During the past decade, the advancement of High-Throughput Sequencing technologies have provided unprecedented insights into the diversity, spatial community composition, temporal patterns, and functional and ecological processes affected by marine unicellular Eukaryotes. A recent study attempted to link the taxonomic diversity of Protistan taxa detected with High-Throughput Sequencing with 30 different traits in samples from ecosystems across the coasts of North Atlantic Ocean, suggesting that small taxa are characterized by broader taxonomic and functional diversity. Furthermore, previously underestimated trophic strategies, such as parasitism and decomposition, have been recognized as central to marine planktonic assemblage structure and ecosystem function. In addition, the environmental effects and biotic relationships in unicellular Eukaryotic communities have been explored, using classical and modern ecological tools. It is evident that the plethora of the genetic and ecological information produced provide a better understanding of marine global ecosystem processes. In particular, coastal areas are systems with high risk of pronounced modification in view of climate change and coastal urbanization leading to increased eutrophication. Thus, there is an increase in the attention of marine microbiologists thanks to the development of High-Throughput Sequencing tools. Either by focusing on specific taxonomic groups, such as the parasitoids Perkinsozoa and Labyrinthulomycetes, the cosmopolitan and conspicuous Tintinnid Ciliates, the newly described Vampyrellida (Rhizaria), or by investigating the overall marine protistan diversity, temporal dynamics, function, biotic interactions, relationships with abiotic variables, effects of climate change, and potential activity, marine microbiologists have attempted to answer the classical questions of who, when, why, and where in coastal systems. However, few High-Throughput Sequencing-based studies have investigated marine unicellular Eukaryotic assemblages in degraded eutrophic coastal areas, with high anthropogenic impacts. These studies, albeit scarce, have revealed high and novel diversity, and have attempted to provide insights of the trophic relationships between known groups and previously overlooked links.
Aplanochytrium sp., a parasitic marine Protist in the Class Labyrinthulomycetes, noted for the production of an extension of the plasma membrane and the ectoplasm called the ectoplasmic net. Aplanochytrium cells are not embedded in the ectoplasmic net but can move by gliding on ectoplasmic threads. Celeste Leander/Tree of Life Project/Wikimedia Commons.
In a paper published in the journal Diversity on 21 March 2020, Savvas Genitsaris of the School of Science and Technology at the International Hellenic University, and the Department of Botany at the Aristotle University of Thessaloniki, Natassa Stefanidou and Maria Moustaka-Gouni, also of the Department of Botany at the Aristotle University of Thessaloniki, Ulrich Sommer of the Geomar Helmholtz Centre for Ocean Research Kiel, and George Tsipas, also of the School of Science and Technology at the International Hellenic University, present the results of a study which used High-Throughput Sequencing to investigate the marine unicellular eukaryotic diversity and spatial distribution throughout a year of frequent samplings in Thessaloniki Bay, a coastal eutrophic Mediterranean urban area.
The High-Throughput Sequencing study was used as a follow-up to a previous project examining via classical microscopy tools the microbial plankton communities in the same area and the same sampling period, which was characterized by marked harmful Algal blooms, red tides, and mucilage aggregate incidents. The Thessaloniki Bay is the inner part of Thermaikos Gulf, located in northern Greece, and has been accepting for decades a large volume of domestic and industrial wastes from the one million residents of the city of Thessaloniki. Although wastewater treatment has been implemented, decreasing the effects of anthropogenic eutrophication, the restricted water circulation and shallowness of the Bay in combination with high nutrient inputs, lead to harmful Algal blooms, apparent red tides, and mucilage aggregates, leading to growing concerns of the citizens and authorities on the water quality of the Bay and particularly of the urban front. Data on the abundance and dynamics of plankton community (both Phyto- and Protozooplankton) in the urban part of the Gulf obtained by classical microscopy, have attempted to identify and characterize the marine Protist abettors and perpetrators responsible for these phenomena. These studies have shown the contribution of taxa known to thrive due to eutrophication and to cause harmful events, such as the Diatoms Cylindrotheca closterium, Skeletonema costatum, Chaetoceros spp., and the Dinoflagellates Noctiluca scintillans and Dinophysis acuminata. Taking into consideration the continuous increase of eutrophication in coastal areas worldwide, there is a need to identify the causative organisms of these events and implement strategies to mitigate their severe impacts to ecosystem and public health. High-Throughput Sequencing-based studies investigating the unicellular Eukaryotic diversity, dynamics, spatial distributions, and environmental effects in coastal eutrophic areas could contribute towards the early and accurate characterization of the microbes responsible for harmful bloom events and red tides.
