Seasonal Assemblages of picocyanobacteria in the subtropical segment of the Kuroshio Current

doi:10.21203/rs.3.rs-3121110/v1

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Seasonal Assemblages of picocyanobacteria in the subtropical segment of the Kuroshio Current

https://doi.org/10.21203/rs.3.rs-3121110/v1

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A long-term observation has been conducted on the community composition diversity of picoplankton in the subtropical segment of Kuroshio Current from 2009 to 2015. The results showed that Synechococcus mainly distributed in the surface water layer regardless of seasonal changes, and the number of cells ranged from 10⁴ to 10⁵ cells mL^− 1. In contrast, the maximum value of Prochlorococcus was maintained at more than 10⁵ cells mL^− 1 throughout the year, and the stirring effect caused by the monsoon determined their distribution in the water layer. In the summer and the autumn, Prochlorococcus were mainly concentrated at the water layer near the bottom of the euphotic zone. In the spring and the winter, they were evenly distributed in the euphotic zone. In addition, the results of 16S rRNA gene diversity analysis showed that the seasonal changes in the relative abundance of Synechococcus and Prochlorococcus in the surface water of each station accounted for 20 to 40% of the total reads. Proteobacteria and Actinobacteria approximately accounted for 45% and 10% of the total picoplankton in the four seasons. The clade-II of Synechococcus and the High-light II of Prochlorococcus were the dominant strains in the waters all year round. These data will be important basic parameters for us to study the impact of global environmental changes on marine biogeochemical cycles in the western boundary currents in the future.

Picophytoplankton, including picocyanobacteria and picoeukaryotes, are the phytoplankton whose cell size was smaller than 2 µm. Because of their high surface area to volume ratio, picophytoplankton prevail in diverse marine environments, especially thriving in oligotrophic oceans. Generally, marine phytoplankton contribute approximately half of global primary production, which is approximately 46 to 50 pentagrams per year. Of these, picophytoplankton are responsible for approximately 24% of marine phytoplankton production [1, 2]. In particular, picophytoplankton were the major primary producers in some marine provinces. For example, picophytoplankton respond to greater than 70% of the total primary production in the tropical Pacific Ocean [3]. Furthermore, 31–92% of the primary productivity is provided by picophytoplankton in the Mediterranean [4]. The biomass of picophytoplankton is rapidly consumed by heterotrophic microorganisms and in turn enters the grazing food chain or the microbial cycle. Several studies have predicted that ocean warming may cause the fraction of small phytoplankton (picophytoplankton) to increase over that of the larger group [5, 6]. This relatively rapid change might alter the phytoplankton communities toward picophytoplankton sizes. Then, it may further change the functioning and biogeochemistry of pelagic ecosystems. Therefore, picophytoplankton are important primary producers not only in marine ecosystems but also in global oceanic biogeochemistry cycles [7].

Synechococcus and Prochlorococcus are the major members of picocyanobacteria. The abundances of Synechococcus and Prochlorococcus in the global oceans are approximately 10⁴ to 10⁶ cells mL^− 1 [8–11]. However, due to their different biological characteristics, Synechococcus and Prochlorococcus have been suggested to occupy diverse marine provinces. Synechococcus is distributed in the global oceans [8, 12]. Based on genetic information, Synechococcus has been divided into fifteen clades and twenty-eight subclades [13]. Clades I and IV were found in cold and nutrient-rich waters, whereas Synechococcus clades II, III, V, VI and VII frequently appeared in tropical and subtropical waters [9]. Prochlorococcus usually inhabit the euphotic zone of tropical and subtropical oligotrophic waters between latitudes 40°N and 40°S [14, 15]. Based on their distribution in the water column, Prochlorococcus species are categorized into two groups: high-light (HL)- and low-light (LL)-adapted ecotypes [13]. These two ecotypes present distinct distributions across different depths [16–18]. HL-adapted ecotypes have a lower divinyl chlorophyll b/divinyl chlorophyll a (Chl b/Chl a) ratio and are typically found in the surface waters of the open ocean [19]. LL-adapted ecotypes have a higher Chl b/Chl a ratio and usually grow in deep waters, which have much lower light intensities. In addition to the different distributions in the water column, the appearance of two Prochlorococcus ecotypes also exhibits distinct seasonal dynamics. For example, the abundance of LL-adapted ecotypes has no clear seasonal distribution in the northern Red Sea; in contrast, HL-adapted ecotypes have a clear seasonal distribution [20]. Moreover, the HL-adapted ecotypes are composed of six clades. HL clade II was found to dominate at low and midlatitudes but was changed by HL clade I at latitudes above 30° [15, 21]. Furthermore, it has been demonstrated that special conditions, such as Asian dust and typhoons, shift the picophytoplankton community composition [22, 23].

