DEVELOPMENT OF PROTOPLASTS FROM HOLDFASTS AND VEGETATIVE THALLI OF MONOSTROMA LATISSIMUM (CHLOROPHYTA, MONOSTROMATACAE) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock and subsequently induce them to form algal filaments to act as an algal "seed" stock. Protoplasts of the alga were isolated enzymatically with 4% cellulase Onozuka R-10 and 2% Macerozyme R-10. The highest number of protoplasts was obtained on a 50-rpm shaker with 1.2 M of sorbitol after 6 h of incubation, with a yield of 9 × 106 protoplasts·g−1 of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments, and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May, about 1% of protoplasts isolated from vegetative thalli developed directly to form tubular thalli, and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, whereas protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than 3 years at 24° C under the low irradiance of 66μmol photons·m−2·s−1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons·m−2·s−1 at 18°–30° C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons·m−2·s−1 and at 18°–22° C. Therefore, the filaments could serve as"seed" stock for algal mass culture.     

DEVELOPMENT OF PROTOPLASTS OF ULVA FASCIATA (CHLOROPHYTA) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate protoplasts of the green alga Ulva fasciata Delile and subsequently induce them to form a microthallus suspension for algal seed stock. The protoplasts were covered with secreted mucilage following 6 h of culture when viewed with SEM. The mucilage fused to form thick layers during day 1 of culture. Microfibrillar cell walls were deposited into the thick layers of mucilage on the 5th day of culture. An average of about 10% of the freshly isolated protoplasts began to divide at 6–14 days. These protoplasts subsequently developed varied morphologies, depending on the time of collection during the year. Protoplasts isolated from U. fasciata collected in March to June developed frond thalli or microthalli when they were cultured in low or high densities (cells/area), respectively. The microthallus suspension was cultured for more than two years at 10–40 μ mol·m^{− 2} ·s^{− 1} . Frond thalli formed when the suspension was cultivated at 100–160 μ mol·m^{− 2} ·s^{− 1} . Therefore, microthallus suspension can serve as a seed stock of U. fasciata .     

GAMETOGENESIS AND GAMETE RELEASE OF ULVA MUTABILIS AND ULVA LACTUCA (CHLOROPHYTA): REGULATORY EFFECTS AND CHEMICAL CHARACTERIZATION OF THE “SWARMING INHIBITOR”1

Gametophytes of Ulva mutabilis Føyn and Ulva lactuca L. were artificially induced to form gametangia by removal of sporulation inhibitors. After this treatment, U. mutabilis gametes were ready for swarming on the third morning after induction, while U. lactuca gametangia needed 1–2 d longer for maturation. Release of gametes of U. lactuca was dependent solely upon exposure to the first light in the morning. Gametangia of U. mutabilis, however, also required sufficient dilution of the swarming inhibitor (SWI). SWI was excreted transiently by both Ulva species early during gametogenesis. While the SWI concentration in U. mutabilis medium remained above the inhibitory concentration until the gametangia were mature, the concentration of U. lactuca‐SWI dropped rapidly below this level. In the presence of sufficient SWI, mature gametes of U. mutabilis remained motionless within the gametangia despite light and open exit pores. However, using SEM, an additional seal was detected within these pores, which probably prevented premature swarming until dilution of SWI and exposure to light. Observations by time lapse microscopy and experiments with the myosin kinase inhibitor BDM suggest that the gametes may be either extruded by the gametangium or leave the exit pore by active gliding motion, driven by a myosin‐like motor protein. The SWIs were purified from both Ulva species, and mass spectral analysis showed their molecular masses (292 Da) were identical.     

