Morphological and molecular characterization of free-floating and attached green macroalgae Ulva spp. in the Yellow Sea of China

During the summer of 2008 and 2009, massive algal blooms repeatedly broke out in the Yellow Sea of China. These were undoubtedly caused by the accumulations of one or more species in the macroalgal genus Ulva. In previous reports, morphological observation indicated that the species involved in this phenomenon is Ulva prolifera but molecular analyses indicated that the species belongs to an Ulva linza–procera–prolifera (LPP) clade. Correct identification of the bloom species is required to understand and manage the blooms, but the taxonomic status of the bloom species remains unclear. In the current study, the taxonomic status of 22 selected specimens from the Yellow Sea was assessed by using both morphological and molecular (ITS and rbcL sequences) data. In addition, 5S rDNA analyses were performed for those samples clustering in the LPP clade, and phylogenetic tree and ribotype analyses were constructed for determining the possible origin of the bloom. Three free-floating and two attached Ulva species were distinguished and described: Ulva compressa Linnaeus and Ulva pertusa Kjellman were found in free-floating samples; U. linza Linnaeus was found on rocks; and U. prolifera O.F. Müller was found in both habitats. Diversity in free-floating Ulva of the Yellow Sea appears to be greater than previously thought. The dominant free-floating Ulva species, U. prolifera, was not closely related to local populations attached to rocks but was closely related to populations from Japan.     

Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area,China

In this paper, species compositions and seasonal variations of attached Ulva species on Porphyra aquaculture rafts and free floating Ulva species at Rudong coastal area, Jiangsu Province of China were investigated during 2010–2011. Based on the sequences analysis of nuclear-encoded ITS (including 5.8S rDNA regions) and 5S rDNA spacer regions, dominant species of both attached and free-floating Ulva samples were identified as Ulva compressa, Ulva linza, Ulva prolifera and Ulva flexuosa. Phylogenetic tree based on sequences of ITS and 5S rDNA spacer regions for attached and free-floating Ulva species was constructed, respectively. Species compositions of the Ulva population attached on aquaculture rafts varied with seasons, and U. prolifera was only found on aquaculture rafts in March 2011 during the 2010–2011 Porphyra yezoensis cultivation season, which had the same sequences of ITS and 5S rDNA spacer regions as that of the dominant species bloomed in the Yellow Sea of China in 2008. Dominant species of the free-floating Ulva population at the early stage of the green tide were U. compressa, U. flexuosa, and U. linza. Free-floating U. prolifera appeared in the middle of May, 2011. ITS sequence similarity rates of U. compressa and U. flexuosa between the attached and free-floating species were 100%. And ITS and 5S rDNA spacer sequences of the attached and the free-floating U. prolifera population also showed no differences. Further study showed that there were two types of free-floating U. prolifera population (Type 5S-A and Type 5S-B) based on 5S rDNA spacer sequences. The present study would provide some useful information for clarifying the outbreak mechanism of green tides occurred in the Yellow Sea, China.     

Tracing the origin of green macroalgal blooms based on the large scale spatio-temporal distribution of Ulva microscopic propagules and settled mature Ulva vegetative thalli in coastal regions of the Yellow Sea,China

From 2008 to 2016, massive floating green macroalgal blooms occurred annually during the summer months in the Yellow Sea. The original source of these blooms was traced based on the spatio-temporal distribution and species composition of Ulva microscopic propagules and settled Ulva vegetative thalli monthly from December 2012 to May 2013 in the Yellow Sea. High quantities of Ulva microscopic propagules in both the water column and sediments were found in the Pyropia aquaculture area along the Jiangsu coast before a green macroalgal bloom appeared in the Yellow Sea. The abundance of Ulva microscopic propagules was significantly lower in outer areas compared to in Pyropia aquaculture areas. A molecular phylogenetic analysis suggested that Ulva prolifera microscopic propagules were the dominant microscopic propagules present during the study period. The extremely low biomass of settled Ulva vegetative thalli along the coast indicated that somatic cells of settled Ulva vegetative thalli did not provide a propagule bank for the green macroalgal blooms in the Yellow Sea. The results of this study provide further supporting evidence that the floating green macroalgal blooms originate from green macroalgae attached to Pyropia aquaculture rafts along the Jiangsu coastline of the southern Yellow Sea.     

