Spring protistan communities in response to warming in the northeastern East China Sea.

The northeastern East China Sea is a highly dynamic marine ecosystem influenced by seasonally varying water mass properties. However, despite being among the world's fastest-warming ocean, there has been limited investigation into the impacts of warming on protistan communities. We collected seawater from two stations (E42 and E46) with different natural protist communities and environmental attributes to investigate the acclimation of the two communities to artificially elevated temperatures (ambient T, +2, and +4 °C). Nutrient and Chl-a conditions reflected oceanographic differences, providing insights into protistan community dynamics. Notably, small-sized autotrophic protists prevailed in the phosphate-deficient E42 community, with mid-incubation heterotrophic conversions. Higher temperatures exacerbated the effects of the P deficiency on the E42 community. While the proportions of Bacillariophyta increased only in the nutrient-balanced E46 communities, those of mixotrophic dinoflagellates increased with elevated temperature, regardless of P deficiency, suggesting that mixotrophy likely aids adaptation in changing marine environments. In summary, the findings of this microcosm study illuminate the potential modulation of spring protistan communities in the northeastern East China Sea under anticipated future warming.

Food web structure for high carbon retention in marine plankton communities.

Total annual net primary productions in marine and terrestrial ecosystems are similar. However, a large portion of the newly produced marine phytoplankton biomass is converted to carbon dioxide because of predation. Which food web structure retains high carbon biomass in the plankton community in the global ocean? In 6954 individual samples or locations containing phytoplankton, unicellular protozooplankton, and multicellular metazooplankton in the global ocean, phytoplankton-dominated bottom-heavy pyramids held higher carbon biomass than protozooplankton-dominated middle-heavy diamonds or metazooplankton-dominated top-heavy inverted pyramids. Bottom-heavy pyramids predominated, but the high predation impact by protozooplankton on phytoplankton or the vertical migration of metazooplankton temporarily changed bottom-heavy pyramids to middle-heavy diamonds or top-heavy inverted pyramids but returned to bottom-heavy pyramids shortly. This finding has profound implications for carbon retention by plankton communities in the global ocean.

Historical trends of traditional, emerging, and halogenated polycyclic aromatic hydrocarbons recorded in core sediments from the coastal areas of the Yellow and Bohai seas.

Historical trends of polycyclic aromatic hydrocarbons (PAHs) contamination were reconstructed from eleven sediment cores located in intertidal zones of the Yellow and Bohai seas for a period encompassing the last 80 years. The analysis encompassed 15 traditional PAHs (t-PAHs), 9 emerging PAHs (e-PAHs), and 30 halogenated PAHs (Hl-PAHs), including 10 chlorinated PAHs (Cl-PAHs) and 20 brominated PAHs (Br-PAHs). Concentrations of target PAHs were highest in industrial and municipal areas situated along the coast of the Bohai Sea, including Huludao, Yingkou, Tianjin, and Dandong, constituting a substantial mass inventory. All target PAHs showed increasing trends since the 1950s, reflecting the development history of South Korea and China. High molecular weight PAHs accumulated in sampling sites more than low molecular weight PAHs. A positive matrix factorization model showed that the PAH sources were coal and gasoline combustion (35%), diesel combustion (33%), and biomass combustion (32%). Over the last 80 years, the contribution of coal and gasoline combustion increased in all regions, while diesel combustion and biomass combustion varied across regions and over time. Toxicity equivalence values were highest for t-PAHs (>99% contribution), followed by Cl-PAHs, Br-PAHs, and e-PAHs. Concentrations of t-PAHs in Eastern Asia seas have increased since the 1900s, particularly in intertidal areas compared to subtidal areas. The intertidal zone removed 83% of the total flux of PAHs originating from land and thus appears to serve as a buffer zone against marine pollution. Overall, this study provides novel knowledge on the historical trends and sources of PAHs on a large scale, along with insights for future coastal management.

Assessing the utility of mitochondrial gene markers in the family Suessiaceae (Dinophyta) with phylogenomic validation.

