THE ULTRASTRUCTURE OF MULTILAYERED STRUCTURES ASSOCIATED WITH FLAGELLAR BASES IN MOTILE CELLS OF TRENTEPOHLIA AUREA1

An ultrastructural study of motile cell development in the green alga Trentepohlia aurea has revealed the presence of multilayered structures (MLS) associated With flagellar bases. These MLS are ultrastructurally similar to MLS described in pteridophyte and bryophyte sperm and in the zoospore of the green algae Coleochaete and Klebsormidium. However, 2 MLS are found in each biflagellate motile cell of T. aurea, while other previously described MLS occur singly in biflagellate motile cells. In addition, the MLS of T. aurea consist of fewer microtubules and are structurally simpler than most other MLS described. The MLS of Trentepohlia may represent a stage in the evolutionary development of the MLS of land plants. The presence or absence of the MLS in motile cells of green algae may be a useful character in phylogenetic studies.     

ULTRASTRUCTURE OF CEPHALEUROS VIRESCENS (CHROOLEPIDACEAE; CHLOROPHYTA). II. GAMETES

Transmission electron microscopic examination of Cephaleuros virescens Kunze growing on leaves of Camellia sp. indicates that gametes are similar to those of Trentepohlia aurea. The gametes bear two, smooth isokont “keeled” flagella containing typical “9 + 2” axonemes and lacking scales. Flagellar insertion is apical and the parallel basal bodies overlap laterally. Each basal body is associated with a separate multilayered structure and component microtubular spline. The latter extends posteriorly beneath the plasmalemma. A nucleus, mitochondria, chloroplasts, and cytoplasmic haematochrome droplets are present. Pyrenoids and eyespots are absent. The subcellular components of C. virescens gametes are comparable to those found in gametes of T. aurea; however, the arrangement of basal bodies and multilayered structures differs slightly from that in T. aurea. Comparison of the fine structure of gametes from Cephaleuros, Phycopeltis, and Trentepohlia clearly indicates that the (1) mode of flagellar insertion, (2) morphology, number, and arrangement of multilayered structures, and (3) keeled flagella are common to these three genera and, thus far, unique among the green algae. Although flagellar insertion is apical, it is not bilaterally symmetrical (sensu stricto), nor is it asymmetrical (cf. Chara and Nitella sperms). The arrangement may be termed “reversed bilateral symmetry” and standardization of the terminology is recommended.     

DIFFERENTIATION OF THE GREEN ALGA CODIUM FRAGILE

Clonal cultures of Codium fragile were established from both swimming cells and vegetative filaments. In the laboratory axis primordia differentiate from heterotrichous juveniles only when cultures are agitated on a reciprocating shaker. The shear forces created by mechanical agitation are essential both for initiation and maintenance of primordia. Contact guidance of growing coenocytic filaments indicates mutual adhesion of filaments as the basis for the differentiation process.     

GENETIC DIVERSITY WITHIN BALTIC SEA POPULATIONS OF NODULARIA (CYANOBACTERIA)1

The determination of the genetic structure of microbial populations has, until recently, required the establishment of many independent clonal cultures for genotypic analysis. In such studies it has been necessary to assume that isolates able to grow in laboratory culture are representative of the full range of diversity within the natural population. In order to test this assumption we used the polymerase chain reaction (PCR) to amplify the intergenic spacer region of the Phycocyanin operon (PC-IGS) from filaments of Nodularia taken both from clonal cultures and from natural populations in the Baltic Sea. Analysis of the nucleotide sequences revealed more variation among 16 cultured isolates than within 23 single filaments sampled from a natural population. As a means of rapidly determining population genetic structure we designed and used mixtures of allele-specific amplification primers in diagnostic PCRs to identify which PC-IGS allele was present in single filaments from natural cyanobacterial assemblages. Using this method, we determined the PC-IGS genotype of 156 filaments from 9 sampling stations throughout the central basin of the Baltic Sea in July 1996. Our results show that two distinct genotypes of Nodularia are present in the population at all stations. Although the two types were present in approximately equal numbers, they were not distributed uniformly.     

