The green tide in Yingkou, China in summer 2021 was caused by a subtropical alga— Ulva meridionalis (Ulvophyceae,Chlorophyta)*

Xiaoqian LÜ , Hao XU , Sheng ZHAO , Fanzhou KONG , Tian YAN , Peng JIANG

1 CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China

2 Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao),Qingdao 266237, China

3 University of Chinese Academy of Sciences, Beijing 100049, China

4 North China Sea Environmental Monitoring Center, State Oceanic Administration, Qingdao 266033, China

5 CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences,Qingdao 266071, China

6 Laboratory for Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, China

Abstract The large-scale green tide caused by Ulva has occurred successively in the Yellow Sea since 2007, and new events of green tide also continued to appear in nearby sea areas, indicating an undergoing rapid development of occurrence patterns for harmful macroalgal blooms (HMBs) along coastal China. In August 2021, a green tide occurred for the f irst time in Bayuquan sea area of Yingkou city, Liaoning Province in the Bohai Sea. In this study, morphological and molecular approaches were used to identify the causative species as U. meridionalis, an alien subtropical alga previously found to dominate green tides in the South China Sea. According to the hydrological data of Bayuquan in summer 2021, combined with morphological and developmental observations for this alga, we hypothesized that the disturbance caused by the typhoon In-Fa might have detached the local U. meridionalis from substrates, especially for those thalli with poorly developed holdfasts, and the ensuing wave-free period with unusually high temperature, which fell in the reported optimum growth temperature for U. meridionalis, might have provided the favorable conditions for the f inal bloom of the f loating seaweeds. This is the f irst report on the bloom of subtropical U. meridionalis in the north temperate sea zone, indicating that the ecological risk of causing green tides in the future by this rapidly spreading species deserves high attention.

Keyword: Bohai Sea; green tide; holdfast; alien species; Ulva meridionalis; Yingkou

1 INTRODUCTION

Harmful macroalgal blooms (HMBs) refer to the overgrowth and accumulation of f loating macrophytes,which has occurred more and more frequently worldwide in recent decades (Smetacek and Zingone,2013). The green tide is one type of HMBs, mainly caused by green seaweeds fromUlva(Ulvophyceae,Chlorophyta) (Fletcher, 1996). These green algae are typical opportunistic seaweeds that can grow rapidly especially when nutrients and water temperature conditions are favourable. In addition to damage to the marine environment (Valiela et al., 1997), once the green tide occurs on the urban seashore, it will have a serious impact on aquaculture (Wang et al., 2011),tourism economy, and even daily life.

Fig.1 Sampling site in the coastline of Yingkou, North China

In August 2021, a green tide event occurred in the coastal waters of Bayuquan, Yingkou City, Liaoning Province, located in Liaodong Bay, Bohai Sea.According to the reports from the website of the local government management department, on August 9, it was f irst noticed that there was only a small amount of green seaweed gathering on the shore of Shanhai Square beach. However, from August 10 to 12, a large amount of algae suddenly appeared in the same area,f loating in the seawater or stranded on the sand beach.Based on the on-site aerial survey with unmanned aerial vehicle (UAV), this green tide was distributed along the coastal zone. The sea area covered by f loating green seaweeds was about 1 000 m long and 50 m wide, with a maximum coverage area of about 0.05 km3, mainly covering the intertidal zone. The local government management department urgently organized environmental investigation and sample analysis, and tentatively identif ied the causative species asU.proliferaO. F. Müller informally (Department of Natural Resources of Liaoning Provincial, 2021a).This green tide event lasted 15 days in total. On August 23, it was reported that this green tide had basically disappeared and the sea area returned to normal after timely cleaning the f loating algae in the seawater and those stranded on the beach (Department of Natural Resources of Liaoning Provincial, 2021b).

