HYALOPYRENIA JAPONICA,A PECULIAR NEW PYRENOCARPOUS LICHEN GENUS FROM JAPAN

Hyalopyrenia japonicaHarada (lichenized Ascomycotina) is described as a new genus on the basis of a specimen from friable rock in forests in Chiba-ken, central Japan. It is characterized by a poorly developed crustose thallus, aTrentepohliaphycobiont, immersed perithecia with hyaline walls, simple paraphyses and periphyses, non-amyloid bitunicate asci with an ocular chamber, and transversely septate hyaline spores.Hyalopyreniais monotypic, and its taxonomic position is uncertain.     

中国地衣新记录属——裂衣属

报道了中国文字衣科地衣一新记录属——裂衣属（Chapsa A.Massal.）及其2新记录种,即印度裂衣（C.indica A.Massal.）和斑果裂衣[C.leprocarpa（Nyl.）A.Frisch],标本来自海南和广西。该属主要特征是：地衣体壳状、树皮生;具子囊盘类或色盘衣类的子囊果,固有果壳融合或不明显,具侧生侧丝;具横隔或砖壁型胞室的子囊孢子。     

An unusual <Emphasis Type="Italic">Lophodermium</Emphasis> species on needles of <Emphasis Type="Italic">Pinus taiwanensis</Emphasis> from China

An interesting fungus, which appeared to resemble a contaminated or infected Lophodermium species, is described and illustrated in this paper. The circinate apex of paraphyses, which intertwined with each other, and the broad slit exposing the unusual-white hymenium distinguished the new species from other known Lophodermium species. Evidence from two markers including internal transcribed spacer (ITS) region and the partial Actin gene indicated that L. pini-taiwanensis was in a robust and separate clade. It is, therefore, described as a new Lophodermium species.     

<Emphasis Type="Italic">Aspicilia tibetica</Emphasis>, a new terricolous species of the Himalayas and adjacent regions

A new species, Aspicilia tibetica Sohrabi & Owe-Larss., is described from Tibet, where it grows on soil and plant debris at altitudes between 4,600 and 5,400 m, and where it seems to be rather commonly distributed. It is characterized by a crustose, white thallus, 8-spored asci with small, globose to ellipsoid ascospores, a brown epihymenium, and non-moniliform to submoniliform paraphyses. It lacks secondary substances. The new species is compared with other terricolous Aspicilia species. Morphological, chemical, and phytogeographical differences between the non-vagrant terricolous species are summarized.     

A comparison of the structural organization of amplified ribosomal DNA from Xenopus mulleri and Xenopus laevis.

Secondary structure maps of long single strands of amplified ribosomal DNA from two closely related species of frogs, Xenopus laevis and X. mulleri, have been compared. The secondary structure pattern of the gene region is identical in both ribosomal DNAs while the patterns in the non-transcribed spacers² differ. In X. mulleri, the spacer shows an extended region without any secondary structure adjacent to the 28 S ribosomal RNA sequence. In contrast, the same region in the X. laevis spacer has extensive secondary structure. A comparison of secondary structure maps and denaturation maps of these two ribosomal DNAs (Brown et al., 1972) reveals that the portion without secondary structure in the X. mulleri spacer corresponds to an early melting A + T-rich region. As in X. laevis ribosomal DNA, Escherichia coli restriction endonuclease (EcoRI) makes two cuts in each repeating unit of amplified ribosomal DNA from X. mulleri. The position of the cleavage sites is identical in the two species as judged from secondary structure mapping of the two classes of EcoRI fragments generated. The small fragments of X. mulleri ribosomal DNA are homogeneous in size with a duplex molecular weight of 3.0 × 10⁶, and contain about 85% of the 28 S ribosomal RNA gene and about 17% of the 18 S ribosomal RNA gene. The large fragments are heterogeneous in size with molecular weights ranging from 4.2 to 4.9 × 10⁶, and contain the remaining portions of the gene regions and the nontranscribed spacer. Heteroduplexes made between large fragments of different lengths show only deletion loops. The position of these loops indicates that the length heterogeneity resides in the non-transcribed spacer region. Electrophoretic analysis of EcoRI digests of chromosomal ribosomal DNA from X. mulleri demonstrates that this DNA is heterogeneous in length as well.     

