DNA cloning demonstrates high genetic heterogeneity in populations of the subaerial green alga Trentepohlia (Trentepohliales,Chlorophyta)

Mats of the green alga Trentepohlia, a genus widely distributed in the tropics as well as temperate regions, have always been perceived as homogeneous (i.e., formed by only one species). As such, their general nature and specific feature play a supportive role in the species delimitation. However, the presence of morphologically dissimilar thalli was observed under the light microscope and when cultivating a piece of a single mat. To address this, we performed DNA cloning of the rbcL gene on mat fragments of T. abietina, T. annulata, T. jolithus and T. umbrina sampled in Europe to reveal if they are composed of one or more species. We revealed that more Trentepohlia haplotypes may coexist in a single mat. In consideration of this, we conclude that the use of material isolated in unialgal culture will be almost mandatory for a taxonomic reassessment of this complicated genus. Another direct implication of this problem is that herbarium specimens consisting of field‐collected material should not be used for direct sequencing. We further hypothesize the reasons why multiple haplotypes of Trentepohlia occur more frequently in the tuft‐like mats. Possibly, fragments and/or cells of other microalgae, including other species of Trentepohlia, might be retained in such mats more easily than in the crustose trentepohlialean mats.     

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.     

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).     

Phragmoplastins,the Trentepohliales,and the Evolution of the Cytokinesis in Green Plants

Representatives of the monophyletic Trentepohliales are widely distributed in the tropics, subtropics, and temperate regions worldwide. They grow in soil, or are epilithic, epiphytic or endophytic. The family comprises approximately 70 species placed in at least four genera (Trentepohlia, Cephaleuros, Phycopeltis and Stomatochroon), with Trentepohlia sensu lato (including Printzina and Physolinum) accounting for half of the species in this family. PCR amplification and sequencing of the 18 SSU rDNA of 18 isolates of the Trentepohliaceae were used to assess the monophyly of the genus Cephaleuros and to determine the phylogenetic relationships among species of Trentepohlia sensu lato. Distance, Parsimony, and Maximum Likelihood analyses indicate that Trentepohlia sensu lato is basal and includes two species recently transferred to Phycopeltis (P. umbrina) and Printzina (P. lagenifera). In contrast, Cephaleuros is a derived monophyletic clade. Analysis of isolates of C. virescens, from the USA, Taiwan, and South Africa indicate that this taxon may consist of different species sharing a convergent morphology. The results of this study have implications for the taxonomy of the genera.     

A METHOD OF OBTAINING AXENIC CULTURES OF TRENTEPOHLIA SPP. (CHLOROPHYTA)1

Axenic cultures of Trentepohlia species are necessary for the study of growth and hysiological characters of the algae. We describe the use of a Sherman micromanipulator to isolate filaments from samples of T. aurea and T. odorata collected from their natural habitats. These filaments were then used as inocula for the establishment of axenic cultures. In the case of T. aurea, further treatment with lactic acid was necessary.     

Unusual carbohydrate pattern in Trentepohlia species

Four Trentepohlia species and the related Cephaleuros virescens (Chroolepidaceae, Trentepohliales, Chlorophyceae) photosynthesize and accumulate mannitol, arabinitol, erythritol and glycerol, while Trentepohlia spp. additionally synthesize a second pentitol, ribitol (adonitol). T. umbrina also contains small amounts of a heptitol, volemitol.     

Methods for the Induction of Reproduction in a Tropical Species of Filamentous Ulva

The green seaweed Ulva is a major fouling organism but also an edible aquaculture product in Asia. This study quantified for the first time the effect of key factors on the reproduction of a tropical species of filamentous Ulva (Ulva sp. 3). The controlled timing of release of swarmers (motile reproductive bodies) was achieved when experiments were initiated in the early afternoon by exposing the thalli to a temperature shock (4°C) for 10 min and subsequently placing them into autoclaved filtered seawater under a 12 h light: 12 h dark photoperiod at 25°C. The release of swarmers then peaked two days after initiation. In contrast, segmentation, dehydration, salinity or time of initiation of experiments had no effect of any magnitude on reproduction. The released swarmers were predominantly biflagellate (95%), negatively phototactic and germinated without complementary gametes. This indicates that Ulva sp. 3 has a simple asexual life history dominated by biflagellate zoids.     

Two different genetic strains of stalked-Ulva (Ulvales, Chlorophyta) grow on intertidal rocky shores in Ebisujima, central Japan

Stalked‐Ulva has been recognized as an ecologic form of Ulva pertusa Kjellman with narrow and extensive stipes that grows on rocky shores with strong wave action. However, it is possible that stalked‐Ulva includes more than two taxa, because it has been shown previously that some isolates of stalked‐Ulva did not cross with U. pertusa. Therefore, further crossing tests, observations of swarmer behavior, culture studies and comparison of DNA sequences were made to clarify whether or not all stalked‐Ulva are included within U. pertusa. Crossing tests showed that stalked‐Ulva contained two different types, one that crossed with U. pertusa (Up strain) and one (N strain) that did not. The biflagellate swarmers of N strains showed negative phototaxis and were slightly larger than male and female gametes of Up strains. Thalli cultured from the swarmers of N strains released the same type of swarmers again. The N strains have an asexual life history, reproducing solely by biflagellate swarmers. Internal transcriber spacer sequence analysis indicated that these two entities are U. pertusa (Up strain) and U. fasciata Delile (N strain). In culture both strains differ morphologically from wild thalli with stipes; it seems that the two different taxa both show a petiolate morphology when growing under the same environmental conditions.     