In recent years, coastal China has been widely threatened by green tides. The f irst report came from the Yellow Sea green tide in 2007 (Jiang et al., 2008),which occurred in successive years and ranked f irst on scale in the world (Liu et al., 2009). Genetic analysis showed that this event was dominated by a unique“f loating ecotype” ofU.prolifera, which is genetically diff erent from attached populations widespread in China (Zhao et al., 2013, 2015; Jiang and Zhao,2018), and later was identif ied asU.proliferasubsp.qingdaoensis(Cui et al., 2018). Moreover, it was suggested that the driftingU.proliferafrom the Yellow Sea may expand to the East China Sea, resulting in the green tides in the Gouqi Island since 2011 (Zhang et al., 2015). In the Bohai Sea,U.proliferahas also been reported to cause summer green tides since 2015 (Song et al., 2019a), but whether it belonged to the same “f loating ecotype” was unclear. For green tides in the South China Sea, three alien species,U.meridionalis,U.tepida, andU.chaugulii, were identif ied as causative species in 2018 (Xie et al.,2020). Since the f loating alga in Yingkou has been tentatively identif ied asU.prolifera, in addition to the local origin, there are at least two possible origins that need special attention: one is from the bloomingU.proliferain Qinhuangdao which might be driven east by a near typhoon In-Fa (National Meteorological Center, 2021); second, considering the north limit of drifts (Li et al., 2020), and possible main settlement region in previous years (Geng et al., 2019; Zhao et al., 2022), the driftingU.proliferafrom the Yellow Sea may also move further northward to the Bohai Sea as shown in Fig.1.

To determine the origin of this event, accurate identif ication on both species and intraspecies level is very necessary and the most priority. In this study,the blooming seaweeds in Yingkou were collected for molecular investigation with multi genetic markers,and detailed morphological characterization was also combined. These results provide basic data to resolve the process and mechanism for this event.

2 MATERIAL AND METHOD

2.1 Seaweeds collection and culture

During the blooming period of green tide in August 2021, all samples of f loating green seaweeds were collected in the coastal waters near Bayuquan of Yingkou city, Liaodong Bay, Bohai Sea (40°13ʹ13ʺN, 122°4ʹ37ʺE) (Fig.1), where there were large concentrations of blooming macrophytes.After algal samples were transported back to the laboratory in a container with ice packs, they were washed with sterilized seawater to eliminate any epiphytes and contaminants. Then, some intact, clean,and healthy individuals were selected for culture in separate Petri dishes with Von Stosch’s Enriched Medium (VSE medium) which was renewed once a week. All seaweeds samples were cultured at 16 °C with a 12-h꞉12-h light (L)꞉dark (D) photoperiod and illumination intensity of 75 μmol photons/(m2·s).

2.2 Molecular identif ication

To prepare genomic DNA templates for polymerase chain reaction (PCR) detections, approximately 5 mm2of thallus for each individual was cut off with a sterilized blade for DNA extraction, using a Plant Genomic DNA Extraction Kit (Tiangen Biotech Co. Ltd., Beijing,China) following the provided instruction. The PCR mixtures contained 10 μL of 2× Taq polymerase(Novoprotein Scientif ic Inc., Suzhou, China), 0.4 μL of each reverse and forward primer (10 μmol/L), 2 μL of genomic DNA, and 7.2 μL of ultrapure water, for a total volume of 20 μL. A total of f ive pairs of primers and their corresponding PCR prof iles, including ITS-a and ITS-d for internal transcribed spacer (ITS, including ITS1, 5.8S and ITS2) (Leskinen and Pamilo, 1997),rbcL-RH1 and rbcL-1385r forrbcL (Manhart, 1994),tufGF4 and tufAR fortufA (Saunders and Kucera,2010), 5S-F and 5S-R for 5S rDNA spacer (Shimada et al., 2008), and YSF-F and YSF-R for sequence characterized amplif ied region (SCAR) marker which was specif ic to the f loating ecotype ofU.proliferadominating the green tides in the Yellow Sea (Zhao et al., 2015), were used in this study.

