Floral scent emission and pollinator attraction in two species of <Emphasis Type="Italic">Gymnadenia</Emphasis> (Orchidaceae)

We investigated scent composition and pollinator attraction in two closely related orchids, Gymnadenia conopsea (L.) R.Br. s.l. and Gymnadenia odoratissima (L.) Rich. in four populations during the day and night. We collected pollinators of both species using hand nets and sampled floral odour by headspace sorption. We analysed the samples by gas chromatography with mass spectrometry to identify compounds and with electroantennographic detection to identify compounds with physiological activity in pollinators. In order to evaluate the attractiveness of the physiologically active compounds, we carried out trapping experiments in the field with single active odour substances and mixtures thereof. By collecting insects from flowers, we caught eight pollinators of G. conopsea, which were members of four Lepidoptera families, and 37 pollinators of G. odoratissima, from five Lepidopteran families. There was no overlap in pollinator species caught from the two orchids using nets. In the scent analyses, we identified 45 volatiles in G. conopsea of which three (benzyl acetate, eugenol, benzyl benzoate) were physiologically active. In G. odoratissima, 44 volatiles were identified, of which seven were physiologically active (benzaldehyde, phenylacetaldehyde, benzyl acetate, 1-phenyl-2,3-butandione, phenylethyl acetate, eugenol, and one unknown compound). In field bioassays using a mixture of the active G. odoratissima compounds and phenylacetaldehyde alone we caught a total of 25 moths, some of which carried Gymnadenia pollinia. A blend of the active G. conopsea volatiles placed in the G. odoratissima population did not attract any pollinators. The two orchids emitted different odour bouquets during the day and night, but G. odoratissima showed greater temporal differences in odour composition, with phenylacetaldehyde showing a significant increase during the night. The species differed considerably in floral odour emission and this differentiation was stronger in the active than non-active compounds. This differentiation of the two species, especially in the emission of active compounds, appears to have evolved under selection for attraction of different suites of Lepidopteran pollinators.     

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.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Three new species of <Emphasis Type="Italic">Stigmidium</Emphasis> and <Emphasis Type="Italic">Sphaerellothecium</Emphasis> (lichenicolous ascomycetes) on <Emphasis Type="Italic">Stereocaulon</Emphasis>

Sphaerellothecium stereocaulorum sp. nov., Stigmidium beringicum sp. nov., Stigmidium stereocaulorum sp. nov. and goniocysts are described on Stereocaulon species from the northern Holarctic. Endococcus nanellus is reported new to Alaska and Mongolia. Taxonomical novelties Sphaerellothecium stereocaulorum Zhurb. & Triebel, Stigmidium beringicum Zhurb. & Triebel, Stigmidium stereocaulorum Zhurb. & Triebel.     

New species of <Emphasis Type="Italic">Chalybea</Emphasis> and <Emphasis Type="Italic">Huilaea</Emphasis> (Melastomataceae)

One new species each is proposed in Chalybea and Huilaea (Melastomataceae: Blakeeae). Chalybea peruviana has elliptic, 5-plinerved leaves with entire, revolute margins, inflorescences with 33–39 flowers, and is endemic to Peru. Huilaea calyptrata has inflorescences with 15–17, irregularly calyptrate flowers, anthers with a warty connective in the shape of an inverted hand fan, and is endemic to Ecuador. A key to the eight species of Huilaea is provided.     

New species in <Emphasis Type="Italic">Barleria</Emphasis> sect. <Emphasis Type="Italic">Stellatohirta</Emphasis> (<Emphasis Type="Italic">Acanthaceae</Emphasis>) from Africa

Summary  Three new species are described in Barleria L. sect. Stellatohirta M. Balkwill from tropical Africa: B. aristata from south-central Tanzania, B. aenea from south-western Tanzania and northeast Zambia, and B. purpureotincta from south-western Zambia. Their affinities and conservation status are discussed.     