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.     

Trentepohlia umbrina on Scots pine as a bioindicator of alkaline dust pollution

The present study was driven by the wish to broaden the selection of ecological indicators for estimating the alkaline dust pollution. The abundance of the algal species Trentepohlia umbrina was studied on Pinus sylvestris trunks, an acidic substrate that it normally does not occupy. The study was carried out in northern Estonia in the surroundings of four major limestone quarries, considerable local-level sources of dust pollution. The cover of T. umbrina on tree trunks was clearly higher near the quarries, evidently due to the elevated bark pH, its maximum values reaching ca. 30% (mean of five trees studied per sample plot). The cover of the species decreased steeply at the distance of 800–900 m from the quarries; further than 1000 m from the quarries the maximum cover was already less than 4%, and further than 2000 m less than 1%. The correlation between the cover of T. umbrina and measured bark pH values indicated a steep increase in cover at about pH 3.8–3.9; the cover values varied between 0 and 4% below and between 10 and 31% above that pH. These results confirm that T. umbrina could be used as an indicator species of alkaline dust pollution. The species forms reddish powdery coating on tree trunks, and in case of high abundance can be very conspicuous looking, being easily detectable at field.     

An ultrastructural re-examination of putative multilayered structures inTrentepohlia aurea

Summary Due to recent interest in the significance of multilayered structures (MLSs) and MLS-like structures in green algal and land plant systematics and evolution, the putative MLSs ofTrentepohlia aurea swarmers were re-examined using conventional TEM of serial thin sections, and high-voltage electron microscopy (HVEM) of thick sections. Features not previously reported to occur in trentepohlialean algae include a structure connecting MLSs to adjacent basal bodies, the lamellar nature of the sub-microtubular layer of the MLS, and the presence of appendages on microtubules overlying the lamellar strip. Reasons are given for concluding that the MLSs ofTrentepohlia and other members of theTrentepohliales are possibly homologous to MLSs ofGlaucophyceae and the MLS-like structures ofFriedmannia. It is proposed that increased attention to the details of MLS structure may be crucial to an understanding of the divergence of main lines of green algal evolution.     

Asexual life history by quadriflagellate swarmers of Ulva spinulosa (Ulvales, Ulvophyceae)

This is the first report of a Ulva species reproducing asexually solely by quadriflagellate swarmers. Ulva spinulosa Okamura et Segawa specimens were collected from Ukibuchi on the Pacific coast of Kochi Prefecture, southern Japan. Quadriflagellate swarmers were released from these specimens. The swarmers showed negative phototaxis before settlement. Thalli cultured from these swarmers also released quadriflagellate swarmers in culture. Microspectrophotometric studies demonstrated equivalent DNA in nuclei of vegetative cells in thalli of U. spinulosa and in sporo‐phytes of the other Ulva species with sexual life history (U. fasciata Delile). Furthermore, the quadriflagellate swarmers of U. spinulosa had the same DNA value, demonstrating that the quadriflagellate swarmers are produced without meiosis.     

Minutes of the Ninth Annual General Meeting

In unialgal culture, Gymnodinium pseudopalustre Schiller (G.p.) and Woloszynskia apiculata sp. nov. (W.a.) multiply respectively by binary fission in the motile state and by motionless zoosporangia, releasing 2, 4 or 8 zoospores. Both species are isogamous, but G.p. is homothallic, W.a. heterothallic. Fusion of the planogametes leads to long-lived planozygotes, which retain two posterior flagella and, while enlarging, assume specific morphologies. The motile stage of the zygotes is terminated by formation of hypnozygotes (resting spores), globular and spiny in G.p., grossly fusiform (‘horned’) and tubercled in W.a. The composition of the hypnozygote walls is described. After their dormancy has been broken by a cold treatment of several weeks in the dark, hypnozygotes of both species germinate when brought back to higher temperature and light. In so doing, those of G.p. excyst one posteriorly biflagellate swarmer as a meiocyte, which, after a stage of nuclear cyclosis or, in karyological terms, zygotene through postzygotene, undergoes two steps of binary fission in the motile stage, separated by several days. In W.a., cyclosis as well as the first meiotic cell division occur inside the closed wall of the hypnozygote (now a meiocyte); thereafter either two swarmers escape and undergo the second meiotic division in a separate zoosporangium or, alternatively, second meiotic nuclear and cell divisions also take place inside the spore wall and four swarmers are finally excysted. Some aspects of dinoflagellate life cycles and taxonomic questions are discussed.     