The Diatom Cylindrotheca closterium, a solitary, motile species which forms mucilaginous aggregates under high salinity conditions and produces allelopathic chemicals that suppress growth of other species. Gert Hansen/Scandinavian Culture Collection of Algae and Protozoa/University of Copenhagen/Nordic Microalgae and Aquatic Protozoa.
The aim of Genitsaris et al.'s study was to investigate and describe the overall marine unicellular Eukaryotic assemblages of different sites of the urban front of Thessaloniki throughout a year in short sampling intervals via High-Throughput Sequencing tools, following a previous study concerning the same area and sampling period, where different periods of plankton blooms were identified and analysis of the environmental variables was presented. The sites are located in close proximity, thus allowing us to examine the variability of unicellular Eukaryotic diversity and dynamics in small temporal and spatial scales. The main questions were: (i) Do the assemblages include previously undetected taxa in the area? (ii) How do these taxa contribute to the phenomena of blooms, red tides, and mucilage aggregates? (iii) What are the temporal and spatial distributions of the unicellular eukaryotic assemblages? (iv) Are they affected by nutrients and how?
A 'red tide' of toxic unicellular Algae covering the entire waterfront of Thessaloniki in June 2017. Greek Reporter.
Samples were collected weekly from March 2017 to February 2018 at an urban site in Thessaloniki Bay (Thermaikos Gulf), namely the White Tower station. Simultaneously, samples were taken from White Tower and three other adjacent sites (Aretsou Beach, Music Hall Coast, and Harbour) every four weeks. The stations were located along the coastline with a distance of about 3 Km between the closest consecutive ones and 10 Km between the most distant ones, which were located at the edges of the sampling area. All sampling sites had a maximum depth of 4 m. In total, 83 water samples were collected.
For all samples, in situ measurements of water temperature and salinity were made Water samples of 2 L were collected from the surface layer of 1 m, and subsamples of 100–250 mL (depending on plankton and particulate matter density) were immediately filtered onto 0.7 μm prewashed filters, and the filters were stored in −20 °C for particulate organic nutrient and Chlorophyll a measurements. Moreover, subsamples of 50 mL were filtered through 0.2 μm cellulose acetate filters and were kept in −20 °C for dissolved inorganic nutrient measurements. Finally, the rest of the water volume was prefiltered through a 200 μm sterile mesh, and subsamples of 100–200 mL from each water sample were subsequently filtered through 0.2 μm nucleopore filters and similarly were kept in −20 °C for molecular analysis.
Study area in Thermaikos Bay, indicating the location of the four sampling sites (*). WT: White Tower; AR: Aretsou; MH: Music Hall; HB: Harbour. Genitsaris et al. (2020).
The seawater temperature during the year of the study ranged from 9.6 °C on 24 January 2018 at the White Tower site, to 29.7 °C on 09 August 2017 at the same site. Salinity varied and was higher during the cold months with highest value recorded on 15 March 2017 at White Tower (38.8 g/L), and the lowest on 19 July 2017, also at White Tower (35.3 g/L). Nutrients, i.e., silicate, phosphate, nitrate, nitrite, ammonia, and particulate organic phosphorus, fluctuated considerably throughout the sampling period, and few extreme values were measured (e.g., on 22 March 2017, at White Tower). High values were also recorded for all nutrients on 28 July 2017 at the White Tower and Music Hall sites, 20 September 2017 in all sites, and 10 January 2018 in White Tower. Chlorophyll a, also showed marked variability, ranging from 0.27 μg L−1 on 27 December 2017 at White Tower, to 17.28 μg L−1 on 13 December 2017 also at White Tower.