THE EFFECTS OF BACTERIA ON THE GROWTH AND REPRODUCTION OF OEDOGONIUM CARDIACUM1

Isolates of male and female Oedogonium cardiacum for which defined media had been established were subsequently found to be contaminated with a species of Corynebacterium which failed to grow in the nutrient broth used, to test for contamination. After the cultures were rendered, axenic through treatment with, penicillin G, they failed to develop oogonia or sperm except occasionally at a very low level. The addition of small amounts of the bacterium increased the development of the reproductive structures; however a much more striking increase was obtained by constantly infecting the algal cultures with Pseudomonas putida. Neither of the bacteria increased growth as measured by dry weight; however the P. putida resulted, in the growth of very long filaments in contrast to the short filaments characteristic of both the axenic cultures and those infected with Corynebacterium sp.     

DIVERSITY,LIFE HISTORY,AND ECOLOGY OF TRENTEPOHLIA AND PRINTZINA (TRENTEPOHLIALES,CHLOROPHYTA) IN URBAN HABITATS IN WESTERN IRELAND 1

On the basis of field and culture investigations, five species of the genera Trentepohlia and Printzina were found to occur in urban habitats in western Ireland: Trentepohlia abietina (Flotow) Hansgirg, T. aurea (Linnaeus) Martius, T. iolithus (Linnaeus) Wallroth, T. cf. umbrina (Kützing) Bornet, and Printzina lagenifera (Hildebrandt) Thompson et Wujek. These species formed perennial populations on a variety of substrata. T. abietina occurred on bark of trees; T. cf. umbrina occurred on stone walls; and P. lagenifera grew on several substrata, mainly cement and asbestos sheeting. T. aurea and T. iolithus were found on old concrete and cement walls; in particular, the latter species formed characteristic, extensive, deep‐red patches on many buildings. In culture, best growth and reproduction of these species were observed at 10 and 15° C, 16:8 h light:dark. Both in culture and in the field, reproduction took place by release of biflagellate swarmers behaving as asexual spores, germinating to produce new plants without any evidence of sexual fusion; release of biflagellate swarmers in the field was generally observed in all seasons throughout a whole annual cycle. Confirmation of the occurrence of sexual reproduction in Trentepohlia was not obtained.     

GROWTH PATTERNS AND MOTILITY OF SPIROGYRA SP. AND CLOSTERIUM ACEROSUM1,2

Spirogyra and Closterium exhibit active motility. This motility is associated with the secretion of pectic mucilage from the cells. The gliding of these cells is not directed toward light but photosynthesis is the energy source for it. The secretion of mucilage causes older Closterium cultures to become thick gelatinous clusters. Spirogyra filaments when undisturbed grow to form thick multistranded rings. This growth pattern might result from the tendency of the filaments to rotate on their long axis.     

Biochemical characterization of carotenoids in two species of <Emphasis Type="Italic">Trentepohlia</Emphasis> (Trentepohliales,Chlorophyta)

Two species of Trentepohlia, i.e., Trentepohlia aurea and Trentepohlia cucullata were collected from walls and tree bark, respectively, at two different seasons in a year. The total carotenoid content in both the species is very high during winter but decreases significantly during summer. By spectroscopic analysis, it was found that. T. aurea and T. cucullata growing in natural habitats are rich sources of carotenoids. The individual carotenoids were separated, identified, and estimated by HPLC, and identified as β-carotene along with some other carotenoids, i.e., neoxanthin, lutein, β-cryptoxanthin, β,γ-carotene, β,ε-carotene (absent during summer).     