NOTES ONPOLYSPORINAVĚZDA,WITH A DESCRIPTION OF A NEW SPECIES FROM TASMANIA

The new species,Polysporina terricolaKantvilas from south-eastern Tasmania, is described and compared to other species in the genus. It is well-characterized by a range of morphological, anatomical and ecological characters, especially by the presence of oil paraphyses and oil droplets in the hymenium, features unknown in other members of the genus. Species ofPolysporinaare best separated by the following characters: size and morphology of the apothecia, size of ascospores, thickness of paraphyses and height of the hymenium. The combinationsP. ferruginea(Lettau) M. Steiner andP. pusilla(Anzi) M. Steiner are validated. A key to the taxa studied is provided.     

The population and community structure of Hawaiian fringing-reef crustose Corallinaceae (Rhodophyta,Cryptonemiales)

Measurements of cover, relative density, and frequency are given for the major reefbuilders on the Waikiki fringing reef. Crustose coralline algae cover 39% of the reef surface and exceed all other organisms as the major builders and consolidators of reef materials. An unidentified coralline (melobesioid C) covers the greatest area (17 %), but Hydrollthon reinboldii (Weber-van Bosse & Foslie) Foslie (11 % cover) because of its thicker thalli and higher relative density (45 %) and frequency (68 %) values is the primary limestone former. Melobesioid C ranks second and Sporolithon erythraeum (Rothpletz) Kylin (6 % cover) third in relative importance. Porolithon onkodes (Heydrich) Foslie (3 % cover), although shown by its low density (4 %) and frequency (6 %) to have a comparatively restricted distribution, is more important than P. gardineri (Foslie) Foslie (2 % cover). P. onkodes maintains and provides the surf-resistant reef edge and is, therefore, of great ecological importance. Coelenterate corals cover less than 1 % of the total area and are relatively unimportant on the fringing reef. The hypothesis is developed that the high ratio (200 : 1) of crustose corallines to corals at Waikiki may be partly due to increases in eutrophication.Experimental evidence shows that P. onkodes can withstand intense illumination and is thereby unique among Hawaiian crustose Corallinaceae. Sporolithon erythraeum is more typical of other crustose corallines since it is physiologically adapted to low-light habitats.     

DIFFERENTIAL LIFE HISTORY PHASE EXPRESSION IN TWO COEXISTING SPECIES OF SCYTOSIPHON (PHAEOPHYCEAE) IN NORTHERN CHILE1

The identity of two phaeophycean taxa that monopolized the middle‐lower rocky intertidal zone of a coastal area chronically exposed to copper mine wastes in northern Chile was unraveled. One of them was preliminarily identified as the gametophytic stage of Scytosiphon lomentaria (Lyngbye) Link. The other, a dark crust, resembled the alternate stage of some Scytosiphon species. Comparative analysis of morphology, life history, and DNA sequences strongly suggests that crusts corresponded to sporophytic S. tenellus Kogame and confirm that erect thalli belonged to S. lomentaria. A clear segregation of erect and crustose thalli was found using internal transcribed spacer region 1 and RUBISCO spacer sequences. Furthermore, whereas crusts always grouped with S. tenellus, erect thalli always grouped with S. lomentaria. Life history studies failed to connect the two entities. First, field‐collected S. tenellus produced progeny that either recycled the crust, which reproduced by unilocular zoidangia, or developed into erect thalli. The latter, unlike typical gametophytic S. lomentaria, developed patchy sori of plurilocular zoidangia. Second, S. lomentaria displayed a direct‐type life cycle, in which progeny from erect individuals only developed into erect thalli and produced only plurilocular zoidangia. This constitutes the first experimental study on Scytosiphon from the Pacific coast of South America and the first report of S. tenellus on this coast. It is also the first report of the crustose stage of Scytosiphon appearing as a perennial and dominant algal species in a temperate rocky intertidal system.     