The Kuroshio Current is one of the western boundary currents located in the Northwest Pacific Ocean. The westward-flowing North Equatorial Current runs into the Philippine coast and then bifurcates into northward (i.e., the Kuroshio Currents) and southward streams (i.e., the Mindanao Current). The Kuroshio Current passes the east coast of Taiwan and southeast coast of Japan and then continues as the North Pacific current [24, 25]. The width of the Kuroshio mainstream in eastern Taiwan is approximately 100 kilometers, the depth is approximately 800 to 1000 meters, and the mainstream speed is approximately 1 to 1.5 m s^− 1. The Kuroshio water is high-temperature and high-salinity. In the subtropical segment of the Kuroshio, its temperature and salinity have no significant seasonal variation and remain at 26°C to 30°C and approximately 34.5 psu, respectively [26]. The Kuroshio water in the euphotic zone is characterized as ultraoligotrophic [27, 28]. However, when the Kuroshio current flows near the east coast of Taiwan, upwelling along the coastal region transports deep nutrients into the surface [27, 29]. The uplift of deep seawater provides a large amount of nutrients to the Kuroshio euphotic zone when the Kuroshio invades the East China Sea shelf [30]. In addition, terrestrial material injection and vertical mixing caused by typhoons or the northeast monsoon provide nutrients to the Kuroshio current. These nutrient sources support primary productivity in the Kuroshio region [30–32]. The surface Kuroshio is oligotrophic and has low chlorophyll a (Chl a) concentrations of less than 0.2 mg m^− 3 [27, 33–36]. The yearly primary production in the subtropical Kuroshio current is approximately 0.91 ± 0.47 mgC/m³/day [33]. Several studies have presented a significantly positive correlation between Chl a and the abundance of picophytoplankton, implying that picophytoplankton are the major primary producers in the Kuroshio [36, 37]. A variety of global circulation models have predicted that the abundance of picophytoplankton will increase significantly under global climate change scenarios [38]. However, data on in situ observations of picoplankton composition and distribution are still lacking. In this study, the seasonal dynamics of picophytoplankton in the subtropical Kuroshio current were revealed. This information will expand our understanding of picophytoplankton’s contribution to marine ecosystems and biogeochemical cycles under global climate change.

Sampling scheme

This study is a long-term observation of phytoplankton community composition in the subtropical Kuroshio Sea from 2009 to 2015. Intensive sampling in four seasons was conducted from 2012 to 2013. A total of nine stations across the Kuroshio current were visited onboard the research vessels Ocean Researcher I and Ocean Researcher II in October (defined as the autumn voyage) 2012, January (defined as the winter voyage) 2013, April (defined as the spring voyage) 2013 and July (defined as the summer voyage) 2013 (Fig. 1). Temperature and salinity were continually recorded by a conductivity/temperature/depth recorder (CTD) (SBE9/11 plus) (Sea-Bird Electronics). To determine the concentrations of chlorophyll a (Chl a) and inorganic nutrients and the abundance of picophytoplankton, water samples were collected using 20-L GO-Flo bottles (General Oceanics) mounted on the CTD rosette sampler. Light intensity in water was continually measured by a photosynthetically active radiation irradiance sensor (PAR sensor) (Chelsea Technologies) equipped with a CTD. The surface water (depth = 5 m) and the water at the euphotic zone bottom, which was defined as the depth of 1% surface light penetration, at Stations K2, K4, K5, K6 and K8 were collected to isolate environmental DNA for analyzing the phylogenetic diversity of the 16S ribosomal RNA (rRNA) gene.