Several conspicuous plankton blooms were recoreded in the study area during the period of the study. A bloom from March to June 2017 was found to be comprised largely of the Cocolithaphores Leptocylindrus danicus, and Leptocylindrus minimus. A red tide from 22 March to 29 March 2017 was caused by the Dinoflagellate Noctiluca scintillans (sometimes known as the Sea Sparkle as it is bioluminescent when disturbed). Noctiluca scintillans caused further red tides on 12 April 2017 and 14–21 June 2017. From 28 June to 4 July 2017 the area suffered a mucilage aggregates phenomenon (massive accumulation of gelatinous material at and below the water surface). From July to September 2017 the area had a bloom of the Diatoms Chaetoceros spp., Cylindrotheca closterium, and Skeletonema costatum. In November 2017 there was a bloom of the Dinoflagellate Dinophysis cf. acuminata. From December 2017 to 17 January 2018 there was a bloom of the Ciliate Mesodinium rubrum. Finally in January–February 2018 there was another bloom of the Diatoms Cylindrotheca closterium, and Skeletonema costatum.
The Sea Sparkle, Noctiluca scintillans, a bioluminescent Dinoflagellate which caused several red tides in Thessaloniki Bay during the summer of 2017. Maria Antónia Sampayo/Instituto de Oceanografia/Universidade de Lisboa/Plankton Net/Wikimedia Commons.
In the 47 samples from the White Tower site, a total of 2256 Operational Taxonomic Units (clusters of organisms, grouped by DNA sequence similarity of a specific taxonomic marker gene; a pragmatic proxy for species in the absence of a full taxonomic study) were recovered. The highest number of Operational Taxonomic Units (378) was detected on 23 August and the lowest (44) on 22 March. Alpha-diversity estimators fluctuated during the study, with extremely low values on 22 March 2017 at the White Tower site, indicating high dominance by one (or few) taxa, and high variation between taxa abundances within the community. Indeed, Operational Taxonomic Unit 2, closely related to the Dinoflagellate Noctiluca scintillans, comprised of over 99% of the total number of reads in that sample.
The cumulative number of Operational Taxonomic Units rose steadily throughout the study. On average, 45.9 new Operational Taxonomic Units were introduced in each sample from White Tower. The most prominent introductions of newly detected White Tower were recorded at 29 March 2017 with 152 newly introduced Operational Taxonomic Units, at 4 July 2017 (99 Operational Taxonomic Units), at 27 September 2017 (78 Operational Taxonomic Units), and at 17 January 2018 (180 Operational Taxonomic Units). These spikes of newly introduced Operational Taxonomic Units coincided with the ending of two red tides (29 March 2017 and 17 January 2018, the end of the conspicuous mucilaginous aggregate of late June 2017, and the end of a summer Diatom bloom (September 2017). Overall, the most diverse groups exhibited temporal variations in abundance. Even so, Dinoflagellata in all cases comprised more than 30% of the number of reads per sample, reaching almost 99% of the number of reads on 22 March 2017 at White Tower, when Operational Taxonomic Unit 2, closely affiliated to Noctiluca scintillans, was over-dominant.
The proportions of each group of unicellular Eukaryotes recovered over the course of the study. Genitsaris et al. (2020).
During the monthly sampling, common to the four sites, the total number of Operational Taxonomic Units corresponding to 12 samplings for each site, was 1153, 1195, 1362, and 1071 for White Tower, Aretsou Beach, Music Hall Coast, and Harbour, respectively. The highest average number of Operational Taxonomic Units was calculated at the Music Hall Coast site (262.4), and the lowest at the Harbour site (210.3). Overall, 560 Operational Taxonomic Units were found to be common in all four sampling sites. Concerning each site individually, 172, 231, 351, and 178 unique Operational Taxonomic Units were detected at White Tower, Aretsou Beach, Music Hall Coast, and Harbour, respectively. The average values of the a-diversity estimators were generally similar between all four sites.
Dinoflagellata in all cases comprised over 30% of the number of reads per sample, while during the warm months of summer, Bacillariophyta (Diatoms) showed higher relative abundance reaching over 50% of the total number of reads at all sites except Harbour. Furthermore, over 40% of the total number of reads were affiliated to Bacillariophyta in November 2017 and February 2018 at White Tower and in November 2017 at Music Hall Coast, while also Ciliophora-related Operational Taxonomic Units (Ciliates) were recorded in high relative abundances (over 40% of the total number of reads) in January 2018, again at Music Hall Coast.