High intra-individual sequence variation in the nuclear rDNA LSU-5S intergenic spacer in the Sargassaceae (Fucales, Phaeophyceae)

Of four species of Sargassaceae, representing the genera Carpophyllum, Cystoseira, Landsburgia, and Sargassum, the intergenic spacer of the ribosomal cistron was amplified using a forward primer annealing at the 3′-end of the large subunit (LSU) of the ribosomal cistron and a reverse primer annealing at the 5S rRNA gene. The PCR products were cloned and the DNA sequences of multiple clones were determined. Almost each clone showed a unique DNA sequence. Intra-individual variation of this LSU-5S intergenic spacer was extremely high and was characterized by great length variation and a high number of short tandem repeats. Sequences were unalignable and therefore it was concluded that the LSU-5S intergenic spacer is unsuitable for phylogenetic and phylogeographic studies of sargassacean taxa.     

Phylogeny and taxonomy of the ‘manna lichens’

We present ataxonomic revision of the ‘manna lichens’ based on morphological, chemical, ecological and molecular data. A large number of herbarium specimens and fresh collections were examined. Phylogenetic analyses were performed using nuclear ribosomal (nrITS, nrLSU) and mitochondrial small subunit (mtSSU) sequences. Some notable phenotypic characters were plotted on the phylogenetic tree, and the analysis reveals that some of these characters are useful for genus and species level distinction of certain ‘manna lichens.’ Phylogeny of the Megasporaceae was revised using a combined data set of nrLSU and mtSSU and performing parsimony and Bayesian analyses. Five genera (Aspicilia, Circinaria, Lobothallia, Megaspora and Sagedia) are recognized. Further, the relationships of five presumably closely related genera of ‘manna lichens’, namely Agrestia (vagrant), Aspicilia (crustose) Circinaria (crustose), Chlorangium (vagrant) and Sphaerothallia (vagrant) with different growth forms were analysed. The analyses revealed that ‘manna lichens’ do not form a monophyletic group but occur in different clades within the genus Circinaria. The genera Agrestia, Chlorangium and Sphaerothallia are assigned as new synonyms under the genus Circinaria and no vagrant or erratic species remain in the genus Aspicilia. The analyses also show that five new erratic, vagrant and crustose species can be recognized. In this study two ‘manna lichens’, viz. Circinaria rostamii sp. nov. (Azerbaijan, Iran and Turkey), and Circinaria gyrosa sp. nov. (Armenia, Azerbaijan, Iran, Turkey, Turkmenistan and Spain) are described as new to science. Three potentially new species with crustose and erratic forms need additional study. Aspicilia fruticolosofoliacea is reduced to synonymy under C. alpicola, and a lectotype is designated for C. aspera. Thirteen new combinations in Circinaria are presented. The phenomenon of vagrancy is briefly discussed, and the biogeography of the ‘manna lichens’ is outlined. Illustrations, distribution maps, and an identification key to the species are provided.     

Genetic diversity of Pseudo-nitzschia brasiliana (Bacillariophyceae) from Malaysia

To clarify the genetic diversity of a potentially toxic pennate diatom, Pseudo-nitzschia brasiliana found in Malaysian waters, 30 strains of P. brasiliana were established into clonal culture since May 2008. The ultrastructure of these strains was examined for confirmation of species identification. The genetic marker, internal transcribed spacer (ITS) of nuclear-encoded ribosomal DNA was used to examine the genetic diversity of P. brasiliana isolated from different geographical localities. The ITS sequences of P. brasiliana were highly conserved in their secondary structures, with five helices in the first internal transcribe spacer (ITS1) and four universal helices in the second internal transcribe spacer (ITS2) with a pseudo-helix. No compensatory base change was observed among the strains examined. Genetic divergences among the Malaysian strains ranged from 0.07 to 0.54%. The present study revealed a high genetic homogeneity of Malaysian P. brasiliana strains.     