A new natural allotriploid,Lycoris × hubeiensis hybr. nov. (Amaryllidaceae), identified by morphological,karyological and molecular data

A new natural hybrid, Lycoris × hubeiensis K. Liu, is confirmed and described based on morphological, karyological and molecular data. The new natural hybrid displays remarkable morphological differences relative to the putative parents L. radiata (2n = 22 = 22A) and L. aurea (2n = 14 = 8m + 6T), and has a karyotype of 3n = 29 = 4m + 22A + 3T. Two chloroplast intergenic regions of ndhF‐rpl32R and rpl32F‐trnL were sequenced with three haplotypes found in the three taxa. The putative hybrid and L. radiata shared the same haplotype (H1), while L. aurea had two other haplotypes (H2 and H3). Combined with the morphological, karyological and molecular data, we conclude that the hybrid is an allotriploid with L. radiata being the maternal parent offering an unreduced gamete and L. aurea being the male parent.     

Cytogenetic characterization and mapping of rDNAs,core histone genes and telomeric sequences in <Emphasis Type="Italic">Venerupis aurea</Emphasis> and <Emphasis Type="Italic">Tapes rhomboides</Emphasis> (Bivalvia: Veneridae)

We describe the chromosomal location of GC-rich regions, 28S and 5S rDNA, core histone genes, and telomeric sequences in the veneroid bivalve species Venerupis aurea and Tapes (Venerupis) rhomboides, using fluorochrome staining with propidium iodide, DAPI and chromomycin A3 (CMA) and fluorescent in situ hybridization (FISH). DAPI dull/CMA bright bands were coincident with the chromosomal location of 28S rDNA in both species. The major rDNA was interstitially clustered at a single locus on the short arms of the metacentric chromosome pair 5 in V. aurea, whereas in T. rhomboides it was subtelomerically clustered on the long arms of the subtelocentric chromosome pair 17. 5S rDNA also was a single subtelomeric cluster on the long arms of subtelocentric pair 17 in V. aurea and on the short arms of the metacentric pair 9 in T. rhomboides. Furthermore, V. aurea showed four telomeric histone gene clusters on three metacentric pairs, at both ends of chromosome 2 and on the long arms of chromosomes 3 and 8, whereas histone genes in T. rhomboides clustered interstitially on the long arms of the metacentric pair 5 and proximally on the long arms of the subtelocentric pair 12. Double and triple FISH experiments demonstrated that rDNA and H3 histone genes localized on different chromosome pairs in the two clam species. Telomeric signals were found at both ends of every single chromosome in both species. Chromosomal location of these three gene families in two species of Veneridae provides a clue to karyotype evolution in this commercially important bivalve family.     

Analysis of protochlorophyllide reaccumulation in the phytochrome chromophore-deficient aurea and yg-2 mutants of tomato by in vivo fluorescence spectroscopy

The aurea and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum) are unable to synthesize the phytochrome chromophore from heme resulting in a block of this branch of the tetrapyrrole pathway. We have previously shown that these mutants also exhibit an inhibition of protochlorophyllide (Pchlide) synthesis and it has been hypothesised that this is due to feedback inhibition by heme on the synthesis of 5-aminolevulinic acid (ALA). In this study we have investigated Pchlide reaccumulation in cotyledons from etiolated wild-type (WT), aurea and yg-2 seedlings using low-temperature fluorescence spectroscopy. WT cotyledons showed two characteristic Pchlide emission maxima at 630 nm (F630) and 655 nm (F655) respectively, while the aurea and yg-2 mutants contained only phototransformable Pchlide F655. Following a white-light flash to WT cotyledons, reaccumulation of phototransformable Pchlide F655 in the first 30 min was absolutely dependent on the presence of Pchlide F630 before the flash. Reaccumulation of Pchlide F630 was not apparent until at least 2 h after the phototransformation. In contrast, Pchlide F630 never accumulated in aurea cotyledons. The relative rates of both Pchlide F655 and total Pchlide synthesis were approximately twice as high in WT compared to aurea. Measurement of ALA synthesis capacity during this period showed that the reduced rate of Pchlide reaccumulation in aurea was due to an inhibition at this step of the pathway. In addition, feeding of ALA resulted in a substantial and equal increase of non-phototransformable Pchlide in both WT and aurea indicating that aurea cotyledons are capable of accumulating high levels of Pchlide that is not associated to the active site of NADPH:Pchlide oxidoreductase (POR). The implications of these results for the mechanism of inhibition of Pchlide synthesis in phytochrome chromophore-deficient mutants and the role of non-phototransformable Pchlide F630 during plastid development are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