Overall, 36 Operational Taxonomic Units were detected having relative abundance of more than 1% of the total number of reads in each sampling site (instantly abundant), representing the most dominant Operational Taxonomic Units during the study. The majority of these (17/36) belonged to the Dinoflagellata, followed by Ciliophora (6/36) and Bacillariophyta (5/36). Additionally, present among the most dominant OTUs were the groups were Chlorophyta, Marine Stramenopiles, Cercozoa, Acantharia, and Cryptophyta. The closest relatives of these Operational Taxonomic Units were known marine taxa, including the Dinoflagellates Scrippsiella trochoidea, Noctiluca scintillans,
Gonyaulax fragilis, Gymnodinium aureolum, Alexandrium margaelefii, and Peridinium quinquecorne, the Diatoms Chaetoceros tenuissimus, Thalassiosira sp., and Skeletonema pseudocostatum, the Cryptophyte Teleaulax sp., and the Cliates Strobilidium sp., Strombidium biarmatum, and Tintinnopsis sp., with cosmopolitan distribution, but also previously undetected in the area taxa.
Among the above taxa, Operational Taxonomic Unit 1, Operational Taxonomic Unit 2, Operational Taxonomic Unit 4, Operational Taxonomic Unit 5, Operational Taxonomic Unit 6, and Operational Taxonomic Unit 8 exhibited relative abundances of over 10% of a sample’s total number of reads, representing arbitrarily bloom forming Operational Taxonomic Units, in at least one sample in all four sites. In particular, Operational Taxonomic Unit 1, with closest relative the Dinoflagellate Scrippsiella trochoidea, followed by Operational Taxonomic Unit 2 (the Dinoflagellate Noctiluca scintillans), were detected in relative abundances of over 10% of a sample’s total number of reads for long periods during the study, including before and during the mucilage aggregate event, especially in White Tower for Operational Taxonomic Unit 2. It is noteworthy that Operational Taxonomic Unit 4, closely affiliated to the Diatom Chaetoceros tenuissimus, was frequently detected during the warm months and was strongly positively related to the water temperature, while the Operational Taxonomic Unit 5 (the Dinoflagellate Gonyaulax fragilis) had high abundances during the mucilaginous aggregate phenomenon in June 2017. Other dominant Operational Taxonomic Units were observed in peak abundances in individual samples throughout the study. On the other hand, Operational Taxonomic Units closely related to known harmful species (such as the Dinoflagellate Dinophysis cf. acuminata, which was found as a rare overall Operational Taxonomic Unit) were found in low abundance (number of reads), suggesting the presence of taxa with potential negative impacts on human health.
The study area is considered a heavily modified marine water body according to the EU Water Framework Directive, one of the most polluted coastal areas of Greece, which is influenced by both anthropogenic activity and freshwater inputs. During the study period nitrogen and phosphorus concentrations were among the highest recorded for eutrophic coastal areas of the Mediterranean Sea. Moreover, environmental variability between the closely located sampling stations was reported as nonsignificant for the majority of the parameters examined.
In this nutrient-rich coastal environment, high biodiversity and abundance of marine unicellular Eukaryotes has been previously reported based on classical microscopy observations. The most abundant and diverse planktonic groups in these observations comprised of Bacillariophyta (Diatoms) and Dinoflagellata, while less diverse groups included Haptophyta, Cryptophyta, Chlorophyta, Euglenozoa, etc. Evidenced by High-Throughput Sequencing, the dominant taxonomic groups of unicellular Eukaryotes belonged to the 'usual suspects', frequently found in the area, i.e., Dinoflagella, Bacillariophyta, and Ciliophora, but also previously undetected groups were revealed, belonging to Cercozoa, the Marine Alveolates group, the Marine Stramenopiles group, Labyrinthulea, Picozoa, Oomycota, Fungi, and other marine unicellular Eukaryotes. At the White Tower site, with higher sampling frequency (weekly samplings) a higher diversity was detected, in comparison to the monthly samplings. It is reasonable to assume that with higher sampling efforts, diversity estimates will increase, as rarely occurring taxa have better chances to be detected. Moreover, because of the short generation times and high turnover rates of marine microbes, rare taxa may have low residence and detection times in the marine environment. Accordingly, frequent temporal (e.g., one week sampling interval) and highly resolved spatial samplings can provide with higher diversity coverage for planktonic organisms.