Assessment of Four Molecular Markers as Potential DNA Barcodes for Red Algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae,Rhodophyta)

DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment.     

Paternal inheritance of plastids in interspecific hybrids of the genus Actinidia revealed by PCR-amplification of chloroplast DNA fragments

RFLPs (restriction fragment length polymorphisms) of PCR (polymerase chain reaction) -amplified fragments were used to trace the pattern of plastid DNA inheritance in the genus Actinidia. A total of 51 progeny originating from interspecific crosses between three A. arguta cultivars and A. deliciosa, the kiwifruit, and 12 progeny originating from the cross between A. kolomikta and A. chinensis were analysed together with their parents. No reciprocal crosses could be tested since they all failed to set viable seeds. Attempts to rescue immature embryos failed in all cases as well. The A. arguta×A. deliciosa crosses were checked for the RFLP patterns of a sequence encoding part of the Rubisco large subunit (rbcL), using either AluI or MseI, and for a sequence encoding part of the photosystem II D1 protein (psbA), using HinfI. The A. kolomikta×A. chinensis cross was checked for the RFLP patterns of sequences encoding the spacers between trnT and the 5′-trnL exon (a-b spacer DNA) and the trnL 3′ exon and trnF (e-f spacer DNA), respectively. The first spacer revealed a natural polymorphism between the two parent species due to a large deletion occurring in A. kolomikta detectable without further restriction enzyme treatment. The e-f spacer DNA was digested with HinfI. The comparison of the RFLP patterns in the parents and their progeny showed a strictly paternal inheritance of chloroplast DNA in Actinidia, with no exception found in any of the crosses examined. As the reciprocal crosses were not available, we do not know whether paternal inheritance of plastids is restricted to the crosses we analysed or if this is the general rule for plastid inheritance in the genus Actinidia. Actinidia is dioecious and is the first purely outbreeding species for which a paternal plastid inheritance has so far been documented.     

An unusual evolutionary behaviour of a sea urchin histone gene cluster

DNA sequences of cloned histone coding sequences and spacers of sea urchin species that diverged long ago in evolution were compared. The highly repeated H4 and H3 genes active during early embryogenesis had evolved (in their silent sites) at a rate (0.5-0.6% base changes/Myr) similar to single-copy protein-coding genes and nearly as fast as spacer DNA (0.7% base changes/Myr) and unique DNA. Thus, evolution in the major histone genes conforms to a universal evolutionary clock based on the rate of base sequence change. By contrast, the H4 and H3 coding sequences and a non-transcribed spacer of the DNA clone h19 of Psammechinus miliaris show an exceptionally low rate of sequence evolution only 1/100 to 1/200 that predicted from the clock hypothesis. According to the classical model of gene inheritance, the h19 DNA sequences in the Psammechinus genome require unusual conservation mechanisms by selection at the level of the gene and spacer sequences. An alternative explanation could be recent horizontal gene transfer of a histone gene cluster from the very distantly related Strongylocentrotus dröbachiensis to the P. miliaris genome.     

Endolithic chlorophyll d-containing phototrophs

Cyanobacteria in the genus Acaryochloris are the only known oxyphototrophs that have exchanged chlorophyll a (Chl a) with Chl d as their primary photopigment, facilitating oxygenic photosynthesis with near infrared (NIR) light. Yet their ecology and natural habitats are largely unknown. We used hyperspectral and variable chlorophyll fluorescence imaging, scanning electron microscopy, photopigment analysis and DNA sequencing to show that Acaryochloris-like cyanobacteria thrive underneath crustose coralline algae in a widespread endolithic habitat on coral reefs. This finding suggests an important role of Chl d-containing cyanobacteria in a range of hitherto unexplored endolithic habitats, where NIR light-driven oxygenic photosynthesis may be significant.     

Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA

We have investigated the anomalously weak binding of human papillomavirus (HPV) regulatory protein E2 to a DNA target containing the spacer sequence TATA. Experiments in magnesium (Mg²⁺) and calcium (Ca²⁺) ion buffers revealed a marked reduction in cutting by DNase I at the CpG sequence in the protein-binding site 3′ to the TATA spacer sequence, Studies of the cation dependence of DNA-E2 affinities showed that upon E2 binding the TATA sequence releases approximately twice as many Mg²⁺ ions as the average of the other spacer sequences. Binding experiments for TATA spacer relative to ATAT showed that in potassium ion (K⁺) the E2 affinity of the two sequences is nearly equal, but the relative dissociation constant (K_d) for TATA increases in the order K⁺ < Na⁺ < Ca²⁺ < Mg²⁺. Except for Mg²⁺, K_d for TATA relative to ATAT is independent of ion concentration, whereas for Mg²⁺ the affinity for TATA drops sharply as ion concentration increases. Thus, ions of increasing positive charge density increasingly distort the E2 binding site, weakening the affinity for protein. In the case of Mg²⁺, additional ions are bound to TATA that require displacement for protein binding. We suggest that the TATA sequence may bias the DNA structure towards a conformation that binds the protein relatively weakly.     

Clustering of transfer RNA genes of Xenopus laevis

The arrangement of the reiterated DNA sequences complementary to transfer RNA has been studied in Xenopus laevis. Prehybridization of denatured DNA with an excess of unfractionated tRNA results in a small but well-defined increase in the buoyant density of fragments which contain sequences homologous to tRNA. The density increase is smaller than that found for 5 S DNA, but is the same or nearly so for all tRNA coding sequences examined. These results indicate that the majority of tRNA genes are clustered together with spacer DNA, the average size of which is estimated to be approximately 0.5 × 10⁶ daltons (native) DNA.In high molecular weight native DNA preparations, the sequences homologous to unfractionated tRNA, tRNA^Val, tRNA₁^Met and tRNA₂^Met band in CsCl at 1.707, 1.702, 1.708 and 1.711 g cm^?3, respectively. The mean buoyant densities are constant at all molecular weights examined but they do not correspond to the base compositions of the complementary tRNA species. These results indicate that isocoding genes are linked to spacer DNA in separate and extensive gene clusters, and that the different clusters contain different spacer DNA sequences. These clusters form well-defined cryptic DNA satellites which are potentially separable from each other as well as from other chromosomal DNA.     

Fungal specific primers for PCR‐amplification of mitochondrial LSU in lichens

Fungal specific primer sequences for the amplification of the large subunit of the mitochondrial ribosomal DNA (mtLSU) are presented in this paper. Fungal specific primers make the separation of fungal and algal cells prior to DNA‐extraction from lichens unnecessary. This is especially useful in crustose and small foliose and fruticose lichens. An example from a complex of closely related species of the crustose lichen genus Biatora shows the usefulness of mtLSU‐sequences for studies of infraspecific variability and lower level systematics of lichenized ascomycetes.     

Length heterogeneity in a region of the human ribosomal gene spacer is not accompanied by extensive population polymorphism

We carried out a restriction enzyme analysis of human ribosomal DNA structure on total placental DNA using the Southern (1975) method. Studies on a single individual using HindIII, PstI, HpaI and BglII revealed that a region of the non-transcribed spacer near the 3′ end of the 28 S gene was heterogeneous in size. Four fragment classes were detected in this individual. Adjacent classes differed in size from one another by about 0·8 × 10³ bases. Analysis of 19 additional placental samples and four cell lines revealed no fragment classes other than those detected in the original sample. Mixing experiments carried out with all 20 placental DNA samples provided further evidence for the discrete nature of the population polymorphism in the length of this region of the spacer. This finding contrasts sharply with the almost continuous nature of the population polymorphism in the length of a region of the non-transcribed spacer in Xenopus laevis.     

CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5′ handle (8 nt) and a 3′ handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5′ handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.