PHYLOGENY OF THE GENUS COLEOCHAETE (COLEOCHAETALES,CHAROPHYTA) AND RELATED TAXA INFERRED BY ANALYSIS OF THE CHLOROPLAST GENE rbcL1

The genus Coleochaete Bréb. is considered to be a key taxon in the evolution of green algae and embryophytes (land plants), but only a few of the approximately 15 species have been studied with molecular phylogenetic methods. We report here the sequences of the gene rbcL from six new cultures of Coleochaete and two of Chaetosphaeridium Klebahn. These sequences were combined with 32 additional sequences, and phylogenetic analyses were performed with maximum likelihood, distance optimality, and parsimony methods. Important subgroups within Coleochaete include two primary lineages, one marked by fully corticated zygotes and the other by naked or weakly corticated zygotes. In the first lineage there is a subclade with tightly joined filaments and distinctive (“T‐shaped”) cell division, an assemblage of strains that resembles the endophytic species Coleochaete nitellarum Jost, and a clade with loosely joined filaments and “Y‐shaped” cell divisions. Consistent with recent multigene phylogenies, these analyses support the monophyly of the Coleochaetales, place the Charales as the sister taxon to land plants, and indicate that Chaetosphaeridium is far more closely related to Coleochaete than to Mesostigma Lauterborn.     

Six‐year demographic study reveals threat of stochastic extinction for remnant populations of a threatened amphibian

Sustained demographic studies are essential for early detection of species decline in time for effective management response. A paucity of such background data hindered the potential for successful conservation during the global amphibian decline and remains problematic today. The current study analysed 6 years of mark‐recapture data to determine the vital demographic rates in three habitat precincts of the threatened frog, Litoria aurea (Hylidae) and to understand the underlying causes of variability in population size. Variability in population size of L. aurea was similar to many pond‐breeding species; however this level of fluctuation is rare among threatened amphibians. Highly variable populations are at greater risk of local extinction and the low level of connectivity between L. aurea populations means they are at a greater risk of further decline due to stochastic extinction events and incapacity to recolonize distant habitat. We recommend that management of this species should encourage recolonization through creation of habitat corridors and reintroduction of L. aurea to areas where stochastic extinction events are suspected.     

Compatibility of Metarhizium anisopliae with Calpurnia aurea leaf extracts and virulence against Rhipicephalus pulchellus

Leaf extracts of Capurnia aurea (Fabaceae) have been reported to attract ticks and can be considered for use in combination with entomopathogenic fungus in an autodissemination approach, whereby ticks that are attracted to baited traps are infected with the inoculum. Bioassays were undertaken to assess the compatibility of aqueous, methanol and acetone extracts of C. aurea with conidia of Metarhizium anisopliae sensu stricto (ss) (Hypocreales: Clavicipitaceae) isolate ICIPE 7 in terms of mycelial growth, conidial production and spore viability. Aqueous extract of C. aurea was compatible with the fungus at all the concentrations tested whereas methanol and acetone extracts inhibited all the fungal growth parameters. The virulence of M. anisopliae formulated in different extracts of C. aurea was also tested against different developmental stages of Rhipicephalus pulchellus in laboratory bioassays. No significant differences in virulence were observed between M. anisopliae applied alone and M. anisopliae formulated in different concentrations of extracts of C. aurea among the larvae, nymphs and adults. However, mortality varied according to the developmental stage, with larval stage being the most susceptible. Aqueous extract was compatible with M. anisopliae and could be therefore mixed together for ‘spot‐spray’ treatments as low cost and environmentally friendly technology for tick control in grazing pasture.     

