Protoplast isolation and regeneration from <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariales,Rhodophyta)

The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL^-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.     

Mitochondrial <Emphasis Type="Italic">cox</Emphasis>1 and plastid <Emphasis Type="Italic">rbc</Emphasis>L genes of <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis> (Gracilariaceae,Rhodophyta)

The mitochondrial cytochrome c oxidase subunit I gene sequence was recently developed for DNA barcoding of red algal species. We determined the 1245 base pairs of the gene from 27 taxa of an agar-producing species, Gracilaria vermiculophylla, and putative relatives and compared the results with rbcL data from the same species. A total of 392 positions (31.5%) were variable, 282 positions (22.6%) were parsimoniously informative, and average sequence divergence was 13% in an ingroup. Within G. vermiculophylla, pairwise divergence of the gene was variable up to 11 bp (0.9%). Seven recognized haplotypes of cox1 tended to be geographically related. In the aligned 1386 bp of rbcL, three haplotypes were recognized. These results suggest that cox1 is a valuable molecular marker within species and will be very useful in haplotype analyses.     

Photoregulation of morphological structure and its physiological relevance in the cyanobacterium <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

The spiral structure of the cyanobacterium Arthrospira (Spirulina) platensis (Nordst.) Gomont was previously found to be altered by solar ultraviolet radiation (UVR, 280–400 nm). However, how photosynthetic active radiation (PAR, 400–700 nm) and UVR interact in regulating this morphological change remains unknown. Here, we show that the spiral structure of A. platensis (D-0083) was compressed under PAR alone at 30°C, but that at 20°C, the spirals compressed only when exposed to PAR with added UVR, and that UVR alone (the PAR was filtered out) did not tighten the spiral structure, although its presence accelerated morphological regulation by PAR. Their helix pitch decreased linearly as the cells received increased PAR doses, and was reversible when they were transferred back to low PAR levels. SDS-PAGE analysis showed that a 52.0 kDa periplasmic protein was more abundant in tighter filaments, which may have been responsible for the spiral compression. This spiral change together with the increased abundance of the protein made the cells more resistant to high PAR as well as UVR, resulting in a higher photochemical yield.     

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.     

Interactions of Algae within the Community of <Emphasis Type="Italic">Gracilaria gracilis</Emphasis> (Rhodophyta)

Interactions among the unattached red alga Gracilaria gracilis, the dominant species of an algal community, and associated algal species Chaetomorpha linum, Enteromorpha prolifera f. prolifera, and Polysiphonia sp. were studied during and after an algal bloom. It was shown that during their bloom the associated algae Enteromorpha and Polysiphonia sp. significantly decreased the photosynthetic rate of G. gracilis but did not affect its growth rate. It is suggested that the inhibition of Gracilaria gracilis photosynthesis is connected to the impact of extracellular metabolites excreted by Chaetomorpha linum, Enteromorpha prolifera f. prolifera, and Polysiphonia sp. In laboratory experiments, the photosynthetic rate of the associated species was enhanced in the presence of Gracilaria. However, no significant alterations were observed in the content of chlorophyll a, growth, and the dark respiration rates of associated algae when they were kept together with Gracilaria. It was suggested that allelopathic interactions observed among algal species during the formation of the monospecific Gracilaria community, as well as during algal blooms, are not determinative.     

<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.     

<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.     

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.     

Structure of reproductive apparatus of <Emphasis Type="Italic">Gracilaria/Gracilariopsis lemaneiformis</Emphasis> (Gracilariaceae,Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial development revealed the similar cytodynamic pattern followed by all the Gracilariaceae members developed from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released.     

Hermes copper (<Emphasis Type="Italic">Lycaena</Emphasis> [<Emphasis Type="Italic">Hermelycaena</Emphasis>] <Emphasis Type="Italic">hermes</Emphasis>: Lycaenidae): life history and population estimation of a rare butterfly

Hermes copper (Lycaena [Hermelycaena] hermes: Lycaenidae) is a rare species endemic to the coastal sage scrub in and around San Diego, CA, USA. This species has experienced substantial habitat loss due to urbanization and recent wildfires. We present data collected from field surveys conducted in 2003 and 2004. The flight season lasted 1–2 months with densities varying among sites and years. We observed adults most often near California buckwheat (Eriogonum fasciculatum) plants and significantly more often on north and west sides of trails or roads. We compared the robustness and statistical power of three indices of population size from the modified Pollard Walk surveys. We recorded the largest single-day count (Max Count), the cumulative number observed throughout the flight season (Pollard) and an estimate based on a four-parameter model (INCA: Insect Count Analyzer). The Pollard estimate was the most robust to sampling error and the most powerful at detecting population changes in simulated data. Improved monitoring techniques, both field methods and statistical estimation, are critical to determine the conservation status of rare butterflies like Hermes copper.     

