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.     

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.     

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.     

Growth characteristics and reproductive capability of green tide algae in Rudong coast, China

Since 2007, green tides have occurred along the coast of the Yellow Sea, China. The green tide extended to 50,000 km² (floating area) within 2–3 months and the calculated covering area was about 400 km² in 2010. These facts implied that the growth and reproduction of the dominant species Ulva prolifera were stimulated. We observed that 1 cm² blades (single layer) released 2.84–6.62?×?10⁶ spores or 1.14–2.65?×?10⁷ gametes and that 91.6–96.4 % of them germinated into younger seedlings. This means that, in theory, 1 g (fresh weight) of blades was able to produce about 2.8?×?10⁸–2.7?×?10⁹ new younger seedlings. From 2009 to 2011, the growth rate of green tide algae was measured in situ in enclosure experiments in Rudong coast, Jiangsu Province and the growth curve of the algae was divided into four phases: lag phase, accelerated phase, stationary phase, and decline phase. Usually, the average daily specific relative growth rate was about 23.2–23.6 % d^?1 for a whole growth period, and it reached up to 56.2 % d^?1 in the accelerated phase. Correspondingly, the morphology of green tide algae in enclosures also showed periodic variation as follows: blades presented new filamentous branches from old thallus in the lag phase, longer filamentous branches in the accelerated phase, tubular and cystic blades in the stationary phase, and folded blades in the decline stage. Those studies may be useful for understanding the green tide blooming mechanism.     

浒苔干粉末提取物对东海原甲藻和中肋骨条藻的克生作用

研究不同溶剂的浒苔干粉末组织提取液对两种赤潮藻—东海原甲藻和中肋骨条藻生长的克生效应。结果表明, 浒苔提取物中确实含有可以影响赤潮藻类生长的克生物质,克生作用具有较明显的浓度效应,添加浓度低时可能会表现为一定的促进生长的作用,添加浓度较高时表现为抑制作用,添加浓度越大,抑制作用越强,即“低促高抑”的特点,这与浒苔提取物对其他微藻的作用相似。其中,蒸馏水提取物对这两种赤潮藻的克生作用小于有机溶剂提取物,有机溶剂中,甲醇和乙酸乙酯提取物对这两种赤潮藻的克生效果最好,正己烷相对较差。根据相似相溶原理,可以初步推断最有效的克生物质应为具有相对较高的极性的有机物。两种赤潮藻对克生物质的敏感程度不同。东海原甲藻对克生物质的敏感性高于中肋骨条藻。蒸馏水、甲醇、乙酸乙酯、正己烷的浒苔干粉末提取物影响东海原甲藻生长的致死作用阈值浓度分别为5.00 g/L、0.50 g/L、0.50 g/L、0.63 g/L（相当于浒苔新鲜藻体浓度为22.00 g-wet/L、2.20 g-wet/L、2.20 g-wet/L、2.75 g-wet/L）;影响中肋骨条藻生长的致死作用阈值浓度则分别为20.00 g/L、1.25 g/L、1.25 g/L、2.50 g/L（相当于新鲜藻体浓度为88.00 g-wet/L、5.50 g-wet/L、5.50 g-wet/L、11.00 g-wet/L）。     

Comparative study on mitogenomes of green tide algae

Since 2007, the annual green tide disaster in the Yellow Sea has brought serious economic losses to China. There is no research on the genetic similarities of four constituent species of green tide algae at the genomic level. We previously determined the mitochondrial genomes of Ulva prolifera, Ulva linza and Ulva flexuosa. In the present work, the mitochondrial genome of another green tide (Ulva compressa) was sequenced and analyzed. With the length of 62,311 bp, it contained 29 encoding genes, 26 tRNAs and 10 open reading frames. By comparing these four mitochondrial genomes, we found that U. compressa was quite different from the other three types of Ulva species. However, there were similarities between U. prolifera and U. linza in the number, distribution and homology of open reading frames, evolutionary and codon variation of tRNA, evolutionary relationship and selection pressure of coding genes. Repetitive sequence analysis of simple sequence repeats, tandem repeat and forward repeats further supposed that they have evolved from the same origin. In addition, we directly analyzed gene homologies and translocation of four green tide algae by Mauve alignment. There were gene order rearrangements among them. With fast-evolving genomes, these four green algal mitochondria have both conservatism and variation, thus opening another window for the understanding of origin and evolution of Ulva.     