Determination of Chl a and inorganic nutrient concentrations

The detailed methods for determining the concentrations of Chl a and inorganic nutrients are described in Chan et al. (2020) [39]. Briefly, one liter of water for Chl a analysis was immediately filtered through a GF/F class filter (Whatman, Maidstone, UK) and stored at -20°C until analysis. The Chl a retained on the filter was extracted in 90% acetone. The Chl a concentration was determined by a fluorometer (10-AU-005) (Turner Design) [40]. To evaluate the fraction of Chl a contributed by picocyanobacteria to the total Chl a concentration, we applied 1.258 fg Chl a cell^− 1 and 1.203 fg Chl a cell^− 1 as the conversion parameters for Prochlorococcus and Synechococcus, respectively, which were determined by the average values of the Chl a concentrations of several algal strains measured by high-performance liquid chromatography (HPLC) [41].

To determine the concentrations of nitrate (NO₃) and phosphate (PO₄), the water sample (100 mL) was placed in a polypropylene bottle, immediately frozen with liquid nitrogen and stored at -20°C until analysis. The NO₃ and PO₄ concentrations were measured by the pink azo dye method and the molybdenum blue method, respectively. The detection limits of NO₃ and PO₄ are 0.3 and 0.01 µM, respectively [42–44]. The nitracline depth was defined as the depth at which the NO₃ concentration difference was 0.5 µM with reference to the surface value [45–47].

Determination of picophytoplankton abundance

The cells were fixed with paraformaldehyde at a final concentration of 0.2% (w/v) and were preserved in liquid nitrogen. Different picophytoplankton populations were categorized with flow cytometry (FACSAria) (Becton-Dickinson) based on cell size and autofluorescence in the range of orange from phycoerythrin (575 ± 15 nm, for Synechococcus) and red from chlorophyll (> 670 nm, for Prochlorococcus and photosynthetic picoeukaryotes) under excitation at 488 nm. A known number of fluorescent beads (TruCOUNT tube) (Becton-Dickinson) were parallelly calculated to convert the original cell abundance in the sample [22]. The putative relationships between the picophytoplankton distribution and ambient hydrographic characteristics were analyzed by redundancy analysis (RDA) and the “envfit” function in the vegan package in R.

Picoplanktonic DNA isolation

Seawater was filtered through a 5-µm mesh nylon net to remove larger plankton. The planktonic cells in the filtrate were collected by 0.2-µm pore size polycarbonate membranes (Nucleopore) (Whatman) under gentle vacuum (≤ 100 mmHg). The membranes were immediately frozen in liquid nitrogen until DNA isolation. The cells retained on the membranes were disrupted with lysozyme (Roche) and proteinase K (Roche) treatments. After purification by hexadecyltrimethylammonium bromide and phenol/chloroform/isoamyl alcohol (25/24/1, v/v/v), the DNA pellet was precipitated using isopropanol and resuspended in Tris-EDTA buffer (pH 8.0) [22]. The DNA concentration and purity were determined by spectrophotometry (NanoDrop) (Thermo Scientific) at wavelengths of 230, 260, 280 and 320 nm.

The diversity of picoplankton composition

DNA product (10 ng) was used as the template for the polymerase chain reaction (PCR) to specifically amplify the V3-V4 region fragments of 16S rRNA genes using the high-fidelity DNA polymerase 2× KAPA HiFi HotStart ReadyMix (Roche) and the forward primer 16SV3V4-F (5’-tcgtcggcagcgtcagatgtgtataagagac AGCCTACGGGNGGCWGCAG-3’) and the reverse primer 16SV3V4-R (5’-gtctcgtgggctcggagatgtgtataagagacAGGACTACHVGGGTATCTAATCC-3’) (The lowercase letters indicate the Illumina adaptor sequences; W = A or T; H = A, T or C; V = A, C or G; N = A, T, G or G) [48]. The amplicons were analyzed by the Illumina MiSeq high-throughput nucleotide sequencing platform (Illumina) using the pair-end method. After the removal of low-quality reads (quality score > Q20) and the trimming of adaptor and primer sequences by Cutadapt software [49], the diversity of the resultant reads was analyzed by DADA2 software [50]. The taxonomic assignment of representative amplicon sequence variants (ASVs) obtained by DADA2 analysis was further conducted with the 16S rRNA reference sequences in the Silva database (version 138).

