Phytoplankton composition from Araçá Bay and São Sebastião Channel, São Paulo, Brazil

Abstract Despite its small area, Araçá Bay (AB) holds cultural, historical, and economic value and displays great benthic biodiversity. Thus, it is crucial to monitor its environmental health, including knowing the main groups of phytoplankton and their temporal variability. The shallow waters of Araçá Bay are continuously modified by the complex hydrography of the adjacent São Sebastião channel (SSC), challenging standard experimental designs for phytoplankton collection. Here we report changes in phytoplankton composition at intervals of five to six weeks from September 2013 to August 2014 in both Araçá Bay and SSC. Samples were collected twice daily for three consecutive days to increase taxonomic resolution. Our goal was to provide an inventory of species occurrences to aid future public policies and environmental management of the area. Analyses revealed high species richness and 166 different phytoplankton taxa. Diatoms and dinoflagellates were always numerically dominant, but taxa occurrence changed markedly. Diatoms of the genera Pseudo-nitzschia were abundant during spring and summer concurrently to signatures of South Atlantic Central Water in the SSC, while Thalassiosira occurred when waters displayed relatively lower salinity. The inventory demonstrated several potentially harmful species of microalgae and cyanobacteria, strongly suggesting investments in monitoring programs in this area that currently experience an increase in population.


Introduction
Phytoplankton communities vary according to the physicochemical conditions of the water (Margalef 1967), but knowledge on the specific composition of these communities remain challenging (Basterretxea et al. 2020).The occurrence and the dominance of a given phytoplankton species reflect its adaptation to the environment (e.g., Anderson et al. 2002, Kremer et al. 2017, Moser et al. 2017, Ryabov et al. 2021).Hence some large-scale generalizations about the taxonomic variability and abundance of phytoplankton can be made in the ocean.Nearshore, however, environmental conditions vary over time scales of hours to days, and the same is true for phytoplankton diversity, for which observations require intense sampling effort.The quantification of species in the world ocean (Sournia, 1991) is a laborious work.Although new instruments and techniques (e.g.Sosik & Olson, 2007) are now available, microscopy analyses remain invaluable for their validation.The availability of phytoplankton species inventories is essential at urbanized coastal sites as they subsidize environmental management actions.
The São Sebastião channel (SSC), located in the north portion of the São Paulo state coast, between the municipalities of São Sebastião and Ilhabela, is partially inserted in the Marine Environmental Protection Area of the North Coast of the State of São Paulo.In the central portion of the channel, a shallow tidal plain (average depth of 1.5 m) limited by rocky flanks (Amaral et al. 2010) is known as Araçá Bay.The bay has an extensive intertidal region, which can be fully exposed to the air and exceeds 300 m in length during low spring tides (Amaral et al. 2018), with a large area of the plain being immersed and submerged within the same tidal cycle (Siegle et al. 2018).Araçá Bay is of esteemed value to the local population because, in addition to harboring high biological diversity (Amaral et al. 2010), it is a stronghold of artisanal fishers who traditionally use small vessels for fishing or leisure (Amaral et al. 2018).
In the past years, the north coast of São Paulo experienced increasing population growth and environmental impacts (Xavier et al. 2016), including the discharge of untreated sewage.Indeed, there is evidence that the interaction of the São Sebastião channel with the continent plays an important role in the exchange of nutrients (Gubitoso et al. 2008) on primary productivity (Regaudie et al. 2017).
Available phytoplankton studies in SSC waters consisted of surveys reporting changes in biomass (i.e., chlorophyll concentration) or relative abundance of major taxonomic groups and their relationships with nutrient concentrations (Muller-Melchers 1955, Gianesella et al. 1999, Saldanha-Corrêa & Gianesella 2003).A review of phytoplankton studies carried out along the São Paulo coast provided a comprehensive inventory of the species present from 1913 to 2006 (Villac et al. 2008).However, no further diversity studies are available.More recent analyses of changes in chlorophyll concentration fractionated by size classes (Giannini & Ciotti 2016) and main taxonomic groups (Ciotti et al. 2018a) derived from efforts during the Araçá Thematic FAPESP project (https://biota-araca.org/index.html),conducted from September 2013 to August 2014 and showed the importance of diatoms when phytoplankton biomass increased.The present study is also derived from the Araçá Project phytoplankton dataset (Tocci 2016) and focuses on detailed taxonomic descriptions of phytoplankton in Araçá Bay and SSC, using light microscopy.The main objective is to update the phytoplankton taxa for this region, report their relative occurrence frequencies, and describe differences between the species found in Araçá Bay and the adjacent waters in the São Sebastião channel.