“INTERNAL THECAE” OF EUNOTIA SOLEIROLII (BACILLARIOPHYCEAE): DEVELOPMENT,STRUCTURE AND FUNCTION AS RESTING SPORES1

The “double thecae” or “internal septa” of Eunotia soleirolii (Kütz.) Rabenh, are shown to represent the thecae of resting spores, as characterized by their physiology, as well as morphology. They differ from all resting spores of centric diatoms by the formation of both their valves as a result of unequal cell divisions; and, from the majority of centric spores by the presence of several girdle bands in both their thecae. Spore formation can be induced by high or low pH, high temperature (24 C), and iron, silica, phosphate or nitrate deficiencies, whereas low temperatures defer it. Spores do not germinate directly, but dormancy can be removed by dark treatments (–2 to 15 C) for a minimum of 4–5 wk. Longer dark treatments result in higher germination rates. At 15 C, a minimum of 2 mo is required and 4 mo is better. Heat treatments (27–42 C) are ineffective, but may shorten the dormancy-breaking subsequent cold period. Instances of secondary dormancy, as well as relative dormancy, were observed. Germination usually occurs in the light between 2 and 21 C. An equal division of the spore is followed by unequal divisions of both new cells with only the two resulting large cells being viable. The experiences in the laboratory aided the discovery of stages of spore germination in nature.     

PHOTOADAPTATION AND THE “PACKAGE” EFFECT IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

In the marine unicellular chlorophyte, Dunaliella tertiolecta Butcher, the spectrally averaged m vivo absorption cross section, normalized to chlorophyll a (so-called a* values), vary two-fold in response to changes in growth irradiance. We used a kinetic approach to examine the specific factors which account for these changes in optical properties as cells photoadapt. Using Triton X-100 to solubilize membranes, we were able to differentiate between “package” effects and pigmentation effects. Our analyses suggest that 43–49% of the variability in a* is due to changes in pigmentation, whereas 51–57% is due to the “package” effect. Further analyses revealed that changes in cell sue did not significantly affect packaging, while thylakoid stacking and the transparency of thylakoid membranes were important factors. Our results suggest that thylakoid membrane protein/lipid ratios change during photoadaptation, and these changes influence the effective rate of light harvesting per unit chlorophyll a.     

AN “ENVIRO‐INFORMATIC” ASSESSMENT OF SAGINAW BAY (LAKE HURON,USA) PHYTOPLANKTON: DATA‐DRIVEN CHARACTERIZATION AND MODELING OF MICROCYSTIS (CYANOPHYTA)1

Phytoplankton and Microcystis aeruginosa (Kütz.) Kütz. biovolumes were characterized and modeled, respectively, with regard to hydrological and meteorological variables during zebra mussel invasion in Saginaw Bay (1990–1996). Total phytoplankton and Microcystis biomass within the inner bay were one and one‐half and six times greater, respectively, than those of the outer bay. Following mussel invasion, mean total biomass in the inner bay decreased 84% but then returned to its approximate initial value. Microcystis was not present in the bay during 1990 and 1991 and thereafter occurred at/in 52% of sample sites/dates with the greatest biomass occurring in 1994–1996 and within months having water temperatures >19°C. With an overall relative biomass of 0.03 ± 0.01 (mean + SE), Microcystis had, at best, a marginal impact upon holistic compositional dynamics. Dynamics of the centric diatom Cyclotella ocellata Pant. and large pennate diatoms dominated compositional dissimilarities both inter‐ and intra‐annually. The environmental variables that corresponded with phytoplankton distributions were similar for the inner and outer bays, and together identified physical forcing and biotic utilization of nutrients as determinants of system‐level biomass patterns. Nonparametric models explained 70%–85% of the variability in Microcystis biovolumes and identified maximal biomass to occur at total phosphorus (TP) concentrations ranging from 40 to 45 μg · L^?1. From isometric projections depicting modeled Microcystis/environmental interactions, a TP concentration of <30 μg · L^?1 was identified as a desirable contemporary “target” for management efforts to ameliorate bloom potentials throughout mussel‐impacted bay waters.     

