Comparing the low‐salinity tolerance of Ulva species distributed in different environments

The green macroalgal genus Ulva (Ulvales, Ulvophyceae, Chlorophyta) is distributed worldwide from marine to freshwater environments. Comparative analyses of hyposalinity tolerance among marine, brackish, and freshwater Ulva species were performed by fluorescein diacetate viability counts. The subtidal marine species Ulva sp., collected from a depth of 30 m, showed the poorest tolerance to low salinity. This species died in 5 practical salinity units (PSU) artificial seawater or freshwater within 1 day. Its closely related species U. linza L. (an intertidal species) and U. prolifera Müller (a brackish species) showed varying tolerances to low salinity. After 7 days of freshwater exposure, the viability of U. linza L. decreased to approximately 20%, while U. prolifera Müller showed nearly 100% viability. The freshwater species U. limnetica Ichihara et Shimada, not yet found in coastal areas, was highly viable in seawater.     

Ulva flexuosa (Ulvaceae,Chlorophyta) inhabiting inland aquatic ecosystems: molecular,morphological and ecological discrimination of subspecies

Species of Ulva (Ulvaceae, Chlorophyta) are relatively common macroalgae in most coastal areas. In this paper, we examine Ulva in inland aquatic ecosystems to determine relationships between the chemical parameters of the water and the Ulva taxa present. Two groups of samples of freshwater Ulva were selected from: (i) habitats with high salinity levels, and (ii) habitats with fresh or slightly saline waters. We showed that these two groups of Ulva, which occupy distinct habitats, represent two separate taxa. The first group represents Ulva flexuosa subsp. pilifera (Kützing) M.J. Wynne, which is very common and regularly encountered in fresh waters, but is equally well represented in slightly saline waters. The second is U. flexuosa subsp. paradoxa (C. Agardh) M.J. Wynne, which is less common, preferring mainly salt-wedge saline waters. We did not find both Ulva flexuosa subspecies together at any of the research sites.

There were significant morphological differences between the Ulva flexuosa subspecies. Ulva flexuosa subsp. pilifera had longer and wider thalli and wider cells than those of subsp. paradoxa. Ulva flexuosa subsp. paradoxa cells were longer and of greater circumference than those of subsp. pilifera. Analyses of the rRNA ITS region and partial rbcL gene sequences demonstrated minor sequence variation within the two subspecies. The results of this study provide detailed information on the characteristics of limnic Ulva habitats and indicate the niches where such populations are likely to occur.     

Freshwater population of Ulva flexuosa (Ulvaceae,Chlorophyta) as a food source for great pond snail: Lymnaea stagnalis (Mollusca,Lymnaeidae)

Cosmopolitan species of the genus Ulva (Ulvaceae; Chlorophyta) that populate the littoral zone of marine habitats constitute a staple diet for a variety of organisms, particularly snails, shellfish, polychaetes, and birds. Occurrence of Ulva species (e.g., U. flexuosa and U . prolifera) has also been observed in freshwater inland ecosystems that have no contact with saline water. However, the influence of the development of macroalgal mats of Ulva on indigenous organisms in limnic ecosystems has not been established. This study investigates the trophic relationships between Ulva flexuosa and one species of snail from freshwater habitats in central Europe. During the summer, the great pond snail (Lymnaea stagnalis) consumed Ulva as a source of nutrition even when other algae and plants were available. Lymnaea stagnalis consumed an average of 100 mg of Ulva thalli per day. This level of biomass exceeded the consumption of an alternative food source, the shoots of Elodea canadensis. Ulva thalli are more actively consumed by great pond snails than Elodea shoots, and this is expressed in terms of the differences of biomass consumption. It was also observed that the interior of the monostromatic tubular thalli of Ulva flexuosa serves as a protective shelter for juvenile great pond snails.     

Examination of Ulva bloom species richness and relative abundance reveals two cryptically co-occurring bloom species in Narragansett Bay,Rhode Island

Blooms caused by the green macroalga Ulva pose a serious threat to coastal ecosystems around the world. Despite numerous studies of the causes and consequences of these blooms, we still have a limited understanding of Ulva bloom species richness and abundance due to difficulties in identifying Ulva species using morphological features. Along the northeastern U.S. coastline, all blooms of distromatic Ulva blades were previously identified as Ulva lactuca. Recent molecular sequencing, however, discovered the presence of additional distromatic Ulva species. Therefore, in order to determine the relative abundance of Ulva species within blooms, we conducted monthly surveys at four Narragansett Bay, RI, sites representing a gradient of bloom severity. We found that the biomass of Ulva within blooms was a mix of Ulva compressa and Ulva rigida, not U. lactuca as previously reported. In contrast, sites not impacted by blooms that were located near the mouth of Narragansett Bay were dominated by U. lactuca. We also observed spatial and temporal differences in Ulva and total macroalgal diversity between bloom-impacted sites, indicating that Ulva bloom composition can be radically different between similar sites within close proximity. We discuss our results in the context of Ulva blooms worldwide, highlighting the need to definitively determine bloom species composition in order to fully understand bloom dynamics.     

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.     

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.     