High-Throughput Sequencing tools have been increasingly used in investigations of marine microbial diversity and have similarly revealed high novel and undetected diversity in coastal areas including the Eukaryotic taxonomic groups detected in Genitsaris et al.'s dataset. Previous studies using High-Throughput Sequencing tools have reported 'new players in the succession scene' of the coast of eastern English Channel, and were associated with Marine Alveolates, Marine Stramenopiles, and Cercozoa, which were also detected in Genitsaris et al.'s study. These new players were suggested to participate in complex interactions, shaping the spatial, and temporal unicellular Eukaryote assemblages in the area. The rare microbial fraction of these assemblages appeared to be particularly active and play significant ecological roles.
During the period of the study, conspicuous, frequent, persistent, and successive Phytoplankton blooms were alternated with red tides and mucilage aggregates. These phenomena were detected by means of microscopy almost continuously throughout the study period and were of great ecological importance for the coastal system. They, also, had significant socio-economic impact to the residents of the city of Thessaloniki, as in several occasions the red tides and harmful Algal blooms were macroscopically visible impacting the touristic urban front of the city centre. The key plankton abettors and perpetrators contributing to the formation of the Phytoplankton blooms were the known mucilage-producing Diatoms Cylindrotheca closterium, Leptocylindrus spp., Skeletonema costatum, Chaetoceros spp., and the Dinoflagellate Gonyaulax cf. fragilis, while the species responsible for the development of the red tides were the Dinoflagellates Noctiluca scintillans and its close relative Spatulodinium pseudonoctiluca, and the photosynthetic Ciliate Mesodinium rubrum. The characterisation of the unicellular Eukaryotic communities with High-Throughput Sequencing tools showed that the dominant biosphere, taking over a high percentage of the reads, was closely related to the abovementioned species. In particular, Operational Taxonomic Units closely related to the red tide forming Noctiluca scintillans, the bloom forming Chaetoceros spp., Skeletonema pseudocostatum, Gymnodinium aureolum and Peridinium quinquecorne, and the mucilage aggregates abettor Gonyaulax fragilis were among the most abundant Operational Taxonomic Units in all samples. Indeed, these Operational Taxonomic Units were detected in high relative number of reads during the same periods that their closest relatives were identified by microscopy.
Furthermore, during these phenomena the system was taken over by the dominant taxa, as evidenced by the hampered rate of newly introduced Operational Taxonomic Units in the system during these periods compared to the nonbloom periods. Among these Operational Taxonomic Units, previously undetected taxa in the area were observed, namely the Dinoflagellates Adenoides eludens and Islandinium tricingulatum, the Haptophyte Haptolina sp., taxa belonging to the Marine Stramenopiles, and the Ciliate Parundella sp., all of which have unusual and complex life cycles and trophic preferences, influential in global processes, and suggesting alternating trophic states and carbon paths. In addition to microscopic data, Operational Taxonomic Units closely related to known harmful species were opportunistically found with high read numbers during bloom/red tide phenomena, such as parasitic Marine Alveolates and Cercozoa, revealing a (previously undetected) significant biodiversity with potentially negative impacts for the unicellular Eukaryotic community and the ecosystem. Marine Alveolates have been detected in high abundances in virtually all studies of marine unicellular Eukaryotes, suggesting their ubiquitous role as parasites of various marine organisms and their significant part as a trophic link in marine systems.
Temperature and salinity were the two environmental variables that seemed to influence most the overall unicellular Eukaryotic communities, but also individually the majority of the dominant Operational Taxonomic Units, as expected. Both water temperature and salinity are among the key components of climate change, which are projected to directly affect marine microbial diversity and functions. Temperature has been found to affect the size, metabolic rate, and activity of microbes, thus influencing marine protistan community and food web structure. Previous studies using High-Throughput Sequencing tools in mesocosm salinity manipulations of Thessaloniki Bay unicellular Eukaryotic assemblages detected a number of Operational Taxonomic Units associated with euryhaline taxa, also found in Genitsaris et al.'s dataset (e.g., Skeletonema, Chaetoceros, Prorocentrum, Gyrodinium, and Gymnodinium-related Operational Taxonomic Units). This has led to the conclusion that in this Mediterranean environment, the standing biodiversity can act as a buffer against environmental fluctuations. Beyond the water temperature and salinity nutrient concentrations were only partly associated with specific Operational Taxonomic Units. In particular, nitrogen was positively correlated with the Operational Taxonomic Unit closely related to the Dinoflagellate Noctiluca scintillans, which was identified as the chief responsible species for the observed red tides during the study. Noctiluca scintillans is among the most frequently observed red tide forming Dinoflagellates worldwide in eutrophic systems, and at water temperatures ranging from 10 to 25 °C, and salinity ranges from 28 to 36 g/L, similar to Thessaloniki Bay. The positive connection between Noctiluca scintillans read abundance and ammonia in Genitsaris et al.'s study area agrees with the reported correlation of its cell abundance and ammonia with microscopy data in the same samples, and might indicate nutrient regeneration by this heterotrophic Dinoflagellate and contribution to the local nutrient pool. In addition, the role of Noctiluca scintillans as a recycler of nitrogen has been previously linked to high concentrations of nitrogen in its cells. Even though microscopy data of the same samples showed strong connections of most nutrients measured in Genitsaris et al.'s study, metabarcoding data did not reveal similar trends with the exception of the positive correlation of ammonia and the Operational Taxonomic Unit closely related to Noctiluca scintillans.