Indonesian Kickxellales: two species of <Emphasis Type="Italic">Coemansia</Emphasis> and <Emphasis Type="Italic">Linderina</Emphasis>

Four species belonging to Kickxellales (Kickxellomycotina) isolated from soil of Indonesia are described and illustrated. Two new species of Coemansia, C. asiatica and C. javaensis, were discovered in South Sulawesi and West Java, and two known species of Linderina, L. pennispora and L. macrospora, were discovered in East Kalimantan and South Sulawesi, respectively. These four species are newly added to the Indonesian mycobiota. A technique for inducing sporulation of C. javaensis and L. macrospora by adding substances derived from invertebrates such as aphids, nereids, or cladocerans to culture media is described.     

Floral ontogeny of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Schisandraceae): implications for androecial evolution within <Emphasis Type="Italic">Schisandra</Emphasis> and <Emphasis Type="Italic">Kadsura</Emphasis>

The organogenesis of staminate and carpellate flowers of Schisandra chinensis (Schisandraceae) was investigated with scanning electron microscopy, with observations on the development of tepals reported for the first time. The results showed that there is no interval between the initiation of the last tepal and that of the first stamen or carpel, and that the shapes of tepal, stamen, and carpel primordia are similar. The tepals and stamens of staminate flowers are initiated acropetally in a continuous spiral Fibonacci phyllotaxis, with no carpel structures observed; the filaments are not connate. The organogenesis of the carpellate flowers is similar to that of the staminate flowers, but with no evidence of stamen development. The carpels are ascidiate without postgenital fusion. Three androecial characters of Schisandra and Kadsura are discussed in a phylogenetic context. The subglobose or obovoid androecium of Schisandra propinqua and Schisandra plena may be homologous with that in sections Kadsura and Sarcocarpon. The plesiomorphic form of the androecium within the two genera is likely to be elongate with more than ten free stamens.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

Pedogamy in <Emphasis Type="Italic">Neidium</Emphasis> (<Emphasis Type="Italic">Bacillariophyceae</Emphasis>)

Single (unpaired) vegetative cells of freshwater pennate diatom Neidium cf. ampliatum differentiated into gametangia and produced a single zygote (auxospore) via a pedogamic process. The gametic nuclei fused after auxospore expansion had begun. The auxospore expanded in parallel to the apical axis of the gametangium.     

<Emphasis Type="Italic">Funastrum rupicola</Emphasis> (<Emphasis Type="Italic">Apocynaceae:</Emphasis><Emphasis Type="Italic">Asclepiadoideae</Emphasis>), a new species from Bolivia

Summary   Funastrum rupicola Goyder, a new species of Apocynaceae: Asclepiadoideae from Bolivia, is described and illustrated. The conservation status of this species is assessed.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Cryptodiscus muriformis</Emphasis> and <Emphasis Type="Italic">Schizoxylon gilenstamii</Emphasis>, two new species of Stictidaceae (Ascomycota)

Cryptodicus muriformis and Schizoxylon gilenstamii (Stictidaceae, Ostropales, Lecanoromycetes, Ascomycota) are described as new to science from collections made in Europe (Sweden, Austria, Switzerland) and North America (Arizona), based on phylogenetic analyses of three loci. Both species show unique morphological characters that distinguish them from closely related species; Cryptodiscus muriformis, growing on decorticated or sometimes corticated branches of Picea abies and Pseudotsuga menziesii, that resembles C. foveolaris and C. tabularum in the yellowish-orange ascomatal disc, but is clearly distinguished by the muriform ascospores. Schizoxylon gilenstamii grows on dead stems of Globularia vulgaris and has a characteristic unique red-purple pigment in the epithecium and upper part of the excipulum that changes to bluish-green with KOH. We also report for the first time the anamorph of Stictis brunnescens.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

<Emphasis Type="Italic">Pluteus magnus</Emphasis> and <Emphasis Type="Italic">Pluteus podospileus</Emphasis> f. <Emphasis Type="Italic">podospileus</Emphasis>, two agaric species new to Japan

Two taxa of the genus Pluteus, i.e., Pluteus magnus and Pluteus podospileus f. podospileus, are newly recorded from Japan. The macroscopic and microscopic features of these two species are described and illustrated.