The indices of richness (abundance-based coverage estimator, ACE) and diversity (Shannon) were estimated by the sequences randomly subsampled (the size of the smallest library was used) from each sample 1,000 times and were expressed as averages to avoid biases generated by differences in the sequencing depth. The beta diversity analysis was performed with hierarchical clustering to visualize the spatiotemporal distribution of the picoplankton community assemblage by PRIMER 6 software with the Bray‒Curtis distance.

Nucleotide sequence deposition

The nucleotide sequences used in this study have been deposited in the Sequence Read Archive (SRA) database under BioProject accession number PRJNA904529.

Based on physical characteristics such as current velocity, temperature and salinity, all stations were subdivided into K1, which was the station with coastal upwelling; Stations K2 to K4, which were located at the mainstream of the Kuroshio current; and Stations K5 and K6, which were close to the Kuroshio, and their hydrological characteristics were affected by it. Stations K7 to K9 were located in the open ocean and were regarded as the reference, where they were not affected by the Kuroshio Current [42, 51]. The hydrography in the upper water column (≤ 100 m) of the Kuroshio is high-temperature, high-salinity and low-nutrient content [42]. In addition to Station K1 being primarily affected by upwelling, the intensity of monsoon blowing determines the mixing grade of water columns at other stations. The northeast monsoon started in the autumn (October) and led to the occurrence of upper water column mixing. This stirring effect was the most vigorous in the winter (January), and the surface water temperatures reached low values of 22 to 24°C. With the weakening of the monsoon, the water mixing gradually moderated in the spring (April). In the summer (July), significant stratification was observed and resulted in a high surface temperature greater than 29°C (Fig. 2A to D). Salinity was maintained above 34.5 at each station throughout the year, although there was a stirring effect derived from the monsoon in the autumn and the winter (Fig. 2A to D).

Seasonal dynamics of nutrient distribution in the subtropical Kuroshio, taking NO₃ as an example, except for the higher concentrations observed at the coastal upwelling stations, the concentrations of both nutrients in the euphotic zones in the Kuroshio mainstream and oceanic province were extremely low (Fig. 2E-2L). In the autumn (i.e., Oct. 2012) (Fig. 2E) and the summer (i.e., Jul. 2013) (Fig. 2H), the depth of the nitracline gradually increased from the coast to the open sea. In contrast, in the winter (i.e., Jan. 2013) (Fig. 2F) and the spring (i.e., Apr. 2013) (Fig. 2G), except for the upwelling stations, the nitracline at oceanic stations was raised and maintained at a depth of approximately 100 m. The seasonal distribution of PO₄ was similar to that of NO₃. The elevation of the nitracline derived from the mixing effect caused by the northeast monsoon in winter and spring would facilitate the upward transport of deep-sea nutrients. The seasonal distribution of chlorophyll was roughly in response to the distribution of nutrients. In the winter and spring, there was an even distribution of chlorophyll in the upper layer (Fig. 2M-2P). However, the maximum chlorophyll layer in the summer and autumn occurred at water depths of approximately 50 m and 100 m, except for that at the upwelling station (K1), which was found at the surface (1.29 mg Chl a m^− 3) (Fig. 2M-2P). Furthermore, the highest content of Chl a (186.36 mg Chl a m^− 3) within the depth ≤ 100 m of most stations was found in the winter.