Discrimination of the common macroalgae (Ulva and Blidingia) in coastal waters of Yellow Sea,northern China,based on restriction fragment-length polymorphism (RFLP) analysis

Since 2007, reoccurring large-scale green algae blooms have caused deleterious effects to the estuarine ecosystem of Yellow Sea, northern China and subsequent economical losses. Previous surveys indicated the green tides were initiated in the coastal water of southern Jiangsu province where Porphyra farming was intensively conducted; however, the main ‘seed source’ of floating green algae is still under debate. Ulva prolifera was confirmed to be the major causative species of green tides. The multiple sympatric ulvoid species in the natural environment has complicated species identification in both field surveys and laboratory studies due to their morphological plasticity. Thus, we developed a genetic identification key based on restriction fragment length polymorphism (RFLP) analysis of the ITS nuclear marker to discriminate the common Ulva and Blidingia species in the Yellow Sea. Ten genetic lineages (1 in Blidingia, 9 in Ulva) were detected along the coast of China through phylogenetic analysis of ITS sequences. They can be separated by virtual restriction digestion using the four selected restriction enzymes (BspT107 I, EcoO109 I, Hin1 I and VpaK11B I). With additional PCR amplification of the 5S spacer region, we were able to discriminate U. prolifera from Ulva linza. Using this genetic key, we screened macroalgal samples collected from the coast of the Yellow Sea, and the results indicated 6 common lineages (U. prolifera, U. linza, Ulva compressa, Ulva pertusa, Clade 6 and Blidingia sp.) in this region, which could be explicitly distinguished by a single enzyme (BspT107 I) coupled with 5S spacer polymorphism. U. prolifera was confirmed to be present on the Porphyra aquaculture rafts with seasonal variation in the species composition. This genetic key will facilitate our long-term field surveys to investigate the origin of the floating U. prolifera and furthermore to explore its bloom dynamics along the coast of the Yellow Sea. It also provided a framework for the future inclusion of more Ulva species, which will expand the usage of this key.     

Molecular and morphological diversity of Narragansett Bay (RI,USA) Ulva (Ulvales,Chlorophyta) populations

Macroalgal bloom‐forming species occur in coastal systems worldwide. However, due to overlapping morphologies in some taxa, accurate taxonomic assessment and classification of these species can be quite challenging. We investigated the molecular and morphological characteristics of 153 specimens of bloom‐forming Ulva located in and around Narragansett Bay, RI, USA. We analyzed sequences of the nuclear internal transcribed spacer 1 region (ITS1) and the chloroplast‐encoded rbcL; based on the ITS1 data, we grouped the specimens into nine operational taxonomic units (OTUs). Eight of these OTUs have been previously reported to exist, while one is novel. Of the eight OTUs, all shared sequence identity with previously published sequences or differed by less than 1.5% sequence divergence for two molecular markers. Previously, 10 species names were reported for Ulva in Rhode Island (one blade and nine tube‐forming species) based upon morphological classification alone. Of our nine OTUs, three contained blade‐forming specimens (U. lactuca, U. compressa, U. rigida), one OTU had a blade with a tubular stipe, and six contained unbranched and/or branched tubular morphologies (one of these six, U. compressa, had both a blade and a tube morphology). While the three blade‐forming OTUs in Narragansett Bay can frequently be distinguished by careful observations of morphological characteristics, and spatial/temporal distribution, it is much more difficult to distinguish among the tube‐forming specimens based upon morphology or distribution alone. Our data support the molecular species concept for Ulva, and indicate that molecular‐based classifications of Ulva species are critical for proper species identification, and subsequent ecological assessment or mitigation of Ulva blooms.     

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.     

