites where monkeys were recorded in the field are not officially protected and there was evidence of hunting and habitat destruction. It is important to improve protection, economic alternatives and environmental education as we move towards an integral solution for the conservation of these species. Validation of the GARP model was done for A. geoffroyi, since we had obtained enough field data for this species; this validation indicated that the predicted distribution of the species was statistically better than expected by chance. Hence, ecological niche modeling is a useful approach when performing an initial assessment to identify distribution patterns, detecting suitable areas for future exploration, and for conservation planning. Our findings provide an improved basis for primate conservation and productive fieldwork in Oaxaca.     

Two new species of <Emphasis Type="Italic">Calathea</Emphasis> (<Emphasis Type="Italic">Marantaceae</Emphasis>) from South-eastern Brazil

Summary   Calathea dryadica and Calathea reginae are described, circumscribed and illustrated. These new species are probably endemic to the Atlantic Forest of Rio de Janeiro State in Southeast Brazil and are considered critically endangered because of the restricted geographic area of occurrence, sometimes enclosed by densely urbanised areas.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Development of SCAR markers linked to powdery mildew (<Emphasis Type="Italic">Uncinula necator</Emphasis>) resistance in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. and <Emphasis Type="Italic">Vitis</Emphasis> sp.)

Sequence-characterized amplified regions markers (SCARs) were developed from six randomly amplified polymorphic DNA (RAPD) markers linked to the major QTL region for powdery mildew (Uncinula necator) resistance in a test population derived from the cross of grapevine cultivars “Regent” (resistant) × “Lemberger”(susceptible). RAPD products were cloned and sequenced. Primer pairs with at least 21 nucleotides primer length were designed. All pairs were tested in the F1 progeny of “Regent” × “Lemberger”. The SCAR primers resulted in the amplification of specific bands of expected sizes and were tested in additional genetic resources of resistant and susceptible germplasm. All SCAR primer pairs resulted in the amplification of specific fragments. Two of the SCAR markers named ScORA7-760 and ScORN3-R produced amplification products predominantly in resistant individuals and were found to correlate to disease resistance. ScORA7-760, in particular, is suitable for marker-assisted selection for powdery mildew resistance and to facilitate pyramiding powdery mildew resistance genes from various sources.     

The impact of time and field conditions on brown bear (<Emphasis Type="Italic">Ursus arctos</Emphasis>) faecal DNA amplification

To establish longevity of faecal DNA samples under varying summer field conditions, we collected 53 faeces from captive brown bears (Ursus arctos) on a restricted vegetation diet. Each faeces was divided, and one half was placed on a warm, dry field site while the other half was placed on a cool, wet field site on Moscow Mountain, Idaho, USA. Temperature, relative humidity, and dew point data were collected on each site, and faeces were sampled for DNA extraction at <1, 3, 6, 14, 30, 45, and 60 days. Faecal DNA sample viability was assessed by attempting PCR amplification of a mitochondrial DNA (mtDNA) locus (∼150 bp) and a nuclear DNA (nDNA) microsatellite locus (180–200 bp). Time in the field, temperature, and dew point impacted mtDNA and nDNA amplification success with the greatest drop in success rates occurring between 1 and 3 days. In addition, genotyping errors significantly increased over time at both field sites. Based on these results, we recommend collecting samples at frequent transect intervals and focusing sampling efforts during drier portions of the year when possible.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Kniphofia leucocephala</Emphasis>: influence of cytokinins

The role of cytokinins in the promotion of flowering in the endangered species Kniphofia leucocephala Baijnath. was investigated using shoots maintained in culture for 3 years. The highest percentage flowering (65%) was obtained on media containing 20 μM benzyladenine (BA). The inclusion of isopentenyladenine and zeatin in the media also resulted in flowering, but these treatments were less effective than BA in inducing flowering. The effect of cytokinins on flowering was dose-dependent, with high concentrations of BA inhibiting flower formation. Treatments that resulted in rooting of explants produced no flowers. The resulting inflorescences in all treatments did not mature and senesced prematurely, even when gibberellic acid (GA₃) was applied post-flower-emergence.     