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.     

A Strategy for the Proliferation of Ulva prolifera,Main Causative Species of Green Tides,with Formation of Sporangia by Fragmentation

Ulva prolifera, a common green seaweed, is one of the causative species of green tides that occurred frequently along the shores of Qingdao in 2008 and had detrimental effects on the preparations for the 2008 Beijing Olympic Games sailing competition, since more than 30 percent of the area of the games was invaded. In view of the rapid accumulation of the vast biomass of floating U. prolifera in green tides, we investigated the formation of sporangia in disks of different diameters excised from U. prolifera, changes of the photosynthetic properties of cells during sporangia formation, and development of spores. The results suggested that disks less than 1.00 mm in diameter were optimal for the formation of sporangia, but there was a small amount of spore release in these. The highest percentage of area of spore release occurred in disks that were 2.50 mm in diameter. In contrast, sporangia were formed only at the cut edges of larger disks (3.00 mm, 3.50 mm, and 4.00 mm in diameter). Additionally, the majority of spores liberated from the disks appeared vigorous and developed successfully into new individuals. These results implied that fragments of the appropriate size from the U. prolifera thalli broken by a variety of factors via producing spores gave rise to the rapid proliferation of the seaweed under field conditions, which may be one of the most important factors to the rapid accumulation of the vast biomass of U. prolifera in the green tide that occurred in Qingdao, 2008.     

High salt stress in the upper part of floating mats of Ulva prolifera,a species that causes green tides,enhances non‐photochemical quenching

Salt stress is a major abiotic stress factor that can induce many adverse effects on photosynthetic organisms. Plants and algae have developed several mechanisms that help them respond to adverse environments. Non‐photochemical quenching (NPQ) is one of these mechanisms. The thalli of algae in the intertidal zone that are attached to rocks can be subjected to salt stress for a short period of time due to the rise and fall of the tide. Ulva prolifera causes green tides and can form floating mats when green tides occur and the upper part of the thalli is subjected to high salt stress for a long period of time. In this study, we compared the Ulva prolifera photosynthetic activities and NPQ kinetics when it is subjected to different salinities over various periods of time. Thalli exposed to a salinity of 90 for 4 d showed enhanced NPQ, and photosynthetic activities decreased from 60 min after exposure up to 4 d. This indicated that the induction of NPQ in Ulva prolifera under salt stress was closely related to the stressing extent and stressing time. The enhanced NPQ in the treated samples exposed for 4 d may explain why the upper layer of the floating mats formed by Ulva prolifera thalli were able to survive in the harsh environment. Further inhibitor experiments demonstrated that the enhanced NPQ was xanthophyll cycle and transthylakoid proton gradient‐dependent. However, photosystem II subunit S and light‐harvesting complex stress‐related protein didn't over accumulate and may not be responsible for the enhanced NPQ.     

Physiological functional traits explain morphological variation of Ulva prolifera during the drifting of green tides

Ulva prolifera green tides, one of the greatest marine ecological disasters, originate in the southern Yellow Sea of China and obtain the highest biomass in Haizhou Bay (latitude around 35° N) during northward drift. U. prolifera shows different morphologies from southern Haizhou Bay (SH) to northern Haizhou Bay (NH). Owing to the distinct nutrient environments between SH and NH, we hypothesized that thalli in NH with poor nutrients increased the surface area to volume ratio (SA:VOL) to better absorb nutrients. Here, we tested this hypothesis by comparing the SA:VOL of thalli in SH and NH. The results showed that the thalli in NH had a lower SA:VOL than those in SH, and SA:VOL had positive relationships with temperature and nutrients, contrary to the general hypothesis. The novel results suggested that morphological differences of U. prolifera were the result of developmental state rather than environmental acclimation. Indicators of reproduction (reproductive allocation ratio) were negatively related to variation in tissue contents of C, N, P, and crude protein, whereas indicators of growth (tissue contents of C, N, P, and crude protein) showed significant positive influences on SA:VOL. The results indicated that a trade‐off relationship between reproduction and growth existed in the northward drift. All the results suggested that physiological functional traits affected morphological variation of U. prolifera in different environmental conditions during the drifting of green tides. This study presents new insights into the opportunist species nature of U. prolifera through morphological variation and associated functional consequences.     