The dominance of Synechococcus and picoeukaryotes was found in the surface water in both the cold and warm seasons (Fig. 3A-D and I-L). The highest abundance of Synechococcus and picoeukaryotes at 10 m at the K1 station (1.5 x 10⁵ cells mL^− 1 and 2.2 x 10⁴ cells mL^− 1) occurred in the summer and spring. However, the maximum total cell numbers of Synechococcus and picoeukaryotes in the upper water column (≤ 100 m) appeared in the winter (9.3 × 10⁸ cells cm^− 2 and 2.3 × 10⁸ cells cm^− 2). In contrast to Synechococcus and picoeukaryotes, Prochlorococcus was mainly found in the summer and autumn (Fig. 3E-H). The total Prochlorococcus concentrations in the upper water column (≤ 100 m) in these two seasons were 3.3 × 10⁹ cells cm^− 2 and 2.4 × 10⁹ cells cm^− 2. In addition, it had the highest number at specific depths (75 m and 50 m) in these two seasons. In contrast, the distribution of Prochlorococcus was more even at depths ≤ 100 m in winter, although the total concentration of Prochlorococcus in the upper water column (≤ 100 m) was the same as that in autumn (2.9 × 10⁹ cells cm^− 2). The distribution of Prochlorococcus was also even, with the lowest abundance in spring of all seasons. According to the conversion factors of Chl a concentration of Synechococcus and Prochlorococcus (1.065 fg Chl a cell^− 1 and 1.51 fg Chl a cell^− 1) [52], their contributions to total Chl a at different sampling sites are shown in Fig. 4. On average, picocyanobacteria were responsible for approximately 30–50% of the total Chl a in the four seasons; the highest percentage was shown in September 2009 and August 2015. Synechococcus contributed the lowest percentage of total Chl a in October (1.5%), while the highest contribution was in September (Fig. 4). Prochlorococcus contributed the highest percentage of total Chl a in the summer (August, 31.7%).

The total abundance of Prochlorococcus, Synechococcus, and picoeukaryotes at depths above 100 m had distinct correlations with biotic and abiotic environmental factors in the RDA (Fig. 5). For example, Prochlorococcus was positively correlated with nitracline depth, while Synechococcus was negatively correlated with water temperature and the depth of the euphotic zone.

A total of 4,410,567 high-quality reads were obtained after eliminating short- and low-quality sequences. The total reads had 1.6 to 9.8 × 10⁵ reads at each station. The overall coverage at each station was greater than 99%. Each station obtained 996 to 3,012 ASVs (Table 1). The diversity and richness indices increased following the distance of the station away from the coast. Open ocean stations had higher diversity and richness indices of picoplankton compared with the stations affected by the Kuroshio current. Furthermore, the open ocean station (K8) had the highest richness and diversity indices, while the lowest indices were at the Kuroshio current station (K4) in summer (Table 1). Hierarchical clustering analysis showed that the bacterial community composition in the surface layer was different from that in the DCM layer (Fig. 6). This result suggested that niche partitioning in the community occurred (ANOSIM, R = 0.68, p = 0.001).

Table 1

The results of 16S rRNA gene V3-V4 sequencing and the indices of species richness (ACE) and diversity (Shannon).
Time	Station	Reads	ASV	ACE	Shannon
Oct.2021	K4	161662	996	997	4.74
	K6	199278	1162	1162	4.95
	K8	319142	1551	1552	5.11
Jan.2013	K2	427192	2082	2083	5.71
	K4	407216	1807	1808	5.01
	K5	976914	3012	3014	5.38
	K6	272391	1590	1590	5.22
	K8	367891	1764	1765	5.36
Apr.2013	K2	291525	1335	1335	5.07
	K4	206446	1233	1233	5.16
	K5	312912	1409	1409	4.73
	K6	294990	1235	1235	4.65
	K8	222253	1201	1201	4.88
Jul.2013	K2	251552	1235	1236	4.97
	K4	283202	1139	1139	4.75
	K5	289476	1134	1134	1.88
	K6	278805	1161	1162	4.85
	K8	626538	1898	1899	5.03

In terms of the community structure of picoplankton at the surface of the Kuroshio current, the ASVs were categorized into the phyla Cyanobacteria, Proteobacteria, Actinobacteria and Bacteroidetes (Fig. 7). Each taxon occupied a consistent ratio in total reads among all stations during the four seasons. In detail, the reads affiliated with Cyanobacteria occupied 20 to 40% of the total reads. The highest cyanobacteria percentage occurred at the K4 station in each season. The maximum percentage of cyanobacteria at the K4 station was in the winter (41.6%). Proteobacteria consistently responded approximately 40–50% (Fig. 7). The major four genera of Proteobacteria were Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria and Flavobacteria. The most dominant genus was Alphabacteria, which accounted for approximately 20–30.8% of the total picoplankton during the four seasons. Actinobacteria accounted for only 6.6 to 14.6% of the total picoplankton in the four seasons.