The dinoflagellate family Suessiaceae comprises cosmopolitan species distributed across polar and tropical waters in both marine and freshwater ecosystems, encompassing free-living forms, symbionts, and parasites. Recently, species diversity within the family has rapidly expanded, now including a few species reported to cause red tides. Despite their ecological and evolutionary importance, classifying them within Suessiaceae is difficult due to the limitations of the existing molecular markers-the highly conserved small subunit ribosomal gene (SSU rDNA) and the presence of two indel regions of sequence fragments of the large subunit ribosomal gene (LSU rDNA)-resulting in poorly resolved phylogenetic relationships. We assessed mitochondrial cytochrome b (cob) and cytochrome c oxidase 1 (cox1) genes to develop robust molecular markers that can reveal the genetic diversity of the family Suessiaceae. The divergences of cob and cox1 sequences among the species in the family were greater than the SSU rDNA but less than the LSU rDNA and the ITS region. Moreover, the distinctive topology inferred from the mitochondrial genes provided high resolution among the suessiacean species. We examined the validity of the genetic markers using phylogenomics based on 2,023 core proteins. The divergence of the cob phylogeny was most consistent with that of the phylogenomic results. Taken together, the cob gene can be a novel marker reflecting topology at the genome-scale within the family Suessiaceae.

Disturbance of sedimentary processes in tidal salt marshes invaded by exotic vegetation.

In situ moorings were conducted at salt marsh and bare flat to reveal the environmental disturbance of sedimentary processes in the intertidal flat. Spartina alterniflora (Spartina), an invasive species, grew up to 156 cm in the Ganghwa tidal flat from June to November 2019. This rapid growth has resulted in the dense salt marsh, which complicated hydrodynamics and associated sediment processes. Stems and leaves could effectively trap fine-grained sediments (17.24-20.42 µm) at the bed, increasing the differences in bed elevation between the two sites to up to 5.11 cm. The cohesive sediments accumulated in Spartina communities were resuspended differently by stem-scale turbulence generated from the disturbance of stems and leaves, depending on wind forcing and vegetation conditions. The vegetated sediments, under low wind speeds (<4 m s-1), were hardly resuspended in the water column, compared to those in the bare flat, resulting in sedimentation. Under high wind speeds (>6 m s-1), stem-scale turbulence was sufficiently strengthened to surpass the sedimentation of suspended sediments; thus, it resuspended additional bed sediments without a loss of the trapped sediment, unlike in the bare flat. The flocculation of suspended sediments in Spartina communities was mainly controlled by stem-scale turbulence. The flocs were confined to a size of 40 µm (settling velocity: 0.17 mm s-1) and developed an approaching spherical shape. After the cut-off of Spartina, a favorable condition for the flocs to grow by 57 µm (settling velocity: 0.23 mm s-1) was established with decrease in stem-scale turbulence. These larger flocs were able to develop into a ramified spherical structure. Despite diminishment of stem-scale turbulence, the disturbed sediments were outflowed, resulting in an abrupt decrease in bed elevation (0.12 cm day-1). The results suggest that the sediment accumulated in Spartina communities remained erodible due to frequent disturbances without sufficient consolidation.

Large-scale sediment toxicity assessment over the 15,000 km of coastline in the Yellow and Bohai seas, East Asia.

The Yellow and Bohai seas have long been contaminated by persistent toxic substances (PTSs) from numerous (un)known anthropogenic sources. In this study, we used Vibrio fischeri bioassay to evaluate ecotoxicological profiles associated with sedimentary PTSs contamination at a large marine ecosystem (LME) scale. A total of 125 surface sediments collected from the coastal areas of the Yellow and Bohai seas were analyzed both for aqueous and organic extracts. Not surprisingly, the results indicated site-dependent toxicities, but most sites were identified as non-toxic to V. fischeri. For aqueous extracts and organic extracts, 13% and 8% of samples, respectively exhibited marginal toxicity, while 0% and 2% of samples exhibited moderate toxicity. However, it should be noted that organic extracts (mean TU = 56) induced stronger toxicities than aqueous samples (mean TU = 0.4). This result generally back-supported the high toxicity potentials associated with sedimentary sink of organic pollutants. Several PTSs measured in the samples indicated a significant contribution to the observed V. fischeri toxicities. Of note, polycyclic aromatic hydrocarbons (PAHs; r = 0.28, p < 0.05), styrene oligomers (r = 0.41, p < 0.01), and alkylphenols (r = 0.38, p < 0.05) showed significant associations to the observed bacterial inhibition. Among PAHs, benzo[a]anthracene and phenanthrene exhibited a significant contribution to the observed V. fischeri toxicities. Meantime, salinity which reflects the distance from the point sources of land-driven pollutants along the rivers and estuaries in the Yellow and Bohai seas was a key environmental variable representing the sample toxicities. Overall, the present study provides baseline information for evaluating the potential sediment toxicity to implement responsible coastal management at an LME scale, and elsewhere.

Stable isotope signatures reveal the significant contributions of microphytobenthos and saltmarsh-driven nutrition in the intertidal benthic food webs.