The dominant <Emphasis Type="Italic">Ulva</Emphasis> strain of the 2008 green algal bloom in the Yellow Sea was not detected in the coastal waters of Qingdao in the following winter

The region of Qingdao, China, experienced the world's largest green tide from May to July 2008. More than one million tons of fresh algal biomass of the green alga Ulva prolifera was harvested, while more was suspected to have sunk to the bottom. The original source of this seaweed was suspected to be from the south as revealed by satellite images. The floating biomass drifted with the water current northward and flourished in nearshore waters around Qingdao. However, direct biological evidence for “seed” source is lacking. It is still unclear whether this alga could survive the Qingdao local coastal environment and pose future danger of potential blooming. Systematic and seasonal sampling of waters in the intertidal zone at six collection sites along the Qingdao coast was conducted from December 2008 to April 2009. Forty-eight water samples were analyzed. From these, nine different morphotypes of Ulva were grown in the laboratory under standard temperature and light regimes. Growth of Ulva was observed in all water samples. However, molecular phylogenetic analyses revealed that the dominant U. prolifera strain of the 2008 bloom was absent in all the water-derived cultures during the sampling period. These results provide evidence that the dominant bloom-forming alga was unlikely able to survive the coastal waters (the minimal surface water temperature in February is 2°C) in winter conditions in Qingdao, even though all the sampling locations were heavily covered by this alga in June 2008.     

Molecular assessment of Ulva (Ulvales,Chlorophyta) diversity in Vietnam including the new species U. vietnamensis

Species diversity of Ulva in Vietnam was investigated using three commonly used genetic markers, the nuclear encoded rDNA ITS region and the plastid encoded rbcL and tufA genes. Single locus species delimitation methods, complemented with morphological and ecological information resulted in the delimitation of 19 species. This diversity is largely incongruent with the traditional understanding of Ulva diversity in Vietnam. Only four species identified in this study, U. lactuca, U. reticulata, U. spinulosa, and U. flexuosa, have been previously reported, and seven species, U. ohnoi, U. tepida, U. chaugulii, U. kraftiorum, U. meridionalis, U. limnetica, and U. aragoënsis, are recorded for the first time from Vietnam. Seven genetic clusters could not be associated with species names with certainty. A new species, U. vietnamensis, is described from marine to brackish coastal areas from southern Vietnam based on its morphological and molecular distinctiveness from the currently known Ulva species. A comparison with recent molecular-based studies of Ulva diversity showed that species composition in Vietnam is similar to that of adjacent countries, including Japan, China, as well as Australia. Our study emphasizes the importance of molecular data in the assessment of Ulva diversity, and indicates that a lot of diversity may still remain to be discovered, especially in tropical regions.     

Molecular analysis of green-tide-forming macroalgae in the Yellow Sea

In the summer of 2008, free-floating green algae bloomed in the Yellow Sea. Samples were collected in a wide area (119°32′-122°00′E, 32°25′-36°49′N). We calculated the sequence divergences of nuclear ITS, chloroplast rbcL, and psbA data of free-floating samples collected from the Yellow Sea and Ulvaceae from Europe and Japan. In the ITS sequence, 19 out of the 21 Yellow Sea samples of 2008 were identical to those of a sample taken at Qingdao in 2007. A low divergence (0.2%) was found in remaining two samples. Similar evidence was shown by pairwise distances of rbcL and psbA gene sequence data, implying the uniformity of the Yellow Sea blooms in 2007 and 2008. The ITS sequence of the Yellow Sea samples differed 8.1-10.8% from free-floating Enteromorpha or Ulva reported worldwide. ITS-based molecular phylogenetic results and rbcL sequence data grouped the free-floating alga in the Yellow Sea into one clade with Enteromorpha procera, Enteromorpha linza and Enteromorpha prolifera. Furthermore, both morphological characteristics and ribotype network of the ITS sequences imply that the blooming algae in 2007 and 2008 were E. prolifera. The haplotypes of the Yellow Sea free-floating E. prolifera are closely related to those from the Japanese coast but less to European and American algae.     

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.     

A Molecular Survey of Ulva (Chlorophyta) in Temperate Australia Reveals Enhanced Levels of Cosmopolitanism