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.     

THE SEASONAL DISTRIBUTION,ABUNDANCE AND DIVERSITY OF DESMIDS (CHLOROPHYTA) IN A SOFTWATER,NORTH TEMPERATE STREAM1

Suspended and benthic algal communities from a mildly acidic, third-order Rhode Island stream were examined to determine the seasonal distribution, abundance and diversity of the lotic desmids. Within a one-year sampling period, 148 species and 202 subspecific taxa of desmids were identified, representing 23 genera. Species of Cosmarium and Closterium accounted for approximately 70% of the desmids present, and were the most diverse and abundant taxa during all seasons except spring, when Hyalotheca dissiliens was the dominant desmid species. Average abundance and species richness generally were greatest during summer for both suspended and benthic desmids. Most desmids occurred in benthic habitats, and were randomly distributed among substrata. Average seasonal abundance was 7.4 × 10⁴ cells·g^?1 dry wt substratum, among 13 types of substrata. Highest desmid abundance was measured among substrata with intricate morphologies, such as Fontinalis spp., which was associated with 1.2 × 10⁶ desmid cells·g^?1 dry wt substratum, or 1.7 × 10³ cells·cm^?2 substratum. Cell division was observed for 70 desmid taxa, and average seasonal reproduction (based on cell numbers) among all substrata ranged from 4% in winter to 20% during summer. In addition, sexually produced zygospores were found occasionally for H. dissiliens. Desmids were distributed among most substrata examined in this stream, with abundance comparable to reported estimates from softwater lakes and acid bogs. In contrast to established dogma, lotic desmids are not incidental drift organisms, but rather comprise a viable and persistent component of the stream periphyton.     

VEGETATIVE REPRODUCTION OF THE FRESHWATER DINOFLAGELLATE GLOEODINIUM MONTANUM1

The vegetative life cycle of Gloeodinium montanum Klebs was examined. In unialgal cultures G. montanum divided predominantly by binary fission once every 2-3 weeks. Nuclear division was followed by a delayed cytokinesis producing non-motile G. montanum cells. When placed in fresh media 2-4 biflagellated swarmers were formed. The swarmers, although similar in appearance to those of Hemidinium ochraceum Levander (1900), differ from that species in their dimensions. During vegetative reproduction swarmers developed directly into non-motile vegetative cells.     

Lichen-like colonies of pure Trentepohlia on limestone monuments

Biofilms were collected on discoloured limestone samples and on adhesive tape from historic buildings at the Mayan site of Edzna, in Campeche, Mexico. Grey, brown, and black areas were colonised predominantly by coccoid and colonial cyanobacteria, also detected as endoliths. The major biomass on the pink stone surface was Trentepohlia. At low power, surface micropits containing crustose-lichen-like black colonies were seen within these pink areas. These were apparently pure colonies of T. umbrina; no filamentous fungi were detected here. The black colour was produced by dense, amorphous pigment deposits in the colony centres. Insolation levels were high (400 μmphotons m⁻² s⁻¹) and the moisture content of the stone was 0.7%. The high carotenoid:chlorophyll-α ratio of Trentepohlia biofilms (3.9:1) is an adaptation protecting against environmental stress. Trentepohlia caused severe local erosion (pitting) and, when present as a more uniform biofilm, the well-known pink surface discoloration.     

Über die Entwicklung des marinen parasitischen PhycomycetenLagenisma coscinodisci (Lagenidiales)

This paper concentrates on the study of vegetative development and sexual reproduction inLagenisma coscinodisci Drebes. Before infecting a diatom cell (Coscinodiscus granii), the freshly released zoospores pass through two different cyst stages. The primary zoospores are kidney-shaped and laterally biflagellated. They form a primary cyst with a spiny cyst wall which is left by isomorphic secondary zoospores. The latter form a secondary cyst (sometimes perhaps repeatedly), which is smooth-walled. The secondary cyst germinates and infects a new diatom cell by means of an infecting tube which enters the cell through the gap between epi- and hypotheca and develops a new thallus. Sexual reproduction is induced by ageing of cultures. Two kinds of isomorphic, kidney-shaped, laterally biflagellated swarmers are produced as in zoosporogenesis. Female-determined swarmers settle down near the host cell and encyst with a more or less smooth wall (oogonia). Male-determined swarmers are obviously attracted by the oogonia and encyst close to them to form a more or less smooth-walled antheridium which drives a thin fertilization tube to the oogonium. During plasmogamy the oogonium develops a thick, short hypha. The cytoplasm with the two nuclei moves completely into this hypha, concentrates near the hyphal tip, and surrounds itself with a thick wall to become a resting spore (oospore) in which karyogamy takes place. The walls of cysts, tubes, hyphae and spores react positively with zinc-chlor-iodide. In the spines and walls of the primary cysts the network of 2 to 3 nm thick fibrils is more distinct than in the other walls.