All PCR products were visualized by gel electrophoresis in a 1.5% agarose gel stained with the dye Super GelRed (US Everbright Inc., Suzhou,China), and purif ied by a Gel Extraction Kit (200)(OMEGA Bio-Tek, Georgia, USA) following the manufacturer’s instructions. After commercial sequencing (Ruibio BioTech Co. Ltd., Qingdao,China), partial obtained representative sequences were uploaded to the GenBank database. The phylogenetic analysis was performed with the DNA sequences including those of samples and references from the GenBank, with those fromUlvariaorBlidingiaas outgroups. The maximum likelihood (ML)phylogenetic trees was constructed using MEGA 6.0(Tamura et al., 2013), with 1 000 bootstrap replicates.

2.3 Morphological characterization

Intact, clean, and healthy samples were selected to record the gross morphology of the specimens,including the shape, size, branches, and holdfast of the thalli. Photographs of the morphological features of the specimens were taken by a Canon PC2152 digital camera (Canon Inc., Tokyo, Japan).Morphologically typical and well-formed samples were selected and pressed on herbarium sheets for making specimens, and deposited in the Marine Biological Museum of Chinese Academy of Sciences(MBMCAS) at the Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China. Random sampling was used for statistical works to analyze the branching characteristics and the proportion of individual plants with holdfasts (n=100). According to the protocols described previously (Ma et al.,2020), two indexes of branching morphology, i.e.,the top branching order and the intensity of primary branches, were measured for statistical analysis(n=100).

A BH2 light microscope (Olympus Corp.,Tokyo, Japan) was employed for examination of microscopic characters of samples, such as the chloroplasts number and location, as well as the shape and arrangement of surface cells. The thalli were crosscut to observe the cell location in transverse view, or stained with Lugol’s iodine solution to observe the number of pyrenoids. All microscopic photographs of the specimens were taken by a CCD camera (Scope Tek MDC200,Mingshi, Ningbo, China) mounted on the microscope. In order to compare the diff erences or stability of some morphological features between the thalli from f ield collection and those from laboratory culture, some samples were selected to induce reproduction with a described method(Hiraoka and Enomoto, 1998). After the formation,release, and germination of germ cells, new plantlets were cultured in Petri dishes under the same conditions mentioned above.

2.4 Analysis of hydrological data from Bayuquan

The daily sea surface temperature (SST) and wave height data of Bayuquan sea area from July to September in 2020 and 2021 were collected from the website of the Municipal Government of Yingkou City (http://www.yingkou.gov.cn), and Microsoft Excel software (Microsoft Excel 2019) was used for data statistics and analysis.

Fig.2 Maximum likelihood (ML) phylogenetic tree based on DNA sequences of ITS

3 RESULT

3.1 Molecular identif ication

A total of 54 nucleotide sequences of ITS, including 24 samples from this investigation and 30 reference sequences downloaded from GenBank, were used to construct the ML tree for species identif ication and phylogenetic analysis. As shown in Fig.2, all sequences from samples were highly identical and gathered into a cluster which consisted of all reference sequences fromU.meridionalis, including all available sequences from samples outside China, and those from specimens that have caused green tides in the South China Sea in 2018 (Xie et al., 2020). Besides,it was found that all sample ITS sequences were highly identical (99.21%) to the short ITS2 sequence(AB598807) from the holotype ofU.meridionalis(Horimoto et al., 2011). On the contrary, all sample sequences were clearly in a diff erent clade from that ofU.prolifera, and the genetic distances between them was approximately 8.6%–9.0%. Similar results were shown in thetufA-ML tree which was constructed with 19 sample sequences and 20 references (Fig.3).Besides, an ML tree based onrbcL sequences was also constructed and similar topological structure was shown, in which there was a high identity between sequences from our sample and the holotype ofU.meridionalisfrom Japan (Supplementary Fig.S1).Since this causative species had been tentatively identif ied asU.prolifera, the PCR detection with 5S or SCAR were also performed, but no amplif ied products were obtained. Combined with all data of molecular identif ication with multiple markers, it can be determined that the single dominant species causing this green tide event wasU.meridionalisR.Horimoto & S. Shimada, rather thanU.prolifera.