The green algal genus Ulva includes a speciose group of marine macroalgae inhabiting shallow seas worldwide. Although algal blooms in Asia highlight the opportunistic nature of several “nuisance” species, recent research clearly reveals important positive benefits of Ulva. Applied research requires accurate, reliable, and rapid identification, however, identification of Ulva spp. has met with con‐siderable difficulty. Consequently, many have turned to molecular markers to aid in taxonomy. Previous studies of plants and algae have relied heavily on ITS and rbcL. Recently, tufA has been presented as a suitable barcoding gene to facilitate species‐level identification of green macroalgae and it is used here to explore the diversity of Ulva spp. in temperate Australia. Ninety Ulva specimens collected from 38 sites across five states were sequenced for this gene region with exemplars from each genetic group also sequenced for rbcL to test for congruence. Collections of Australian Ulva spp. were compared to samples from Asia and North America and exhibited trends consistent with recent studies in terms of species relationships. Results support an overwhelmingly cosmopolitan flora in temperate Australia that contrasts with other Australasian surveys of Ulva that report a greater number of endemics and new species. Four new records, as well as numerous range extensions for taxa already known from the country, are documented. Evidence for three nonindigenous Ulva species in temperate Australia is discussed.     

Examination of prezygotic and postzygotic isolating barriers in tropical Ulva (Ulvophyceae,Chlorophyta): evidence for ongoing speciation

Phylogenetic clades based on DNA sequences such as the chloroplast rbcL gene and the nuclear ITS region are frequently used to delimit algal species. However, these molecular markers cannot accurately delimit boundaries among some Ulva species. Although Ulva reticulata and Ulva ohnoi occasionally bloom in tropical to warm‐temperate regions and are clearly distinguishable by their reticulate or plain blade morphology, they have few or no sequence divergences in these molecular markers and form a monophyletic clade. In this study, to clarify the speciation and species delimitation in the U. reticulata‐ohnoi complex clade, reproductive relationships among several sexual strains from the Philippines and Japan including offspring that originated from the type specimen of U. ohnoi were examined by culturing and hybridization in addition to the ITS‐based analysis. As a result, both prezygotic and postzygotic reproductive isolation were revealed to occur between genetically perforated U. reticulata and imperforate U. ohnoi. They were also separated on the basis of sequence analysis of the ITS region. That strongly supports that the two taxa are independent biological species. Although no prezygotic barrier among the Philippine and Japanese strains of U. reticulata was observed, unexpectedly zoospores produced by hybrid sporophytes in some of their combinations mostly failed to develop, indicating partial formation of a postzygotic barrier despite a 0.2% divergence in the ITS sequence. These findings suggest speciation is still ongoing in U. reticulata.     

Molecular phylogenetic analyses of Ulva (Chlorophyta, Ulvophyceae) mats in the Xiangshan Bay of China using high-resolution DNA markers

Intertidal Ulva mats occur annually in winter and spring in the Xiangshan Bay (29°26′–29°34′ N, 121°27′–121°50′ E) of China. Thousands of tons of Ulva biomass have been harvested as edible seaweeds for human consumption for several decades in this region. This investigation was designed to quantify Ulva microscopic propagules associated with the mat, identify species composition, and to analyze intra-species relationships using three molecular markers. Phylogenetic analysis based on the nuclear encoded rDNA internal transcribed spacer region, the plastid encoded large subunit of the ribulose 1,5-bisphosphate carboxylase gene, and the 5S rDNA spacer region showed that the mat was principally composed of Ulva prolifera and Ulva flexuosa. Their propagules were detected in both the water column and sediment. Based on phylogenetic analyses of the 5S rDNA spacer region, mat samples of U. prolifera and U. flexuosa were genetically distinct from the green tide samples in the Yellow Sea and U. flexuosa samples from Jiangsu coasts, respectively, revealing that isolated geographical position of the Xiangshan Bay might result in the maintenance of a distinct Ulva population. The results demonstrate that high-resolution DNA markers have great potential in identification and discrimination at and below the species level.     

Abundance and distribution of Ulva microscopic propagules associated with a green tide in the southern coast of the Yellow Sea

The presence of Ulva microscopic propagules may play an important role in the rapid development of high-biomass blooms of green algae in the Yellow Sea. Six cruises were conducted, to determine the abundance and distribution of Ulva microscopic propagules associated with a green tide that developed in the southern coastal waters of the Yellow Sea from April to August, 2012. Results indicated that Ulva microscopic propagules were widespread in these waters, with the highest density being up to 4800 ind. L⁻¹, prior to the appearance of the green tide in April. High densities were also widely distributed along the coast during May and June, after the appearance of the floating green tide. The quantity of Ulva microscopic propagules significantly decreased when the floating green tide declined in July, reaching densities of up to 162 ind. L⁻¹, following the disappearance of the floating green tide in August. Quantitative studies on the distribution patterns of Ulva microscopic propagules along the southern coast of the Yellow Sea indicated a significant correlation between density and salinity, turbidity and nutrient concentrations. Temporal and geographical distribution patterns of Ulva microscopic propagules were also significantly affected by the presence of a large biomass of attached, or floating, Ulva species algae.     