The High-Throughput Sequencing tools implemented in Genitsaris et al.'s study showed a high diversity of known, frequently occurring Protists, also detected with microscopy in previous studies of the area. It further revealed several taxa previously undetected in the area, with seemingly important roles in the structure and functioning of the entire unicellular Eukaryotic communities, and key contribution to the phenomena of red tides, harmful Algal blooms, and mucilage aggregates observed during the study. The unicellular Eukaryotic assemblages showed temporal patterns rather than small-scale spatial separation, possibly responding to the water temperature and salinity seasonal variations. The rich species pool along with the eutrophic status of the area facilitates the unicellular Eukaryotic succession, mainly favoring the taxa affected by the established cycles of temperature and salinity. Overall, environmental pressures (eutrophication, nitrogen pollution) under seasonal changes of water temperature and salinity (circulation pattern), are suggested to be the major drivers of the composition changes and the appearance of blooms and red tides in the study area.
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Overall, 36 Operational Taxonomic Units were detected having relative abundance of more than 1% of the total number of reads in each sampling site (instantly abundant), representing the most dominant Operational Taxonomic Units during the study. The majority of these (17/36) belonged to the Dinoflagellata, followed by Ciliophora (6/36) and Bacillariophyta (5/36). Additionally, present among the most dominant OTUs were the groups were Chlorophyta, Marine Stramenopiles, Cercozoa, Acantharia, and Cryptophyta. The closest relatives of these Operational Taxonomic Units were known marine taxa, including the Dinoflagellates Scrippsiella trochoidea, Noctiluca scintillans,
Gonyaulax fragilis, Gymnodinium aureolum, Alexandrium margaelefii, and Peridinium quinquecorne, the Diatoms Chaetoceros tenuissimus, Thalassiosira sp., and Skeletonema pseudocostatum, the Cryptophyte Teleaulax sp., and the Cliates Strobilidium sp., Strombidium biarmatum, and Tintinnopsis sp., with cosmopolitan distribution, but also previously undetected in the area taxa.
The Dinoflagellates Scrippsiella trochoidea, widely distributed bloom-forming species present in Thessaloniki Bay at a high abundance during the study period. Phyto'pedia.
Among the above taxa, Operational Taxonomic Unit 1, Operational Taxonomic Unit 2, Operational Taxonomic Unit 4, Operational Taxonomic Unit 5, Operational Taxonomic Unit 6, and Operational Taxonomic Unit 8 exhibited relative abundances of over 10% of a sample’s total number of reads, representing arbitrarily bloom forming Operational Taxonomic Units, in at least one sample in all four sites. In particular, Operational Taxonomic Unit 1, with closest relative the Dinoflagellate Scrippsiella trochoidea, followed by Operational Taxonomic Unit 2 (the Dinoflagellate Noctiluca scintillans), were detected in relative abundances of over 10% of a sample’s total number of reads for long periods during the study, including before and during the mucilage aggregate event, especially in White Tower for Operational Taxonomic Unit 2. It is noteworthy that Operational Taxonomic Unit 4, closely affiliated to the Diatom Chaetoceros tenuissimus, was frequently detected during the warm months and was strongly positively related to the water temperature, while the Operational Taxonomic Unit 5 (the Dinoflagellate Gonyaulax fragilis) had high abundances during the mucilaginous aggregate phenomenon in June 2017. Other dominant Operational Taxonomic Units were observed in peak abundances in individual samples throughout the study. On the other hand, Operational Taxonomic Units closely related to known harmful species (such as the Dinoflagellate Dinophysis cf. acuminata, which was found as a rare overall Operational Taxonomic Unit) were found in low abundance (number of reads), suggesting the presence of taxa with potential negative impacts on human health.