The ASV reads for Synechococcus and Prochlorococcus in the total cyanobacteria phylum showed different seasonal patterns in both the surface and DCM layers (Fig. 8). In detail, Prochlorococcus accounted for 71.1–92.2% of the total cyanobacteria in summer (Fig. 8D and H) and autumn (Fig. 8A and E) in the surface and DCM layers. In winter, the relative abundance of Prochlorococcus significantly decreased (Fig. 8B and F) in the two layers. In particular, Prochlorococcus in DCM accounted for only 20–30% of the total cyanobacteria when it accounted for 40–80% on the surface. Then, the relative abundance of Prochlorococcus increased again in spring (40–60% of total cyanobacteria) in the two layers. In contrast, Synechococcus had a high percentage in winter and spring, accounting for approximately 20–80% of the total cyanobacteria, while that in summer and autumn accounted for only 2.3–13.8% in the two layers (Fig. 8). Within the Synechococcus group, clades I, II, III, and VII formed the major group in the main Kuroshio current (Fig. 9). Synechococcus clade II had the highest relative abundance of Synechococcus (90%) in four seasons of the surface. Synechococcus clade II was also dominant in January and April of DCM. However, it decreased by approximately 50% in the DCM warm seasons when the relative abundance of clade VII increased. Prochlorococcus was categorized into two groups, the high light group (HL) and the low light group (LL), by the differential Chl a intensity in flow cytometry. The HL-II groups existed in both the surface and DCM and were responsible for more than 90% of the total relative abundance of Prochlorococcus (Fig. 10). The HL-I group was mainly observed in the DCM and had a high relative abundance in the summer. Finally, the LL group only presented DCM. Interestingly, the relative abundance of the LL group started to increase in spring and had a maximum relative abundance in the summer (approximately 20%).

The Kuroshio current is an important western boundary current in the Northwest Pacific Ocean. More than half of Chl a in the Kuroshio current is contributed by picoplankton [36]. However, in comparison with other highly oligotrophic oceanic currents (i.e., the Gulf of Mexico), few studies have focused on prokaryotic picoplankton in the Kuroshio current [53–55]. Thus, in this study, we revealed the detailed community structure of picoplankton and their distribution in the Kuroshio current among four seasons. It would help to understand the interaction between the western boundary current hydrography and the picophytoplankton succession.

In our in situ observation results, the highest abundance of Synechococcus primarily appeared at the K1 station in summer (1.5 x 10⁵ cells mL^− 1) and spring (9.9 x 10⁴ cells mL^− 1), where there was strong nutrient input from upwelling (coastal uplift). On the other hand, obvious stratification was found at other stations. Thus, the nutrients at the other stations were scarcer than those at the K1 station and could not support Synechococcus, which had a high abundance similar to that at the K1 station. Liu et al. (2021) [56] indicated that the growth of Synechococcus in the Kuroshio current was enhanced following increasing temperature by dilution experiments. In addition, under high temperature (surface water temperature + 4°C), a higher growth rate of Synechococcus was observed in nutrient-replete conditions than in nutrient-limited water [56]. Hence, in the summer of the Kuroshio current, sudden nutrient input events could induce Synechococcus to thrive temporarily. It has also been demonstrated that the nutrients brought by dust storms, typhoons and coastal uplift promote the growth of Synechococcus [22, 23, 36]. In addition, deeper nutrients uplifted by upwelling also stimulated Synechococcus to grow in the upwelling area [57, 58].