Effect of Marine Bacterial Isolates on the Growth and Morphology of Axenic Plantlets of the Green Alga Ulva linza

The green marine macroalga, Ulva linza, adopts an “atypical” form when grown in the absence of bacteria. Twenty unique strains of periphytic bacteria, isolated from three species of Ulva, were identified by 16S rDNA sequencing. These isolates were assessed for their effect on the growth and morphological development of axenic plantlets of U. linza. Results showed that the effect of bacterial strains was strain- but not taxon-specific. Thirteen isolates returned the aberrant morphology to normal and of these, five also significantly increased growth rate. One isolate increased growth, but had no effect on morphology. Biofilms of some of these isolates stimulated the settlement of Ulva zoospores but there was no correlation between bacterial isolates that stimulated zoospore settlement and those that initiated changes in morphology and/or growth of the cultured alga.     

cDNA cloning of a lectin-like gene preferentially expressed in freshwater from the macroalga Ulva limnetica (Ulvales, Chlorophyta)

The macroalgal species Ulva limnetica Ichihara et Shimada was investigated to understand the molecular mechanism of its tolerance or adaptation to freshwater. We detected a 19 kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which accumulated in greater amounts in freshwater conditions compared with marine conditions. The band was excised and the partial amino acid sequence was determined by Edoman degradation. Based on the sequences, we isolated the corresponding cDNA by the rapid amplification of cDNA ends (RACE) technique. The constructed, full-length cDNA was 1272 bp in length, consisting of 198 bp 5'-non-coding region, an open reading frame of 840 bp (279 amino acids), 233 bp 3'-non-coding region and poly (A) tail. The protein encoded by the cDNA showed 30% identity and 45% similarity to lectin isolated from Ulva pertusa Kjellman, and we named this gene ULL (encoding Ulva limnetica lectin-like protein). Northern blot analysis demonstrated that the expression level of the ULL in the freshwater-cultured sample was higher than in the seawater-cultured sample.     

Molecular phylogenetic analyses of the Japanese Ulva and Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva

In order to elucidate the species composition of free‐floating Ulva that cause green tide in several bays in Japan, and to clarify the generic status of Ulva and Enteromorpha (Ulvales, Ulvophyceae), the nuclear encoded internal transcribed spacer (ITS) region including the 5.8S gene and the plastid encoded large subunit of ribulose‐1, 5‐bisphosphate carboxylase/ oxgenase (rbcL) gene sequences for 15 species were determined. Both ITS and rbcL analyses indicate that free‐floating Ulva samples are divided into four different lineages that correspond to Ulva lactuca Linnaeus, U. pertusa Kjellman, U. armoricana Dion etal. and U. fasciata Delile. These four species are distinguished by cell morphology including the arrangement of cells, the shape and size of cells and the position of chloroplasts. Molecular data also indicated that Ulva and Enteromorpha are not separated as respective monophyletic groups within a large monophyletic clade and congeneric as shown by previous molecular studies using the ITS sequences alone. This strongly suggests that these genera are congeneric and Enteromorpha should be reduced to the synonym of Ulva.     

SEASONAL VARIABILITY OF PHYSICOCHEMICAL AND RHEOLOGICAL PROPERTIES OF ULVAN IN TWO ULVA SPECIES (CHLOROPHYTA) FROM THE BRITTANY COAST1

The seasonal variability in the extraction yield, physicochemical characteristics, and rheological properties of ulvan from two Ulva species contributing to Brittany “green tides” has been studied. These seaweeds were collected in the water column for Ulva armoricana Dion, de Reviers et Coat and on hard substrata for Ulva rotundata Bliding. The maximum ulvan extraction efficiency was not related to the maximum ulvan content in the seaweeds, but with the active growth period of the seaweeds. Ulvan chemical structure, macromolecular characteristics, and rheological properties were affected by both species and seasons. The proportion of high‐molecular‐weight ulvan was the major factor positively correlated with the gelling properties. Characteristics of ulvan from U. rotundata subjected to tides were more affected by seasons than ulvan from U. armoricana living in a more constant environment. These results point to several useful recommendations concerning Ulva sp. biomass collected with regard to ulvan characteristics and uses.     

TEMPORAL VARIATION IN ISOMORPHIC PHASE AND SEX RATIOS OF A NATURAL POPULATION OF ULVA PERTUSA (CHLOROPHYTA)1

The green macroalga Ulva L. is well known as having an alternation of isomorphic biphases: gametophyte and sporophyte. However, an examination of the temporal alternation in phase dominance has not been carried out. By inducing reproduction of thallus samples in the laboratory, this study reports the temporal changes in the two phases and sex ratios in a natural population of Ulva pertusa Kjellm. in the Seto Inland Sea, Japan, over a period of >3 years. The results showed that a temporal alternation in phase dominance occurred after 11–20 months; seasonal changes in phase dominance were not observed in the Ulva population. The sex ratio of male and female gametophytes remained ～1:1 throughout the investigated period, although skewed sex ratios were observed in several collections.     