The Diatom Chaetoceros tenuissimus, was frequently detected during the warm months and was strongly positively related to the water temperature in Thessaloniki Bay during the study period. BioMarks Data Portal.
The study area is considered a heavily modified marine water body according to the EU Water Framework Directive, one of the most polluted coastal areas of Greece, which is influenced by both anthropogenic activity and freshwater inputs. During the study period nitrogen and phosphorus concentrations were among the highest recorded for eutrophic coastal areas of the Mediterranean Sea. Moreover, environmental variability between the closely located sampling stations was reported as nonsignificant for the majority of the parameters examined.
In this nutrient-rich coastal environment, high biodiversity and abundance of marine unicellular Eukaryotes has been previously reported based on classical microscopy observations. The most abundant and diverse planktonic groups in these observations comprised of Bacillariophyta (Diatoms) and Dinoflagellata, while less diverse groups included Haptophyta, Cryptophyta, Chlorophyta, Euglenozoa, etc. Evidenced by High-Throughput Sequencing, the dominant taxonomic groups of unicellular Eukaryotes belonged to the 'usual suspects', frequently found in the area, i.e., Dinoflagella, Bacillariophyta, and Ciliophora, but also previously undetected groups were revealed, belonging to Cercozoa, the Marine Alveolates group, the Marine Stramenopiles group, Labyrinthulea, Picozoa, Oomycota, Fungi, and other marine unicellular Eukaryotes. At the White Tower site, with higher sampling frequency (weekly samplings) a higher diversity was detected, in comparison to the monthly samplings. It is reasonable to assume that with higher sampling efforts, diversity estimates will increase, as rarely occurring taxa have better chances to be detected. Moreover, because of the short generation times and high turnover rates of marine microbes, rare taxa may have low residence and detection times in the marine environment. Accordingly, frequent temporal (e.g., one week sampling interval) and highly resolved spatial samplings can provide with higher diversity coverage for planktonic organisms.
High-Throughput Sequencing tools have been increasingly used in investigations of marine microbial diversity and have similarly revealed high novel and undetected diversity in coastal areas including the Eukaryotic taxonomic groups detected in Genitsaris et al.'s dataset. Previous studies using High-Throughput Sequencing tools have reported 'new players in the succession scene' of the coast of eastern English Channel, and were associated with Marine Alveolates, Marine Stramenopiles, and Cercozoa, which were also detected in Genitsaris et al.'s study. These new players were suggested to participate in complex interactions, shaping the spatial, and temporal unicellular Eukaryote assemblages in the area. The rare microbial fraction of these assemblages appeared to be particularly active and play significant ecological roles.
During the period of the study, conspicuous, frequent, persistent, and successive Phytoplankton blooms were alternated with red tides and mucilage aggregates. These phenomena were detected by means of microscopy almost continuously throughout the study period and were of great ecological importance for the coastal system. They, also, had significant socio-economic impact to the residents of the city of Thessaloniki, as in several occasions the red tides and harmful Algal blooms were macroscopically visible impacting the touristic urban front of the city centre. The key plankton abettors and perpetrators contributing to the formation of the Phytoplankton blooms were the known mucilage-producing Diatoms Cylindrotheca closterium, Leptocylindrus spp., Skeletonema costatum, Chaetoceros spp., and the Dinoflagellate Gonyaulax cf. fragilis, while the species responsible for the development of the red tides were the Dinoflagellates Noctiluca scintillans and its close relative Spatulodinium pseudonoctiluca, and the photosynthetic Ciliate Mesodinium rubrum. The characterisation of the unicellular Eukaryotic communities with High-Throughput Sequencing tools showed that the dominant biosphere, taking over a high percentage of the reads, was closely related to the abovementioned species. In particular, Operational Taxonomic Units closely related to the red tide forming Noctiluca scintillans, the bloom forming Chaetoceros spp., Skeletonema pseudocostatum, Gymnodinium aureolum and Peridinium quinquecorne, and the mucilage aggregates abettor Gonyaulax fragilis were among the most abundant Operational Taxonomic Units in all samples. Indeed, these Operational Taxonomic Units were detected in high relative number of reads during the same periods that their closest relatives were identified by microscopy.