A tidal saltmarsh supporting marine ecosystem services becomes more recognized, but an understanding of its trophic dynamics is far limited. We investigated the benthic food-web dynamics in Ganghwa tidal flat, a representative tidal saltmarsh of South Korea. The stable carbon and nitrogen isotope ratio values were analyzed for the sixteen species of macrobenthos and their potential food sources such as microphytobenthos (MPB) and various organic matters. A year-round survey was conducted in the three sites at Ganghwa showing the different types of sediment bottom (sandy mud or mud) and/or vegetation (Phragmites or Suaeda). In general, the isotopic signature fairly well demonstrated that trophic structure is primarily influenced by sediment mud content and/or dominated vegetation. Among the four feeding types of consumers (filter feeder; deposit feeder; omnivore; carnivore), the deposit feeders such as mollusk, arthropod, and annelid showed increased dietary dependency on MPB at bare sites. Whilst they actively utilized 13C-depleted organic matters at vegetated sites. Meantime, significantly enriched stable nitrogen isotopic ratio of macrobenthos was evidenced in fall, which reflected the seasonal variation of food sources and physiological processes in survival strategy. Overall, the stable isotopic approach was useful to address the key factors (in)directly influencing the benthic food web structure and its functioning in a typical tidal saltmarsh of the Yellow Sea.

Gyrodinium jinhaense n. sp., a New Heterotrophic Unarmored Dinoflagellate from the Coastal Waters of Korea.

Four unarmored heterotrophic dinoflagellates were isolated from the coastal waters of southern Korea. The rDNA sequences of four clonal cultures were determined, and the morphology of one of the four strains was examined using light and scanning and transmission electron microscopy. The large subunit (LSU) and small subunit (SSU) rDNA sequences of each of the strains differed by 0-0.9% from those of the other strains, and the SSU rDNA sequence of the strain differed by 1.8-4.4% from those of other Gyrodinium species, whereas the LSU (D1-D2) rDNA sequence differed by 12.4-22.2%. Furthermore, phylogenetic trees showed that Gyrodinium jinhaense n. sp. formed a distinctive clade among the other Gyrodinium species. Meanwhile, microscopy revealed an elliptical bisected apical structure complex and a cingulum that was displaced by approximately one-quarter of the cell length, which confirmed that the dinoflagellate belonged to the genus Gyrodinium. However, the cell surface was ornamented with 16 longitudinal striations, both on the episome and hyposome, unlike other Gyrodinium species. Furthermore, the cells were observed to have pusule systems and trichocysts but lacked mucocysts. Based on morphology and molecular data, we consider this strain to be a new species in the genus Gyrodinium and thus, propose that it be assigned to the name G. jinhaense n. sp.

Effects of warming and eutrophication on coastal phytoplankton production.

Phytoplankton production in coastal waters influences seafood production and human health and can lead to harmful algal blooms. Water temperature and eutrophication are critical factors affecting phytoplankton production, although the combined effects of warming and nutrient changes on phytoplankton production in coastal waters are not well understood. To address this, phytoplankton production changes in natural waters were investigated using samples collected over eight months, and under 64 different initial conditions, established by combining four different water temperatures (i.e., ambient T, +2, +4, and + 6 °C), and two different nutrient conditions (i.e., non-enriched and enriched). Under the non-enriched conditions, the effect of warming on phytoplankton production was significantly positive in some months, significantly negative in others, or had no effect. However, under enriched conditions, warming affected phytoplankton production positively in all months except one, when the salinity was as low as 6.5. These results suggest that nutrient conditions can alter the effects of warming on phytoplankton production. Of several parameters, the ratio of initial nitrate concentration to chlorophyll a concentration [NCCA, µM (µg L-1)-1] was one of the most critical factors determining the directionality of the warming effects. In laboratory experiments, when NCCA in the ambient or nutrient-enriched waters was ≥1.2, warming increased or did not change phytoplankton production with one exception; however, when NCCA was <1.2, warming did not change or decreased production. In the time series data obtained from the coastal waters of four target countries, when NCCA was 1.5 or more, warming increased phytoplankton production, whereas when NCCA was lower than 1.5, warming lowered phytoplankton production, Thus, it is suggested that NCCA could be used as an index for predicting future phytoplankton production changes in coastal waters.

Improved real-time PCR method for quantification of the abundance of all known ribotypes of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides by comparing 4 different preparation methods.