Bioaccumulation of alkaline soil metals (Ca,Mg) and heavy metals (Cd,Ni, Pb) patterns expressed by freshwater species of Ulva (Wielkopolska,Poland)

The paper includes results of the first study on the accumulation of selected metals in freshwater populations of Ulva taxa at 16 sites in Poland. The thalli examined contained very high concentrations of Ca and Mg, owing to well‐developed surface incrustations of carbonate. Among the heavy metals investigated, the most significant concentration in the thalli was Ni, whereas the lowest was Pb. The median concentrations of Ni and Cd in the freshwater Ulva thalli were significantly higher than those reported in the available literature for marine taxa of the same genus. Several statistically significant correlations were found when the concentrations of different metals in the thalli and the water were analysed (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Phylogeography of the genus <Emphasis Type="Italic">Ulva</Emphasis> (Ulvophyceae,Chlorophyta), with special reference to the Japanese freshwater and brackish taxa

The nuclear-encoded ITS and associated 5.8S rDNA regions were sequenced for 72 specimens of Ulva collected from 44 rivers across Japan, including U. prolifera Müller from the Shimanto River, Kochi Prefecture, as well as 26 samples originally identified as U. linza L. from 20 coastal marine areas. Sequence data revealed that the samples fall into six distinct clades: the U. flexuosa Wulfen clade (2 samples), the Ulva linza-procera-prolifera (LPP) complex clade (75 samples), Ulva sp. 1 clade (3 samples), Ulva sp. 2 clade (7 samples), Ulva sp. 3 clade (4 samples) and Ulva sp. 4 clade (7 samples). The LPP complex contained a mixture of 26 samples collected from seashores and 49 samples obtained from rivers, including U. prolifera from the Shimanto River, and GenBank data for U. linza and U. procera Ahlner. The samples of the LPP complex differed by only 0–7 substitutions (0–1.149%). Subsequent phylogeographic analyses of the LPP complex based on the 5S rDNA spacer region revealed the presence of two further groupings: a group including 22 strictly marine littoral U. linza samples and a U. prolifera group composed of a mixture of 4 marine samples and all 49 river samples. The monophyly of all river samples indicates that adaptation to low salinity might have occurred only once in the evolutionary history of the LPP complex.     

TRUE IDENTITY OF THE EUROPEAN FRESHWATER ULVA (CHLOROPHYTA,ULVOPHYCEAE) REVEALED BY A COMBINED MOLECULAR AND MORPHOLOGICAL APPROACH1

A set of 18 freshwater and morphologically similar marine samples of Ulva were collected from inland and coastal waters throughout Europe to assess their taxonomic identity and invasive potential. An additional 11 specimens were obtained from herbaria. The material was studied using a combination of classical morphological methods and molecular techniques; the latter included sequencing of the nuclear internal transcribed spacer (ITS) region (ITS1‐5.8S‐ITS2) and the chloroplast RUBISCO LSU (rbcL) gene and comparison of the ITS2 secondary structure predictions. Based on classical methods, all the specimens could be determined as U. flexuosa Wulfen and could be further divided into three groups matching three infraspecific taxa. This pattern was generally well supported by molecular phylogenetic analyses. All sequenced samples formed a monophyletic lineage within Ulva, showing a putative synapomorphy in the ITS2 secondary structure. The individual subspecies corresponded to phylogenetic clusters within this lineage. In freshwater habitats, the dominant taxon was U. flexuosa subsp. pilifera, but subsp. paradoxa was also occasionally recorded. In marine habitats, only U. flexuosa subsp. flexuosa and subsp. paradoxa were located. These findings support the view that U. flexuosa subsp. pilifera is primarily a freshwater alga that probably dominates in Europe. As confirmed by the study of herbarium specimens, U. flexuosa should be regarded as indigenous, although it has a tendency to form blooms under certain conditions. Besides clarifying the identity of prevailing European freshwater Ulva, the study provides novel data concerning the distribution and morphological plasticity within the U. flexuosa complex.     

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.     

Systematics of green algae resembling Ulva conglobata,with a description of Ulva adhaerens sp. nov. (Ulvales,Ulvophyceae)

A phylogenetic and morphological study of green algae resembling Ulva conglobata from Japan was undertaken, along with morphological observations of the original material of U. conglobata Kjellman. The samples resembling U. conglobata included five genetically distinct species: U. fasciata, U. pertusa, U. tanneri, Ulva sp. 1 and Ulva sp. 2. The discovery of marginal denticulations in some of the original material of U. conglobata, made it possible to distinguish those species without denticulations: U. pertusa, U. tanneri and Ulva sp. 2. The morphological characteristics of Ulva sp. 1 matched those of U. conglobata, but Ulva sp. 1 was not clearly identified as U. conglobata owing to the lack of DNA sequence data of the original material. Ulva sp. 2 had lobes adhering to each other by rhizoids. This morphological feature is stable in Ulva sp. 2 and unique among Ulva species. In conjunction with the molecular data, Ulva sp. 2 was described as a new species, U. adhaerens sp. nov. This species features rhizoidal extensions in regions other than the base and an elaborate arrangement of the extensions used for adhesion. It thereby expands our knowledge of the morphogenesis of the morphologically simple genus Ulva.