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.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Application of <Emphasis Type="Italic">Gracilaria lichenoides</Emphasis> (Rhodophyta) for alleviating excess nutrients in aquaculture

A study was conducted in Xiangshan Bay, Ningbo, China, using red alga Gracilaria lichenoides to alleviate nutrient pollution in shrimp (Litopenaeus vannamei) and fish (Epinephelus awoara) culture ponds. Our results showed that G. lichenoides was efficient at absorbing inorganic nitrogen (IN) and inorganic phosphate (IP), and maintained a more stable dissolved oxygen (DO) level. A total of 506.5 kg (1,013 kg ha⁻¹) of shrimp and 210.5 kg (421 kg ha⁻¹) of fish were harvested from the shrimp/algae (SA) and fish/algae (FA) ponds, respectively. Only 53.5 kg shrimp were harvested from the shrimp pond without Gracilaria (S) due to anoxic asphyxia, and 163 kg fish were harvested from the fish culture pond without Gracilaria (F). Compared with using microalgae, bioremediation by macroalgae has no risk of harmful algal blooms (HABs), and it is easy to control seaweed biomass. During the experiment, there was a better environmental condition (lower chemical oxygen demand, IN, IP and chlorophyll a concentrations) in the ponds with Gracilaria. Furthermore, Gracilaria spp. can be used as food for abalone or other aquacultured animals and thus enhance economic return.     

Sociality in <Emphasis Type="Italic">Euglossa</Emphasis> (<Emphasis Type="Italic">Euglossa</Emphasis>) <Emphasis Type="Italic">viridissima</Emphasis> Friese (Hymenoptera,Apidae, Euglossini)

Euglossa viridissima is an orchid bee that forms both solitary and multiple female nests, making it a suitable species for the study of factors leading to diverse degrees of sociality in Euglossines. We conducted observations in eight reused nests (where a first generation of bees had been produced) kept in artificial boxes from the Yucatan Peninsula, Mexico. Five nests were reused (reactivated) by a single female (SFN), two nests reused by a mother and one daughter (MFN1) and one nest reused by the mother and two daughters (MFN2). No single nest was reactivated by unrelated females. The number of foraging trips, their duration and the duration of cell provisioning was not different between SFN and MFN. The overall production of cells per female was not different either between both types of nest. However, in MFN although all females did lay eggs, there was a reproductive skew in favor of the mother (95 and 45% of the brood produced in MFN1 and MFN2 respectively). She showed reproductive control of her daughters through oophagy and displaying threatening behavior when the daughters tried to open a cell where she had laid an egg. Brood losses to parasites (Anthrax sp. (Bom-byliidae) and Hoplostelis bivittata (Megachilidae)) were only found in SFN which possibly reflects and advantage of MFN in this respect. Our results coupled with other studies in Euglossa, reveal that a wide range of social behaviors occur in this genus, from solitary and communal to primitive reproductive division of labor. Multiple factors involving different levels of pressure imposed by food availability and parasites may favor such a diverse range of nesting behaviors. Interestingly, female associations in E. viridissima seem a result of kin selection that is enforced by coercion from mother females on their daughters. More studies are needed to shed light upon the social organization of Euglossa and other Euglossines and on their phylogenetic relationships in order to trace the origins of eusociality in Apidae. Received 12 February 2008; revised 25 June 2008; accepted 17 July 2008.     

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">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">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.     

Genetic diversity of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariaceae,Rhodophyta) from west coast,Peninsular Malaysia based on mitochondrial <Emphasis Type="Italic">cox</Emphasis>1 gene analysis

Mitochondrial cytochrome c oxidase subunit I (cox1) was employed to investigate the intraspecific genetic diversity of Gracilaria changii collected from various localities distributed along the west coast of Peninsular Malaysia. Gracilaria changii is an agarophyte with potential for commercialization in Malaysia as it has high yields of good quality agar with high gel strength for the production of food grade agar and agarose. The phylogeographic aspect of G. changii has not been studied despite its abundance and potential commercialization. In this study, six mitochondrial haplotypes (C1–C6) were revealed from 62 specimens varying by 0–3 bp over 923 bp. Results indicate that haplotype C1 is the common ancestor and the most widespread haplotype due to its prevalence in Morib, Gua Tanah, Middle Banks, Batu Besar, Batu Tengah, Sungai Pulai, and Kuala Sungai Merbok. In this study, Morib was suggested as contributing the highest intra-population diversity with the identification of three haplotypes. The mitochondrial marker cox1 is a highly divergent mitochondrial marker and is applicable for studies on species identification and assessment of genetic diversity of G. changii.     

<Emphasis Type="Italic">Phuphanochloa,</Emphasis> a new bamboo genus (<Emphasis Type="Italic">Poaceae</Emphasis>: <Emphasis Type="Italic">Bambusoideae</Emphasis>) from Thailand

Summary  A new monotypic bamboo genus Phuphanochloa (Poaceae: Bambusoideae) from north-eastern Thailand is described, together with a new species, P. speciosa.