Molecular analysis of the phytochrome deficiency in an aurea mutant of tomato

Summary The au ^w mutant allele of the aurea locus in tomato has previously been shown to cause deficiency for the phytochrome polypeptide (Parks et al. 1987). We have begun to characterize the molecular basis and consequences of this deficiency. Genomic Southern blot analysis indicates that there are at least two and probably more phytochrome polypeptide structural genes in tomato. RNA blot analysis shows that the au ^w mutant contains normal levels of phytochrome mRNA and in vitro translation of au ^w poly(A)⁺ RNA yields a phytochrome apoprotein that is quantitatively and qualitatively indistinguishable on SDS-polyacrylamide gels from that synthesized from wild-type RNA. These results indicate that the phytochrome deficiency in aurea is not the result of lack of expression of phytochrome genes but is more likely due to instability of the phytochrome polypeptide in planta. Possible reasons for such instability are discussed. Analysis of the molecular phenotype of aurea indicates that the phytochrome-mediated increase in the abundance of the mRNA encoding chlorophyll a/b binding protein (cab) is severely restricted in the mutant as compared with wild-type tomato. Thus, the au w strain exhibits defective photoregulation of gene expression consistent with its very reduced level of the phytochrome photoreceptor.     

OBSERVATIONS ON NAVICULA ULVACEA,A RARE FOLIOSE MARINE DIATOM1

Navicula ulvacea (Berkeley ex Kütz.) P. T. Cleve, a marine colonial blade-forming diatom originally discovered in Scotland was collected at North Rustico, Prince Edward Island, during winter 1971. It formed dense stands of Porphyra-like fronds, ranging up to 60 mm long × 20 mm wide. These occurred under ice-covered water on rocks 1–2 m below zero tide. Comparisons made at the light and scanning electron microscope level compared favorably with original Aberdeen material. Clonal cultures established from single cells at first also formed blades, but rarely more than 10 mm long. In numerous serial subcultures made since 1971 no further blades have been formed. Instead, growth consisted of well-separated cells or short filaments scattered over the bottom and sides of the vessels. Older cultures developed prostrate sheets of filaments, embedded in a mucilagenous matrix and occasional bubble-like eruptions. Results of the cultures suggest that N. ulvacea probably occurs in a prostrate form in nature, perhaps more commonly than in the blade form. Literature records since the original collection in 1844 are brought together but are considered doubtful. A discussion on nomenclature is included.     

Characterization and localization of repetitive DNA sequences in the ornamental Alstroemeria aurea Graham

 Three repetitive DNA sequences were isolated from a genomic DNA library of the ornamental Alstroemeria aurea Graham. Two repeats, A001-I and A001-II, were quite homologous and highly A. aurea-specific. A001-I was a 217-bp sequence with several telomeric TTTAGGG repeats at the 5′ end and a unique sequence of 98 bp at the other end. The third repeat, A001-IV, was a 840-bp sequence which contained two sub-sequences of 56 and 74 bp respectively, previously found in chloroplast (cp) DNA of tobacco and spinach and to a lesser extent in the cpDNA of maize and rice. Repeat A001-IV was not species-specific and its hybridization signal was weaker than the other repeats. Fluorescence in situ hybridization (FISH) revealed the A. aurea-specific repeats to be located in the heterochromatic regions of all A. aurea chromosomes. The differences in FISH pattern make them useful tools for karyotype analysis. The non-species-specific sequence A001-IV gave a dispersed signal over all the Alstroemeria chromosomes in an interspecific hybrid. The potential use of these repetitive DNA sequences for the study of phylogenetic relationships within the genus Alstroemeria is discussed. Received: 24 November 1996/Accepted: 20 December 1996     

Development and characterization of microsatellite markers for the tropical tree species Tabebuia aurea (Bignoniaceae)

In this study, we report the development and characterization of 21 polymorphic microsatellite loci for Tabebuia aurea, using genomic library enrichment. Number of alleles per locus and expected heterozygosity ranged from nine to 26 and from 0.808 to 0.955. The high combined probability of genetic identity (1.03 × 10^?37) and probability of paternity exclusion (0.9889) showed that multilocus genotypes are likely to be unique and will allow detailed parentage studies in natural populations of T. aurea. Additionally, a high percentage of transferability was achieved for the four species of the same genus studied.