<Emphasis Type="Italic">In vitro</Emphasis> root induction in weedy <Emphasis Type="Italic">amaranthus</Emphasis> species to obtain mitotic chromosomes

Summary Mitotic chromosome analysis has proven to be an important tool in monitoring the potential for genetic exchange among related plant species. One major obstacle to using mitotic chromosome analysis in any species is obtaining large numbers of clear, well-spread metaphase chromosomes necessary to perform cytological techniques such as chromosome banding and fluorescent in situ hybridization. The ability to obtain good chromosome spreads is in part determined by the number and morphology of the roots, which contain the metaphase tissue. Many Amaranthus species produce very thin, delicate roots. The technique used in the process described herein provides for much more substantial roots, allowing for higher probability of obtaining well-spread metaphase chromosomes. Seeds were planted in a soilless mixture, and then cuttings and leaves were taken from the plants. The cuttings were sterilized and placed in Murashige and Skoog (MS) media, while leaf tissue was analyzed by flow cytometry, both pre-and post-propagation, to obtain DNA contents. No changes in DNA content were observed. The in vitro procedure produced significantly larger roots than were produced in soilless mix. Furthermore, all of the in vitro roots observed had 32 chromosomes of normal morphology. In vitro root propagation allowed large numbers of roots to be obtained from a single plant, thereby resulting in increased probability of obtaining cells with metaphase chromosomes that reflected the original plants' chromosome numbers and therefore may be used for molecular cytogenetic analysis.     

Ectomycorrhizal characterization of an American chestnut <Emphasis Type="SmallCaps">(</Emphasis><Emphasis Type="Italic">Castanea dentata</Emphasis><Emphasis Type="SmallCaps">)</Emphasis>-dominated community in Western Wisconsin

Circa 1900, a farmer from the eastern US planted 11 American chestnut (Castanea dentata) seeds on a newly established farm near West Salem in western Wisconsin. These trees were very successful, producing a large stand of over 6,000 trees. Since this area is well outside the natural range of chestnut, these trees remained free from chestnut blight until 1987. In the West Salem stand, chestnuts are the dominant species of a mixed forest community, reminiscent of the chestnut–oak ecosystems of pre-1900 Appalachia. To identify putative mycorrhizal associates of chestnut in this unique forest, our approach was twofold: (1) an extensive fruiting body survey was conducted for four seasons that yielded approximately 100 putative mycorrhizal species and (2) a belowground molecular approach was used to generate DNA sequences of the internal transcribed spacer region from ectomycorrhizae. Unexpectedly, chestnut did not appear to be the dominant underground ectomycorrhizal-forming plant species. This study highlights the need to identify the plant host species when conducting belowground molecular-based surveys and provides preliminary identification of ectomycorrhizal fungi associated with a disjunct stand of American chestnut. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

Genomic DNA extraction from<Emphasis Type="Italic">Victoria amazonica</Emphasis>

DNA extraction is difficult in many plants because of metabolites that interfere with DNA isolation procedures and subsequent applications such as DNA restriction, amplification, and cloning. We developed the first reliable and efficient method for isolatingVictoria amazonica genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 1.5 M NaCl, 2% polyvinylpyrrolidone (PVP) (Mr 1000), 5% mercaptoethanol, 0.12% sodium sulfite, and an incubation at 65°C for 4 h. The purity of isolated genomic DNA was confirmed by means of high-performance liquid chromatography (HPLC) profile and spectrophotometric analyses (A_260/230 ratio of 1.836, A_260/280 of 1.842). DNA was obtained in the amount of 387 μg per gram of leaf material, and it proved amenable to restriction digestion.     

Characterization of <Emphasis Type="Italic">EamaT1</Emphasis>, a member of <Emphasis Type="Italic">maT</Emphasis> family of transposable elements from the earthworm <Emphasis Type="Italic">Eisenia andrei</Emphasis> (Annelida,Oligochaeta)

The maT family is a unique clade within the Tc1-mariner superfamily, and their distribution is to date known as being limited to invertebrates. A novel transposon named EamaT1 is described from the genome of the earthworm Eisenia andrei. The full sized EamaT1 was obtained by degenerate and inverse PCR-based amplification. Sequence analysis of multiple copies of the EamaT1, which consisted of 0.9 and 1.4 kb elements, showed that the consensual EamaT1 with inverted terminal repeats (ITRs) of 69 bp was 1,422 bp long and flanked by a duplicated TA dinucleotide. The EamaT1 is present in approximately 120–250 copies per diploid genome but undergoes an inactivation process as a result of accumulating multiple mutations and is nonfunctional. The open reading frame (ORF) of the EamaT1 consensus encoding 356 amino acid sequences of transposase contained a DD37D signature and a conserved paired-like DNA binding motif for the transposition mechanism. The result of ITRs comparison confirmed their consensus terminal sequences (5′-CAGGGTG-3′) and AT-rich region on the internal bases for ITRs-transposase interaction.     