PHYLOGENETIC AFFINITIES OF “CHANTRANSIA” STAGES IN MEMBERS OF THE BATRACHOSPERMALES AND THOREALES (RHODOPHYTA)1

This study evaluated the phylogenetic relationship among samples of “Chantransia” stage of the Batrachospermales and Thoreales from several regions of the world based on sequences of two genes—the plastid‐encoded RUBISCO LSU gene (rbcL) and the nuclear SSU ribosomal DNA gene (SSU rDNA). All sequences of “Chantransia macrospora” were shown to belong to Batrachospermum macrosporum based on both molecular markers, confirming evidence from previous studies. In contrast, nine species are now associated with “Chantransia pygmaea,” including seven species of the Batrachospermales and two of the Thoreales. Therefore, the presence of “C. macrospora” in a stream can be considered reliable evidence that it belongs to B. macrosporum, whereas the occurrence of “C. pygmaea” does not allow the recognition of any particular species, since it is associated with at least nine species. Affinities of “Chantransia” stages to particular taxa were congruent for 70.5% of the samples comparing the rbcL and SSU analyses, which were associated with the same or closely related species for both markers. Sequence divergences have been reported in the “Chantransia” stage in comparison to the respective gametophyte, and this matter deserves further attention.     

FLAGELLAR APPARATUS STRUCTURE IS SIMILAR BUT NOT IDENTICAL IN VOLVULINA STEINII,EUDORINA ELEGANS,AND PLEODORINA ILLINOISENSIS (CHLOROPHYTA): IMPLICATIONS FOR THE “VOLVOCINE EVOLUTIONARY LINEAGE”1

The colonial and multicellular members of the Volvocales can be arranged in order of increasing size and complexity as the “volvocine series.” This series is often assumed to reflect an evolutionary progression. The flagellar apparatuses of previously examined algae are not consistent with a simple lineage. The flagellar apparatuses of Astrephomene gubernaculifera Pocock, Gonium pectorale Müller, Platydorina caudata Kofoid, Volvox rousseletii G. S. West, and V. carteri f. weismannia (Powers) Iyengar differ from one another, and there is no apparent progression inflagellar apparatus features from the simple to complex colonial forms. We examined the flagellar apparatuses of Volvulina steinii Playfair, Eudorina elegans Ehr., and Pleodorina illinoisensis Kofoid and found them to be similar to one another. The basal bodies are connected by a distal fiber that is offset to the anti side of the cell. Two microtubular rootlets originate on the inside of the basal bodies and extend toward the syn side. The other two rootlets are oriented perpendicular to the first two and are anti-parallel to each other. A coarsely striated component underlies the four-membered rootlets and extends to the basal bodies. A proximal fiber complex connects the two basal bodies. This complex consists of a branched striated component on the cis side of each basal body. One part extends toward the anti side of the cell, while the other extends into a fibrous component that runs between basal bodies. An additional structure extends in the anti direction from the trans side of each basal body. A fibrous component extends past one basal body in all four species. This component goes past the trans basal body in Volvulina steinii and the cis basal body in E. elegans and P. illinoisensis. The flagellar apparatuses of these organisms are similar to those of G. pectorale and Volvox carteri but different from the other colonial volvocalean algae examined. The algae examined in this study plus G. pectorale and V. carteri probably share a common evolutionary history that postdates the transition from the unicellular to colonial habit. Such a shared evolutionary history is a requirement of the volvocine hypothesis. However, we have not observed progressive changes in the flagellar apparatus correlated with increasing cell number, differentiation, and sexual specialization. Thus, it is possible, but not certain, that G. pectorale, Volvulina steinii, E. elegans, P. illinoisensis, and Volvox carteri may form part of a volvocine lineage.     

REMARKABLE CONSERVATION OF INTERNALLY TRANSCRIBED SPACER SEQUENCES OF ARTHROSPIRA (“SPIRULINA” ) (CYANOPHYCEAE,CYANOBACTERIA) STRAINS FROM FOUR CONTINENTS AND OF RECENT AND 30‐YEAR‐OLD DRIED SAMPLES FROM AFRICA1