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.     

Spatiotemporal variations of inorganic nutrients along the Jiangsu coast,China, and the occurrence of macroalgal blooms (green tides) in the southern Yellow Sea

Large macroalgal blooms (i.e. green tides of Ulva prolifera) occurred in the southern Yellow Sea, China, yearly from 2007 to 2016. They were among the largest of such outbreaks around the world, and these blooms likely originated along the coast of the Jiangsu Province, China. Understanding the roles of nutrients in the onset of these macroalgal blooms is needed to identify their origin. This study analyzes the spatiotemporal variations in dissolved inorganic nitrogen and phosphorus (DIN and PO₄–P) and the N/P ratio along the Jiangsu coast from 1996 to 2014 during late-March to April, the months which corresponds to the pre-bloom period of green tides since 2007. A zone of high DIN and PO₄–P concentrations has developed along the Jiangsu coast, between the cities of Sheyang and Nantong, since 1996. There was an 18-year trend of increasing DIN concentrations during the pre-bloom period as well as a positive correlation between the U. prolifera biomass and DIN concentrations. Nutrient inputs from rivers and mariculture in the Jiangsu Province may have provided nitrogen that contributed the magnitude of macroalgal blooms that subsequently spread into the southern Yellow Sea.     

从藻类生理生态学角度解析绿潮爆发的内在机制

绿潮是在特殊环境条件下,海水中某些大型绿藻爆发性增殖或高度聚集而引发的海洋生态异常现象。自2007年起,黄海已连续15年爆发绿潮,对沿岸生态环境、社会发展、经济建设造成极其严重的危害,解决绿潮问题刻不容缓。绿潮的形成是外因和内因综合作用的结果。目前,国内外学者对绿潮爆发的外因,即环境因子已具有科学的认知,并围绕绿潮藻类对某些环境因子的生理响应展开系列研究,且取得了一定成果。然而,针对绿潮爆发的内因,即绿潮藻类为应对大幅波动的环境因子而产生的极强的生理生态适应性尚未进行深入探讨。本文从藻类生理生态学角度入手,综述绿潮成因种对多种生态因子波动的响应机制,包括光照强度、二氧化碳、温度、盐度与干出、营养盐、重金属及生物因素,归纳了绿潮藻类的光合生长生理、营养生理和繁殖生理对环境因子波动的适应性调节机制,揭示了绿潮藻类爆发的内因。     

Ulva diversity in the Yellow Sea during the large‐scale green algal blooms in 2008–2009

In the Yellow Sea of China, large‐scale green tides have broken out for three consecutive years from 2007 to 2009. As part of the efforts to localize the algal source, two cruises were conducted in the early stage and the outbreak stage of the bloom in 2009. We analyzed the morphological and genetic diversity of drifting Ulva specimens and culture‐derived isolates from seawater sampled in different localities. For phylogenetic analyses, the nuclear encoded ribosomal DNA internal transcribed spacer region (ITS nrDNA) and the plastid encoded large subunit of ribulose‐1, 5‐bisphosphate carboxylase/oxgenase gene (rbcL) were used. Our molecular and morphological data indicate that the dominant free‐floating Ulva species in 2008 and 2009 possibly belonged to a single strain of the U. linza‐procera‐prolifera (LPP) clade. The ITS sequences from bloom‐forming algal samples with dense branches were identical to those from U. linza‐like specimens without branches derived from the Yellow Sea. Microscopic individuals of the dominant Ulva strain were detected in eight stations, revealing that spore dispersal in the water helped to enlarge biomass in the water during the outbreak stage of green tide in the Yellow Sea.     