3.2 Morphological characterization

Fig.3 Maximum likelihood (ML) phylogenetic tree based on DNA sequences of tuf A

Fig.4 Morphological and anatomical characteristics of samples

In order to provide additional evidence for species identif ication and detect the adaptive characteristics under the specif ic life mode of f loating, morphological and anatomical observation was carried out on samples. All samples appeared to beEnteromorphalike species that were tubular and monostromatic thalli. The length of thalli was 5–15 cm, and the breadth of main axis was approximately 1 mm. Two voucher specimens, MBM287241 (Fig.4a) and MBM287242,have been made from typical individuals, and deposited at the MBMCAS. Each thallus had an obvious branched main axis, but only primary branches were detected. The primary branches were roughly evenly distributed along the main axis, with a density of 0.47–2.90/cm (n=100). In freshly collected samples, the tubular algae were common with air bubbles inside, which may have made it easier to f loat during the blooming period in seawater (Fig.4b & c).It should be noted that according to observation and statistics, about 49% of the f iled-collected samples were found to have holdfasts (n=100), but they were generally poorly developed and very weak (Fig.4d &e). This distinct feature was observed repeatedly in the next generation propagated in the laboratory. It was shown that the released and germinated seedlings can develop holdfasts to grow on the bottom of Petri dishes (Fig.4f & g), but the development of holdfasts was so weak that even gently disturbs like renew of media would detach seedlings in bigger sizes from the bottom of Petri dishes (Fig.4h). On the surface view,transverse constrictions were obvious and common on the thalli (Fig.4i). For freshly collected samples, each thallus showed a single-tubular type in transverse view (Fig.4j). However, after a period of culture in the laboratory, along the main axis of thalli, the two opposite layers of cells near the central axis began to approach and bond with each other to form a vesicular structure on both sides (Fig.4k). Therefore, the cross section exhibited a bowknot-shaped double-tubular type (Fig.4l), which was reported highly specif ic toU.meridionalis(Xie et al., 2020).

The anatomical observation of the surface view showed that cells were polygonal with rounded corners, and irregularly arranged throughout the thalli. It is worth noting that for the f iled-collected samples, each cell contained a single chloroplast, and most of the chloroplasts were adherent to one side of the cell (Fig.4m). On the contrary, the chloroplasts were found to f ill the whole cells from the samples of generated seedlings (Fig.4n). The cross section of samples showed that the cell was located in the middle of thalli (Fig.4o). After staining with Lugol’s iodine, each cell was shown to possess 1–4 pyrenoids,and most cells only had 1–2 pyrenoid (one, 30.0%;two, 51.4%; three, 17.2%; four, 1.4%;n=70) (Fig.4p).

3.3 Analysis of hydrological data from Bayuquan

Fig.5 SST and wave height in Bayuquan from July to September in 2020 and 2021

As shown in Fig.5 that based on the data of Bayuquan sea area in 2020 and 2021, the SST generally increased f irst and then decreased from July to September, and obviously higher in 2021 than that in 2020. Every year, there was a high temperature period between about July 15 and August 25, during which the average SST in 2020 was 26.1 ℃ and the highest temperature was 27.4 ℃, while the average SST in 2021 was 29.0 ℃ and the highest temperature was 30.4 ℃, indicating that the warming range and the speed of SST increase and decrease in 2021 were signif icantly higher than that in 2020. During July–September 2021, the dates with higher waves were mainly in July and September, while in August there was a 16-day long period of low waves, accompanied by high temperatures. It should be noted that there were two peaks of wave heights in July, which were formed on July 12 by the Bohai low pressure and July 30 by the typhoon In-Fa respectively (http://www.yingkou.gov.cn). On the contrary, in 2020 the dates with higher waves were mostly in August and September, almost aff ecting the whole hightemperature period.