The photosynthetic Ciliate Mesodinium rubrum, one of the species responsible for the development of the red tides in Thessaloniki Bay during the study period. Phyto'pedia.
Furthermore, during these phenomena the system was taken over by the dominant taxa, as evidenced by the hampered rate of newly introduced Operational Taxonomic Units in the system during these periods compared to the nonbloom periods. Among these Operational Taxonomic Units, previously undetected taxa in the area were observed, namely the Dinoflagellates Adenoides eludens and Islandinium tricingulatum, the Haptophyte Haptolina sp., taxa belonging to the Marine Stramenopiles, and the Ciliate Parundella sp., all of which have unusual and complex life cycles and trophic preferences, influential in global processes, and suggesting alternating trophic states and carbon paths. In addition to microscopic data, Operational Taxonomic Units closely related to known harmful species were opportunistically found with high read numbers during bloom/red tide phenomena, such as parasitic Marine Alveolates and Cercozoa, revealing a (previously undetected) significant biodiversity with potentially negative impacts for the unicellular Eukaryotic community and the ecosystem. Marine Alveolates have been detected in high abundances in virtually all studies of marine unicellular Eukaryotes, suggesting their ubiquitous role as parasites of various marine organisms and their significant part as a trophic link in marine systems.
Temperature and salinity were the two environmental variables that seemed to influence most the overall unicellular Eukaryotic communities, but also individually the majority of the dominant Operational Taxonomic Units, as expected. Both water temperature and salinity are among the key components of climate change, which are projected to directly affect marine microbial diversity and functions. Temperature has been found to affect the size, metabolic rate, and activity of microbes, thus influencing marine protistan community and food web structure. Previous studies using High-Throughput Sequencing tools in mesocosm salinity manipulations of Thessaloniki Bay unicellular Eukaryotic assemblages detected a number of Operational Taxonomic Units associated with euryhaline taxa, also found in Genitsaris et al.'s dataset (e.g., Skeletonema, Chaetoceros, Prorocentrum, Gyrodinium, and Gymnodinium-related Operational Taxonomic Units). This has led to the conclusion that in this Mediterranean environment, the standing biodiversity can act as a buffer against environmental fluctuations. Beyond the water temperature and salinity nutrient concentrations were only partly associated with specific Operational Taxonomic Units. In particular, nitrogen was positively correlated with the Operational Taxonomic Unit closely related to the Dinoflagellate Noctiluca scintillans, which was identified as the chief responsible species for the observed red tides during the study. Noctiluca scintillans is among the most frequently observed red tide forming Dinoflagellates worldwide in eutrophic systems, and at water temperatures ranging from 10 to 25 °C, and salinity ranges from 28 to 36 g/L, similar to Thessaloniki Bay. The positive connection between Noctiluca scintillans read abundance and ammonia in Genitsaris et al.'s study area agrees with the reported correlation of its cell abundance and ammonia with microscopy data in the same samples, and might indicate nutrient regeneration by this heterotrophic Dinoflagellate and contribution to the local nutrient pool. In addition, the role of Noctiluca scintillans as a recycler of nitrogen has been previously linked to high concentrations of nitrogen in its cells. Even though microscopy data of the same samples showed strong connections of most nutrients measured in Genitsaris et al.'s study, metabarcoding data did not reveal similar trends with the exception of the positive correlation of ammonia and the Operational Taxonomic Unit closely related to Noctiluca scintillans.
The High-Throughput Sequencing tools implemented in Genitsaris et al.'s study showed a high diversity of known, frequently occurring Protists, also detected with microscopy in previous studies of the area. It further revealed several taxa previously undetected in the area, with seemingly important roles in the structure and functioning of the entire unicellular Eukaryotic communities, and key contribution to the phenomena of red tides, harmful Algal blooms, and mucilage aggregates observed during the study. The unicellular Eukaryotic assemblages showed temporal patterns rather than small-scale spatial separation, possibly responding to the water temperature and salinity seasonal variations. The rich species pool along with the eutrophic status of the area facilitates the unicellular Eukaryotic succession, mainly favoring the taxa affected by the established cycles of temperature and salinity. Overall, environmental pressures (eutrophication, nitrogen pollution) under seasonal changes of water temperature and salinity (circulation pattern), are suggested to be the major drivers of the composition changes and the appearance of blooms and red tides in the study area.
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