Another interesting finding was that the total cell number of Synechococcus in the upper water column (≤ 100 m) was higher in the winter than in the summer. From a previous study, the growth rates of Synechococcus increased with increasing water temperature. Although the average surface water temperature in summer was 28.7°C, it remained at 26.1°C in the winter season of the Kuroshio current. Following a previous study, the growth rate of Synechococcus at 26°C remained comparable to that at 28°C. Thus, we suspected that the growth rate of Synechococcus remained in these two seasons. Furthermore, because of monsoon-induced vertical mixing, the nutrients in the winter were transported more effectively to the surface layer in the winter. Additionally, there was strong upwelling invasion during cold seasons. The upwelling events were observed not only at costal stations (K1 and K2) but also at open ocean stations (K8 and K9). From the above, these factors likely contributed to a more evenly distributed number of Synechococcus across each station in the winter. In fact, the highest number of Synechococcus in winter reached 9.3 × 10⁴ cells mL^− 1. Consequently, the total cell number of Synechococcus in the upper water column (≤ 100 m) was the highest during the winter season (9.3 × 10⁸ cells cm^− 2). This explains the negative correlation between water temperature and the abundance of Synechococcus in the RDA. Monsoon-induced vertical mixing, upwelling and holding high water temperature in the Kuroshio current caused even distribution and high total Synechococcus abundance in the upper water column (≤ 100 m) in winter.

Offshore of northeastern Taiwan, upwelling often occurs when the branch of the Kuroshio current intrudes the East China Sea shelf at higher latitudes. Chung and Gong (2019) [58] discovered that the surface of upwelling sites exhibited a high abundance of Synechococcus (5.9 × 10⁴ cells mL^− 1), ranging from 1 to 2 x 10⁴ cells mL^− 1 in the surface waters of the sites influenced by the Kuroshio current. The relative abundance of Synechococcus, based on total 16S rRNA amplicon sequencing, was up to 96%. These Synechococcus populations contained highly phylogenetic divergence, including clade II, clade X, and clade XI [58], with clade II being the most dominant (96% of total Synechococcus). In this study, we focused on a more southern Kuroshio current, characterized by low available nutrients in situ. Therefore, the differences found in the abundance of Synechococcus compared with the findings of Chung and Gong (2019) [58] could be attributed to variations in the formation of upwelling caused by the main or branch currents of the Kuroshio current system. Additionally, this study revealed the presence of Synechococcus in clade VII, clade III, and clade X. These variations in clade composition are likely influenced by different depths of upwelling, which might provide distinct nutrient concentrations for microorganisms in situ [59–61].

We hypothesized that similar to Synechococcus, picoeukaryotes may also be stimulated by nutrient input. The high abundance of both Synechococcus and picoeukaryotes in the winter may be due to nutrient input from subsurface layers through mixing. In addition, we found that picoeukaryotes were also particularly abundant on the surface of upwelling stations (K1) during the spring and summer. The RDA revealed a positive correlation between the abundance of picoeukaryotes and Synechococcus (Fig. 5), supporting our hypothesis that the two microorganisms are correlated. Chan et al. (2020) [39] also demonstrated that the abundance of picoeukaryotes in the Kuroshio current was positively correlated with Synechococcus abundance (Pearson correlation, p < 0.01).

Prochlorococcus is dominant in many oligotrophic environments, such as central oceanic gyres and the southern Gulf of Mexico [62, 63]. It has also consistently been observed as the prevailing group upstream of the Kuroshio current [56, 64]. According to our results, the total abundance of Prochlorococcus in the upper water column (≤ 100 m) showed a positive correlation with the nitracline depth (p = 0.031). This indicates that the total Prochlorococcus increased as the nitracline depth increased, which has a scarce nitrate concentration. This relationship was particularly evident during summer and autumn, characterized by a deeper nitracline compared to other seasons (summer: R² = 0.80 and p < 0.05; autumn: R² = 0.75 and p < 0.05). Notably, while Prochlorococcus exhibited a more widespread vertical distribution in other seasons, its distribution was concentrated at specific depths within the DCM during summer and autumn. Furthermore, the relative abundance of the LL and HL-I Prochlorococcus ecotypes, which thrive in low temperature and low light intensity, increased in DCM during summer and autumn (Fig. 10H and E) [65]. In contrast, the LL and HL-I ecotypes displayed similar relative abundances in the winter season, possibly due to well mixing that brought them to the surface (Fig. 10B and F). Recent studies have revealed that Prochlorococcus carries numerous nitrate assimilation genes and is abundant in or near nitracline in oligotrophic marine environments [53, 66, 67]. Therefore, this distinct difference in abundance is likely attributable to a clear stratification in the subsurface and deep nitracline during summer and autumn that allows the two Prochlorococcus ecotypes to accumulate at deeper depths to rapidly utilize nutrient pulses [68].