Red tides by the ichthyotoxic dinoflagellate Cochlodinium polykrikoides have caused large scaled mortality of fish and great loss in aquaculture industry in many countries. Detecting and quantifying the abundance of this species are the most critical step in minimizing the loss. The conventional quantitative real-time PCR (qPCR) method has been used for quantifying the abundance of this species. However, when analyzing >500 samples collected during huge C. polykrikoides red tides in South Sea of Korea in 2014, this conventional method and the previously developed specific primer and probe set for C. polykrikoides did not give reasonable abundances when compared with cell counting data. Thus improved qPCR methods and a new specific primer and probe set reflecting recent discovery of 2 new ribotypes have to be developed. A new species-specific primer and probe set for detecting all 3 ribotypes of C. polykrikoides was developed and provided in this study. Furthermore, because the standard curve between cell abundance and threshold cycle value (Ct) is critical, the efficiencies of 4 different preparation methods used to determine standard curves were comparatively evaluated. The standard curves were determined by using the following 4 different preparations: (1) extraction of DNA from a dense culture of C. polykrikoides followed by serial dilution of the extracted DNA (CDD method), (2) extraction of DNA from each of the serially diluted cultures with different concentrations of C. polykrikoides cultures (CCD method), (3) extraction of DNA from a dense field sample of C. polykrikoides collected from natural seawater and then dilution of the extracted DNA in serial (FDD method), and (4) extraction of DNA from each of the serially diluted field samples having different concentrations of C. polykrikoides (FCD method). These 4 methods yielded different results. The abundances of C. polykrikoides in the samples collected from the coastal waters of South Sea, Korea, in 2014-2015, obtained using the standard curves determined by the CCD and the FCD methods, were the most similar (0.93-1.03 times) and the second closest (1.16-1.33 times) to the actual cell abundances obtained by enumeration of cells. Thus, our results suggest that the CCD method is a more effective tool to quantify the abundance of C. polykrikoides than the conventional method, CDD, and the FDD and FCD methods.

Most Low-Abundance "Background" Symbiodinium spp. Are Transitory and Have Minimal Functional Significance for Symbiotic Corals.

Speculation surrounds the importance of ecologically cryptic Symbiodinium spp. (dinoflagellates) that occur at low abundances in reef-building corals and in the surrounding environment. Evidence acquired from extensive sampling, long-term monitoring, and experimental manipulation can allow us to deduce the ecology and functional significance of these populations and whether they might contribute to the response of coral-dinoflagellate mutualisms to climate change. Quantitative PCR was used here to diagnose the prevalence, seasonal variation, and abundances of Symbiodinium spp. within and between colonies of the coral, Alveopora japonica. Consistent with broader geographic sampling, only one species comprised 99.9 %, or greater, the population of symbionts in every sample. However, other Symbiodinium including the non-mutualistic species, Symbiodinium voratum, were often detected, but at estimated cell densities thousands-fold less than the dominant symbiont. The temporal variation in prevalence and abundances of these "background" Symbiodinium could not be definitively related to any particular environmental factor including seasonality and water chemistry. The prevalence (proportion detected among host samples), but not abundance, of S. voratum may weakly correspond to increases in environmental inorganic silica (SiO2) and possibly nitrogen (NO3). When multiple background Symbiodinium occurred within an individual polyp, the average cell densities were positively correlated, suggesting non-specific processes of cell sorting and retention by the animal. While these findings substantiate the existence of a broader, yet uncharacterized, diversity of Symbiodinium, we conclude that only those species which can occur in high abundance and are temporally stable are ultimately important to coral-dinoflagellate mutualisms. Many transient Symbiodinium spp., which occur only at trace abundances in the coral's microbiome, belong to different functional guilds and likely have little, if any, importance to a coral's physiology. The successful integration between host and symbiont into a stable functional unit should therefore be considered when defining host-symbiont specificity.

Genetics and morphology characterize the dinoflagellate Symbiodinium voratum, n. sp., (Dinophyceae) as the sole representative of Symbiodinium Clade E.