4 DISCUSSION

Based on results of molecular identif ication with multiple markers, it was shown that the sequences of samples from the green tide of Yingkou were highly identical to that of the holotype ofU.meridionalis,and other widespreadU.meridionalisspecimens.Moreover, the 5S rDNA spacer and SCAR signal that should be present in the f loating ecotype ofU.proliferain the Yellow Sea, were not detected. Therefore, it can be determined that the single dominant species causing this green tide event wasU.meridionalisrather thanU.prolifera. Additional evidence supporting this conclusion was also provided from the morphological and anatomical observation. In addition to a previous brief morphological description with the holotype ofU.meridionalisthat collected from brackish water habitat (Horimoto et al., 2011), Xie et al. (2020)supplemented more details to the conspecif ic samples that were from marine habitats. It was shown that for samples in this study, f irst, the transverse constrictions were common on the thalli and only primary branches were detected, which were consistent with the previous description. It was found that two features, i. e., the chloroplasts location in surface view and the number of pyrenoids, were very similar to the description from those in brackish water habitats, but diff erent from those in marine habitats, suggesting that it may result from the lower salinity in Bayuquan area (Song et al., 2017). Secondly, some characteristics, such as the typical bow-shaped double-tubular structure in marineU.meridionalis, were found to be absent in f ield samples but reproducible after subsequent laboratory culture. We speculate that this may be related to the development stages for this species as previously shown (Xie et al., 2020). In addition, some distinct features of our samples were also noticed. It has been reported that the primary branches always occurred in the basal region of the thalli and rarely in the mid or upper regions (Horimoto et al., 2011;Xie et al., 2020). However, they were roughly evenly distributed along the main axis in this study. Based on the identif ication results, it was indicated that the origin of Yingkou green tide was not from the f loating ecotype ofU.proliferain the Yellow Sea, nor from the transport of blooming algae in Qinhuangdao, but probably from the native algae, since nearly half of samples were found to have holdfasts.

Bohai Sea is a semi-closed sea with a low degree of seawater exchange. Due to the increasing impact of human activities, the Bohai Sea receives about one billion tons of wastewater every year (Duan et al.,2010), resulting in increased eutrophication levels.In addition to frequent red tides (Zhao et al., 2004),brown tides (Zhang et al., 2012), jellyf ish blooms(Dong et al., 2010), green tides (Xing et al., 2016;Song et al., 2019a), and even overgrowth of seagrass(Xu et al., 2019) have occurred in recent decades. It indicates that the marine ecosystem in the off shore area has been seriously out of balance. Bayuquan is located at the estuary of Liaohe River in Liaodong Bay, with the highest DIN value in the Bohai Sea due to the main impact of Liaohe River runoff (Wang et al., 2009). It was known that Bayuquan was a sea area with frequent red tides and serious impacts (Yu et al.,2018), but as far as we know, this event is the f irst green tide in this area. Based on the hydrological data of Bayuquan in summer 2021, here we speculated about the possible occurrence process of this event.During the high temperature period, typhoon In-Fa entered the Bohai Sea around July 30 (National Meteorological Center, 2021), which brought greater waves to Bayuquan about twice as high as usual.Due to the weak holdfasts observed in the f ield samples, it was speculated that someU.meridionaliscould be detached from substrates by the wave disturbance. After the typhoon, there was a period of high temperatures and calm for about half a month,which was conducive to the rapid growth of f loatingU.meridionalis. In particular, the average SST during the high temperature period in 2021 is signif icantly increased by 2 ℃ compared with that in 2020, and the highest SST is up to 30.4 ℃. Since it was reported thatU.meridionalisgrown explosively in a narrow range of temperatures of 25–30 ℃ (Hiraoka et al.,2020a), the unusually high temperature might be a critical condition for the rapid proliferation of this subtropical alga in eutrophication waters. Finally, the SST began to drop rapidly from August 20, and with the emergency cleanup, the green tide event quickly ended.