The cyanobacteria covered 20 to 40% of the total picoplankton, according to the results for high-throughput sequencing in this study. A previous study also investigated the prokaryotic community in the same sampling sites from June 2013, March, July, and November 2014, and March 2015 by the V5-V6 region of the 16S rRNA gene [69]. In Cheng et al. (2020) [69], the results showed that cyanobacteria had a very low relative abundance (3%) among total picoplankton when the relative abundances of Proteobacteria and Actinobacteria were 59% and 13%, respectively. This finding contradicts the results from this study. We suspected that different DNA extraction methods might be one of the reasons for the different compositions of the picoplankton. The DNA extraction in this study was conducted by the phenol/chloroform method, while Cheng et al. (2020) [69] used a commercial DNA isolation kit. Indeed, the DNA extraction method for processing of samples can yield different amounts and qualities of DNA products [70–75]. In addition, different DNA extraction efficiencies might be reflected in the subsequent community structures [76, 77]. Thus, various modified methods were developed from previous studies for different types of samples [78, 79]. Further study will be needed to determine whether different DNA extraction methods could alter the composition of picoplankton from the same marine water sample.

The nutrient-scarce, warm, and high-salinity Kuroshio Current has a profound impact on both the marine ecology of the northwestern Pacific Ocean and the global climate [80–82]. It is important to understand the characteristics of the fundamental microorganism community in different regions within the Kuroshio Current. This study revealed that the composition of prokaryotic picoplankton was significantly different between the surface and DCM, except in January, which had a deep mixing zone. Synechococcus (dominated in Clade II) and Prochlorococcus (dominated in HL-II groups) were the major members of picocyanobacteria, which accounted for half of the Chl a in the Kuroshio current. The seasonal dynamics of Synechococcus were caused by water temperature, nutrient input and euphotic zone, whereas Prochlorococcus had a positive correlation with nitracline depth. Thus, nutrients in situ rapidly and highly affected the seasonal dynamics of these fundamental microorganism groups.

Western Boundary Currents are driven by the differences in the density of the water masses on either side of the boundary, and these density differences are largely driven by temperature and salinity gradients in situ. The authors suggest that as the Earth's climate warms, these density differences will weaken, resulting in a slowing of WBCs (including the Kuroshio current) [82]. The potential impact of changes in Western Boundary Currents on the transport of nutrients and other important biogeochemical constituents could ultimately impact the abundance and distribution of picoplankton in these regions. Hence, we propose that long-term monitoring of the variations in picocyanobacteria and their relative biotic and abiotic factors can highlight their importance in the ocean, which is regulated by global warming.

Funding

The study was supported by the research grants MOST103-2611-M-019-011, MOST104-2611-M-019-011, MOST-111-2611-M-031-001, and NSTC-112-2611-M-031-001 from the Ministry of Science and Technology of Taiwan (Now restructured as the National Science and Technology Council of Taiwan). This work was also financially supported by the Center of Excellence for the Oceans, National Taiwan Ocean University from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education of Taiwan.

Conflict of Interest

The authors declare no competing interests.

Ethics Approval

Not applicable.

Author Contributions Statement

CCC and GCG conceived and designed the study and experiments. IJL and CWH collected samples and performed the experiments. CCC and YFC analyzed the data and wrote the manuscript. All authors critically revised the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We thank the captains and crew of the research vessels Ocean Researcher I and Ocean Researcher II for their assistance. We also thank the National Core Facility for Biopharmaceuticals and the National Center for High-performance Computing of National Applied Research Laboratories of Taiwan for providing computational resources and storage resources.

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No competing interests reported.

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Seasonal Assemblages of picocyanobacteria in the subtropical segment of the Kuroshio Current

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Abstract

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Introduction

Materials and Methods

Sampling scheme

Determination of Chl a and inorganic nutrient concentrations

Determination of picophytoplankton abundance

Picoplanktonic DNA isolation

The diversity of picoplankton composition

Nucleotide sequence deposition

Results

Discussion

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References

Additional Declarations

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