Dinoflagellates in the genus Symbiodinium are ubiquitous in shallow marine habitats where they commonly exist in symbiosis with cnidarians. Attempts to culture them often retrieve isolates that may not be symbiotic, but instead exist as free-living species. In particular, cultures of Symbiodinium clade E obtained from temperate environments were recently shown to feed phagotrophically on bacteria and microalgae. Genetic, behavioral, and morphological evidence indicate that strains of clade E obtained from the northwestern, southwestern, and northeastern temperate Pacific Ocean as well as the Mediterranean Sea constitute a single species: Symbiodinium voratum n. sp. Chloroplast ribosomal 23S and mitochondrial cytochrome b nucleotide sequences were the same for all isolates. The D1/D2 domains of nuclear ribosomal DNA were identical among Western Pacific strains, but single nucleotide substitutions differentiated isolates from California (USA) and Spain. Phylogenetic analyses demonstrated that S. voratum is well-separated evolutionarily from other Symbiodinium spp. The motile, or mastigote, cells from different cultures were morphologically similar when observed using light, scanning, and transmission electron microscopy; and the first complete Kofoidian plate formula for a Symbiodinium sp. was characterized. As the largest of known Symbiodinium spp., the average coccoid cell diameters measured among cultured isolates ranged between 12.2 (± 0.2 SE) and 13.3 (± 0.2 SE) µm. Unique among species in the genus, a high proportion (approximately 10-20%) of cells remain motile in culture during the dark cycle. Although S. voratum occurs on surfaces of various substrates and is potentially common in the plankton of coastal areas, it may be incapable of forming stable mutualistic symbioses.

Feeding by heterotrophic dinoflagellates and ciliates on the free-living dinoflagellate Symbiodinium sp. (Clade E).

To investigate heterotrophic protists grazing on Symbiodinium sp., we tested whether the common heterotrophic dinoflagellates Gyrodinium dominans, Gyrodinium moestrupii, Gyrodinium spirale, Oblea rotundata, Oxyrrhis marina, and Polykrikos kofoidii and the ciliates Balanion sp. and Parastrombidinopsis sp. preyed on the free-living dinoflagellate Symbiodinium sp. (clade E). We measured the growth and ingestion rates of O. marina and G. dominans on Symbiodinium sp. as a function of prey concentration. Furthermore, we compared the results to those obtained for other algal prey species. In addition, we measured the growth and ingestion rates of other predators at single prey concentrations at which these rates of O. marina and G. dominans were saturated. All predators tested in the present study, except Balanion sp., preyed on Symbiodinium sp. The specific growth rates of O. marina and G. dominans on Symbiodinium sp. increased rapidly with increasing mean prey concentration < ca. 740-815 ng C/ml (7,400-8,150 cells/ml), but became saturated at higher concentrations. The maximum growth rates of O. marina and G. dominans on Symbiodinium sp. (0.87 and 0.61/d) were much higher than those of G. moestrupii and P. kofoidii (0.11 and 0.04/d). Symbiodinium sp. did not support positive growth of G. spirale, O. rotundata, and Parastrombidinopsis sp. However, the maximum ingestion rates of P. kofoidii and Parastrombidinopsis sp. (6.7-10.0 ng C/predator/d) were much higher than those of O. marina and G. dominans on Symbiodinium sp. (1.9-2.1 ng C/predator/d). The results of the present study suggest that Symbiodinium sp. may increase or maintain the populations of some predators.

Gymnodinium smaydae n. sp., a new planktonic phototrophic dinoflagellate from the coastal waters of Western Korea: morphology and molecular characterization.

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1-5.8S-ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3-10.9 µm long and 5.1-10.0 µm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4-0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1-5.8S-ITS2, and LSU rDNA region differed by 1.5-3.8%, 6.0-17.4%, and 9.1-17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.

Heterotrophic feeding as a newly identified survival strategy of the dinoflagellate Symbiodinium.

Survival of free-living and symbiotic dinoflagellates (Symbiodinium spp.) in coral reefs is critical to the maintenance of a healthy coral community. Most coral reefs exist in oligotrophic waters, and their survival strategy in such nutrient-depleted waters remains largely unknown. In this study, we found that two strains of Symbiodinium spp. cultured from the environment and acquired from the tissues of the coral Alveopora japonica had the ability to feed heterotrophically. Symbiodinium spp. fed on heterotrophic bacteria, cyanobacteria (Synechococcus spp.), and small microalgae in both nutrient-replete and nutrient-depleted conditions. Cultured free-living Symbiodinium spp. displayed no autotrophic growth under nitrogen-depleted conditions, but grew when provided with prey. Our results indicate that Symbiodinium spp.'s mixotrophic activity greatly increases their chance of survival and their population growth under nitrogen-depleted conditions, which tend to prevail in coral habitats. In particular, free-living Symbiodinium cells acquired considerable nitrogen from algal prey, comparable to or greater than the direct uptake of ammonium, nitrate, nitrite, or urea. In addition, free-living Symbiodinium spp. can be a sink for planktonic cyanobacteria (Synechococcus spp.) and remove substantial portions of Synechococcus populations from coral reef waters. Our discovery of Symbiodinium's feeding alters our conventional views of the survival strategies of photosynthetic Symbiodinium and corals.