Ulvameridionaliswas initially identif ied and described in the brackish water habitats from Okinawa, Japan (Horimoto et al., 2011). According to its global distribution pattern, Xie et al (2020)suggested it a subtropical species, and reported that it had caused green tides at low latitudes along the coast of the South China Sea in 2018. Because a unique bowknot-shaped structure found only in the transverse view of this species has never been described in any previous records from China, the current distribution ofU.meridionalisin China was considered a result of recent introduction which was followed by rapid local spreads. In the Bohai Sea,this species was only detected occasionally in 2014(Xie et al., 2020), and was usually absent from most seaweed surveys (Wang and Wang, 2009; Liu et al.,2011; Yu et al., 2017; Song et al. 2019b). In this study,we reported for the f irst time thatU.meridionaliscould also cause green tide in the north temperate sea zone, suggesting that the recent colonization of this species in the Bohai Sea might have reached a certain scale. Recent invasions to China with rapid local spreads ofUlvaspecies have been noticed, and it was considered to be driven by the global warming(Xie et al., 2020; Wei et al., 2022). It was predicted that with ocean warming, tropicalUlvaspecies would spread to temperate regions and cause green tides(Hiraoka, 2021), withU.ohnoiandU.reticulataas typical examples (Hiraoka et al., 2020b). This study demonstrates that subtropicalU.meridionalisalso spreads to the temperate zone to cause green tides,supporting the predictions of the latest studies.

In addition to the known facts thatU.meridionalishas an extremely prominent ability to grow rapidly(Hiraoka, 2012; Hiraoka et al., 2020a; Liu et al., 2020;Tsubaki et al., 2020) and cause blooms (Tsubaki et al.,2017; Xie et al., 2020). Here we suggested that there were at least two other possible reasons for the rapid northward extension of the sea area where this species bloomed along coastal China. First, althoughU.meridionalisgrew at an optimum of 25–30 °C (Hiraoka et al., 2020a), it has been reported to distribute in all four sea areas of China, from 20°01ʹN to 39°55ʹN(Xie et al., 2020), indicating that it was likely an eurythermal species that could tolerate and grow over a wide temperature range. This might help expand its distribution and give it the opportunity to bloom in summer at higher latitudes when temperature and nutrient conditions are favorable. Similar phenomena have been found with the Bohai Sea red tide events which occurred mainly in August or September and usually dominant by eurythermal species or those adapted to high temperature (Zhao et al., 2004;Dou et al., 2020). Second, it seems that the spread ofU.meridionaliswas likely to rely on settlement,i.e., living with holdfasts attached to substrates, to achieve colonization of populations and continuous invasion into new habitats (Xie et al., 2020). Thus, in new habitats, when a certain number of individuals detached from substrates under severe disturbances,they would be possible to bloom in new locations. A negative example came from the Yellow Sea green tide. It was found that the thalli of the f loating ecotype ofU.proliferacan only be detected on nori rafts from the Subei radial sand ridges, but they have been almost completely missing in all intertidal zones over years (Zhao et al., 2015; Zhang et al., 2018). It has been proved that after the blooming stage with longdistance drifting, the wide-spread micro-propagules ofU.proliferawere diffi cult to survive in new habitats(Miao et al., 2018; Zhao et al., 2018, 2022), and this might partly explain why the source of the Yellow Sea green tide has never spread northward. This indicated that although the unattached forms have the ability to build up a large biomass, forming massive green tides(Smetacek and Zingone, 2013; Hiraoka, 2021), the extremely weak f ixation ability reduces the expansion of new habitats for resident populations. Therefore,the current distribution and holdfast development of attached populations ofU.meridionalisin the Bohai Sea need to be further investigated, and the ecological risk of this species causing green tides in the future also deserves high attention.

5 CONCLUSION

In August 2021, a green tide occurred for the f irst time in Bayuquan sea area of Yingkou, Liaoning Province in the Bohai Sea. In this study, morphological and molecular approaches were used to identify the causative species asU.meridionalis, a subtropical alga previously found to dominate green tides in the South China Sea. According to the hydrological data of Bayuquan in summer 2021, combined with morphological and developmental observations for this alga, the possible occurrence process of this event was speculated. This is the f irst report on the bloom of subtropicalU.meridionalisin the north temperate sea zone, indicating that the ecological risk of causing green tides in the future by this rapidly spreading species deserves high attention.

6 DATA AVAILABILITY STATEMENT

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

7 ACKNOWLEDGMENT

We are very grateful to Ms. Xiaoxue MA from Dalian Ocean University for her assistance in algal sample collection.