Photosynthetic activity and proteomic analysis highlights the utilization of atmospheric CO2 by Ulva prolifera (Chlorophyta) for rapid growth

Free‐floating Ulva prolifera is one of the causative species of green tides. When green tides occur, massive mats of floating U. prolifera thalli accumulate rapidly in surface waters with daily growth rates as high as 56%. The upper thalli of the mats experience environmental changes such as the change in carbon source, high salinity, and desiccation. In this study, the photosynthetic performances of PSI and PSII in U. prolifera thalli exposed to different atmospheric carbon dioxide (CO₂) levels were measured. Changes in photosynthesis within salinity treatments and dehydration under different CO₂ concentrations were also analyzed. The results showed that PSII activity was enhanced as CO₂ increased, suggesting that CO₂ assimilation was enhanced and U. prolifera thalli can utilize CO₂ in the atmosphere directly, even when under moderate stress. In addition, changes in the proteome of U. prolifera in response to salt stress were investigated. Stress‐tolerance proteins appeared to have an important role in the response to salinity stress, whereas the abundance of proteins related to metabolism showed no significant change under low salinity treatments. These findings may be one of the main reasons for the extremely high growth rate of free‐floating U. prolifera when green tides occur.     

Diluted seawater promoted the green tide of Ulva prolifera (Chlorophyta,Ulvales)

Ulva prolifera (Müller) J. Agardh is the main causative species of the 2008 Yellow Sea green tide incident. We investigated the influences of diluted seawater on the vegetative growth and reproductive cell formation of the alga. The thalli that were cultivated under low salinities (10‰ and 20‰) and low pH values (pH around 7.0) showed obvious and steady biomass gain, while those cultivated under high salinities (40‰) and relatively higher pH conditions (pH around 8.0) manifested significant biomass loss. The trend was, however, completely the opposite for reproductive cell formation and there were indications that enrichment, over very wide concentration ranges of both nitrogen and phosphate, could significantly promote vegetative growth. Results also indicated that relatively low salinity and low pH regimes boosted vegetative growth but were unfavorable for reproductive cell formation and vice versa. Based on these results, the possible origin and development mechanisms of the green tide event are discussed. Eutrophication in the Changjiang River estuary and adjacent sea areas, as well as extremely high freshwater inflows before, and during, the flood of 2007 – due to the full operation of large‐scale water facilities in the area – may have both played an important role in the formation and development of the green tide event.     

Changes in morphological plasticity of Ulva prolifera under different environmental conditions: A laboratory experiment

The large-scale green tides, consisting mainly of Ulva prolifera, have invaded the coastal zones of western Yellow Sea each year since 2008, resulting in tremendous impacts on the local environment and economy. A large number of studies have been conducted to investigate the physiological traits of U. prolifera to explain its dominance in the green tides. However, little has been reported regarding the response of U. prolifera to changing environmental factors via morphological variation. In our experiments, we found remarkable morphological acclimation of U. prolifera to various temperature (20 and 25 °C) and salinity (10, 20, and 30) conditions. U. prolifera had more, but shorter branches when they were cultured at lower temperature and salinity conditions. To investigate the significance of these morphological variations in its acclimation to changes of environmental factors, physiological and biochemical traits of U. prolifera grown under different conditions were measured. Higher temperature increased the relative growth rate while salinity did not affect it. On the other hand, higher temperature did not enhance the net photosynthetic rate whilst lower salinity did. The increased net photosynthetic rate at lower salinity conditions could be attributed to more photosynthetic pigments—chlorophyll a, chlorophyll b, and carotenoids—in thalli due to there being more branches at lower salinity conditions. Increased numbers of branches and thus an increased intensity of thalli may be helpful to protect thalli from increased osmotic pressure caused by lower salinity, but it led to more shading. In order to capture enough light when being shaded, thalli of U. prolifera synthesized more photosynthetic pigments at lower salinity levels. In addition, higher temperature increased nitrate reductase activity and soluble protein content but variations in salinity did not impose any effect on them. Our results demonstrate conclusively that U. prolifera can acclimatize in the laboratory to the changes of environmental factors (salinity and temperature) by morphology-driven physiological and biochemical variation. We suggest that the morphological plasticity of U. prolifera may be an important factor for it to outcompete other algal species in a changing ocean.