Long-term trend of Ulva prolifera blooms in the western Yellow Sea

Blooms of the green macroalga Ulva prolifera in the western Yellow Sea occurred every year since 2008, and they have been reported and studied extensively using a variety of means including remote sensing. However, to date, long-term bloom patterns have not been reported except for a few case studies showing examples in different years. Here, using MODIS observations and an objective method to perform statistical analysis, mean Ulva coverage in the western Yellow Sea has been derived and analyzed between 2007 and 2015 at both monthly and annual scales. On annual scale, mean Ulva coverage decreased after 2008, but increased rapidly after 2012 from 8 km² in 2012 to 116 km² in 2015 (the largest ever reported in history for this region). In the month of June the mean coverage increased from 18 km² in 2012 to 363 km² in 2015. Other than 2009 and 2010, the month of June showed maximum Ulva coverage in every year. These coverage estimates are significantly lower than previously reported values as they represent “pure” algae coverage after taking into account of partial pixel coverage. Several environmental factors were examined in an attempt to determine the reasons behind such long-term changes, yet the results are inconclusive, suggesting a strong necessity of further coordinated and multi-disciplinary researches.     

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.     

Timing of disturbance,top‐down,and bottom‐up driving on early algal succession patterns in a tropical intertidal community

Herbivory and nutrient enrichment are major drivers of the dynamics of algal communities. However, their effects on algal abundance are under the influence of seasons. This study investigated the effects of herbivory and nutrient enrichment on early algal succession patterns using cages (uncaged and fully caged treatments) and two nutrient levels (ambient and enriched concentrations). To determine seasonal influences, experiment plots on dead coral patches were cleared during both dry and rainy season. Of the 17 algal species recruited in the experiment plots, three were dominant: Ulva paradoxa C. Agardh, Padina in the Vaughaniella stage, and Polysiphonia sphaerocarpa Børgesen. In this succession process, U. paradoxa was the earliest colonizer and occupied the cleared plots within the first month after clearing with the highest percentage of 83.33 ± 1.67% to 88.33 ± 9.28%. Then, it was replaced by the late successional algae, Padina in the Vaughaniella stage, and P. sphaerocarpa. The effects of herbivory and nutrient enrichment on algal abundance varied across algal functional groups and seasons. During the dry season, neither herbivory nor nutrient enrichment affected Ulva cover but during the rainy season, Ulva cover was influenced by nutrient enrichment. However, the abundance of algae in this early stage was not apparently affected by either herbivory or nutrient enrichment. Our results indicated that the timing of disturbance strongly influenced the algal abundance and successional patterns in this tropical intertidal community.     

Genetic variation of <Emphasis Type="Italic">Ulva (Enteromorpha) prolifera</Emphasis> (Ulvales,Chlorophyta)—the causative species of the green tides in the Yellow Sea,China

Ulva (Enteromorpha) prolifera, widely distributed from the intertidal to the upper subtidal zones around the world, was the dominant species of the massive green tides in the Yellow Sea in the summers of 2007, 2008, and 2009. However, little is known about its intra-species genetic diversity. In this study, six attached and seven floating U. prolifera samples collected from different sites distributed from the north of the Yellow Sea to the south of the East China Sea were taken in inter-simple sequence repeat (ISSR) analysis. Based on the results of the 90 polymorphic bands from four ISSR primers, the genetic diversity level of the floating samples (H = 0.1663, I = 0.2608) was found to be lower than that of the attached samples (H = 0.2105, I = 0.3346). Unweighted pair-group mean analysis (UPGMA) and principal coordinate analysis (PCoA) suggested that floating U. prolifera samples in the Yellow Sea from 2007 to 2009 had a close genetic relationship, and the floating samples were separated from the attached samples. Genetic differentiation and limited gene flow among attached U. prolifera populations were indicated by analysis of molecular variance.