Assessing the conservation status of the land snail <Emphasis Type="Italic">Oreohelix peripherica wasatchensis</Emphasis> (Family Oreohelicidae)

Establishing conservation priorities for invertebrate groups has proven difficult as many proposed units of diversity are based on morphological features that do not reflect evolutionary history. This confusion is confounded by poorly defined ranges of proposed endemic and endangered groups, leading to problems formulating adequate conservation management strategies. We examined one such group, Oreohelix peripherica wasatchensis, a land-snail located in the Wasatch Front Range of Utah that is a candidate for protection under the Endangered Species Act. We employed a broad sampling approach to determine the current range of O. p. wasatchensis, including the type locality, several localities in the surrounding Wasatch Mountains, and localities throughout Utah, Nevada, and Colorado. From these samples, we sequenced the mitochondrial DNA loci 12S and Cytochrome Oxidase I (COI) and nuclear loci Internal Transcribed Spacers 1 and 2 (ITS1 and ITS2) and 5.8S. We estimated phylogenetic relationships using maximum likelihood and Bayesian analyses. Molecular data and radula morphology data indicate that the group identified as O. p. wasatchensis falls into two distinct clades. We recommend further ecological and population assessments of these two distinct mitochondrial clades based on the newly defined range to evaluate its endangered status under the IUCN criteria.     

DNA elements modulating the <Emphasis Type="Italic">KARS12</Emphasis> chromosomal replicator in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Eukaryotic chromosomal DNA replication is initiated by a highly conserved set of proteins that interact with cis-acting elements on chromosomes called replicators. Despite the conservation of replication initiation proteins, replicator sequences show little similarity from species to species in the small number of organisms that have been examined. Examination of replicators in other species is likely to reveal common features of replicators. We have examined a Kluyeromyces lactis replicator, KARS12, that functions as origin of DNA replication on plasmids and in the chromosome. It contains a 50-bp region with similarity to two other K. lactis replicators, KARS101 and the pKD1 replication origin. Replacement of the 50-bp sequence with an EcoRI site completely abrogated the ability of KARS12 to support plasmid and chromosomal DNA replication origin activity, demonstrating this sequence is a common feature of K. lactis replicators and is essential for function, possibly as the initiator protein binding site. Additional sequences up to 1 kb in length are required for efficient KARS12 function. Within these sequences are a binding site for a global regulator, Abf1p, and a region of bent DNA, both of which contribute to the activity of KARS12. These elements may facilitate protein binding, protein/protein interaction and/or nucleosome positioning as has been proposed for other eukaryotic origins of DNA replication.     

Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Chloroplast DNA phylogeography of <Emphasis Type="Italic">Pedicularis</Emphasis> ser. <Emphasis Type="Italic">Gloriosae</Emphasis> (Orobanchaceae) in Japan

Phylogeographic analyses using chloroplast DNA (cpDNA) variation were performed for Pedicularis ser. Gloriosae (Orobanchaceae). Eighty-one plants of 18 populations of 6 species (P. gloriosa, P. iwatensis, P. nipponica, P. ochiaiana, P. sceptrum-carolinum and P. grandiflora) were analyzed. Fifteen distinct haplotypes were identified based on six cpDNA regions: the intergenic spacer between the trnT and trnL 3′exon, trnL 3′exon-trnF, atpB-rbcL, accD–psaI, the rpl16 intron and the trnK region (including the matK gene). Via phylogenetic analyses of the haplotypes, two continental species, P. sceptrum-carolinum and P. grandiflora, were placed at the most ancestral position in the trees. The former species is widely distributed in the Eurasian continent, and the latter is distributed in Far East Asia. Two robust major cpDNA clades (clades I and II) were revealed in the Japanese archipelago, although the statistical values of monophyly of these clades were weak. Clade I included the haplotypes (A-1, A-2, B-1, B-2 and J) of three species (P. gloriosa, P. iwatensis and P. ochiaiana), and Clade II included seven haplotypes (C-D, E-1, E-2 and F-H) of P. nipponica. These results suggest that this series originated on the Eurasian continent and that subsequently populations at the eastern edge of the continent differentiated into the two Japanese lineages. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.