The internally transcribed spacer (ITS) sequences of 21 Arthrospira clonal strains from four continents and assigned to four different species (A. platensis, A. maxima, A. fusiformis, A. indica) in the culture collections were determined. Two main clusters, I and II, were differentiated by 49 positions out of 475 nt or 477 nt, respectively. Each cluster was further subdivided into two subclusters. Subclusters I.A and I.B were separated by two substitutions, whereas subclusters II.A and II.B were distinguished by four substitutions. After direct sequencing of the PCR products, three dried samples from Chad aged between 3 months and 35 years yielded a sequence belonging to subcluster I.A, as did a recent commercial product. The strains grown in production plants belonged to the same (sub)clusters as strains from culture collections, mainly I.A and II. PCR primers specific for each cluster and subcluster were designed and tested with crude cell lysates of Arthrospira strains. One dried sample (“dihé” 1) and a herbarium sample from Lake Sonachi (Kenya) only contained I.A sequences, whereas the commercial product was a mixture of the four genotypes and the other two dried samples contained minor polymorphisms characteristic of different clusters. Five clonal Arthrospira strains, thought to be duplicates, showed the simultaneous presence of the two forms of the four diagnostic positions that distinguish subclusters genotype II.A and genotype II.B. This is likely to be caused by multiple copies of the rDNA operon, in a intermediate stage of homogenization between subcluster II.A and subcluster II.B. The high conservation of ITS sequences is in contrast with the assignment to four different species, the great morphological variability of the strains, and their wide geographic distribution.     

SYMBIODINIUM NATANS SP. NOV.: A “FREE‐LIVING” DINOFLAGELLATE FROM TENERIFE (NORTHEAST‐ATLANTIC OCEAN)1

We examined a free‐living Symbiodinium species by light and electron microscopy and nuclear‐encoded partial LSU rDNA sequence data. The strain was isolated from a net plankton sample collected in near‐shore waters at Tenerife, the Canary Islands. Comparing the thecal plate tabulation of the free‐living Symbiodinium to that of S. microadriaticum Freud., it became clear that a few but significant differences could be noted. The isolate possessed two rather than three antapical plates, six rather than seven to eight postcingular plates, and finally four rather than five apical plates. The electron microscopic study also revealed the presence of an eyespot with brick‐shaped contents in the sulcal region and a narrow anterior plate with small knob‐like structures. Bayesian analysis revealed the free‐living Symbiodinium to be a member of the earliest diverging clade A. However, it did not group within subclade A_I (=temperate A) or any other subclades within clade A. Rather, it occupied an isolated position, and this was also supported by sequence divergence estimates. On the basis of comparative analysis of the thecal plate tabulation and the inferred phylogeny, we propose that the Symbiodinium isolate from Tenerife is a new species (viz. S. natans). To elucidate further the species diversity of Symbiodinium, particularly those inhabiting coral reefs, we suggest combining morphological features of the thecal plate pattern with gene sequence data. Indeed, future examination of motile stages originating from symbiont isolates will demonstrate if this proves a feasible way to identify and characterize additional species of Symbiodinium and thus match ribotypes or clusters of ribotypes to species.     

APPLICATION OF PHYLOGENETIC PRINCIPLES TO TESTING EVOLUTIONARY SCENARIOS: A COMMENT ON KACZMARSKA ET AL. “MOLECULAR PHYLOGENY OF SELECTED MEMBERS OF THE ORDER THALASSIOSIRALES (BACILLARIOPHYTA) AND EVOLUTION OF THE FULTOPORTULA”1

While rigorous techniques have usually been used to generate phylogenetic trees from molecular data, morphological analysis has sometimes been more informal. A recent example was a study of the evolution of the fultoportula in the diatom order Thalassiosirales ( Kaczmarska et al. 2006 ). Phylogeny was inferred using modern phylogenetic principles applied to nuclear SSU rDNA sequences, but inferences about morphological character evolution were made using noncanonical reasoning and evolutionary scenario building. The preferred hypothesis posited that marginal fultoportulae evolved from the marginal ridge of Lithodesmiales. A related hypothesis suggested that fultoportulae in the valve center were not homologous with those near the valve margin. Shared symplesiomorphies, shared homoplasies, gaps in the fossil record, and subtle morphological differences between central‐ and marginal‐area fultoportulae were offered as the primary evidence for these scenarios. The literature has demonstrated such arguments to be either irrelevant or logically weaker than inferences made under the tests of similarity, conjunction, and congruence. Five prior hypotheses about the origin and evolution of the fultoportula were examined in this study using these tests. The hypothesis that the areola evolved into the multistrutted process, which evolved into the fultoportula, was best supported.