Materials and Methods
The phytoplankton checklist is composed of samples derived from three oceanographic stations located in the interior of Araçá Bay (AB) and a single station located in the southern portion of the São Sebastião Channel (SSC) at the 15 m isobath (Figure 1).Nine surveys occurred between September 2013 and August 2014 every five to six weeks, in the morning and afternoon of three consecutive days (Ciotti et al. 2018b) to increase the probability of observing the variable hydrodynamics of SSC and rarer phytoplankton taxa.We used a Sontek Castway CTD to vertically profile the temperature and salinity at each station and a 5 L Van Dorn bottle to collect water for analyses of inorganic nutrients, chlorophyll-a, and phytoplankton cell enumeration.Three water samples were combined to represent AB and SSC to increase the representativeness of the occurring taxa analysis.AB samples refer to the combination of three independent stations located at isobaths between 1.5 and 2 m, while SSC refers to three successive deployments of the Van Dorn bottle (Tocci, 2016).The composite samples were further concentrated (2 L to about 100 mL) by reverse filtration with a 5 μm nylon mesh and preserved with formaldehyde neutralized in hexamethylenetetramine (0.4 %).Climatological data on precipitation rates were consulted on the CPTEC-INPE website (http://clima1.cptec.inpe.br/).
Cell enumeration and taxonomic analysis used Üthermol sedimentation chambers of 5 mL or 10 mL and an inverted optical microscope ZEISS-Axio® Observer D1 equipped with phase contrast and differential interference contrast (DIC).Only cells with maximum linear dimension MLD > 5 µm were counted, and identifications reached the lowest possible taxonomic level (genus and species) only for cells with MLD > 10 µm, with the help of specialized literature (e.g., Tomas 1997, Tenenbaum et al. 2004, Tenenbaum et al. 2006, Garcia & Odebrecht 2009, Haraguchi & Odebrecht 2010).Names and synonyms were checked and updated by queries of the Algaebase database (Guiry & Guiry 2021), and the diatom classification followed the work by Medlin & Kaczmarska (2004).The records for Pseudonitzschia followed the nomenclature of Hasle (1965) that divided the colony-forming species of the genus Nitzschia into two complexes: the "delicatissima" -for cells with widths equal to or smaller than 3 µm, and the «seriata» for cells wider than 3 µm.These two complexes were later combined and updated to the genera Pseudo-nitzshia (Hasle 1994).

Results
Seawater temperature varied from 19.4 to 29.4° C in Araçá Bay (AB) and from 16.4 to 29.7°C in São Sebastião Channel (SSC), while salinity ranged from 30.8 to 36.6 in AB and from 30.9 to 35.7 in SSC (Table 1).A mixture of South Atlantic Central Water (SACW, thermohaline     1).During all surveys, we observed smaller volumes of accumulated precipitation compared to the regional climatology.Phytoplankton densities were as high as 10 6 cel L -1 (Table 2).Despite the significance of picoplankton and nanoplankton for Brazilian coastal waters, it is worth mentioning that this inventory covered organisms greater than 5 µm.Diatoms were the predominant taxonomic group at both AB and SSC, with pennate diatoms representing 67% of phytoplankton species in the latter during the sampling period.Comparatively larger abundances of flagellates and armored dinoflagellates were noticeable at AB and SSC, respectively (Table 2).
The diatom genus Thalassiosira predominated during fall and winter (Figure 2), while Pseudo-nitzschia spp.prevailed during October and December 2013 (spring events) and January 2014 (summer) (Figure 2), after periods of high precipitation rates and when surface waters were warm and showed high phosphate concentrations.The highest densities of Pseudo-nitzschia spp. in October 2013 (10 5 10 5 cel.L -1 ) were concurrent with the presence of cold waters (19.4°C, 35.5) near the SSC bottom (data presented in Ciotti et al. 2018a).
Overall, our inventory showed that diatoms and dinoflagellates represented together, over 80% of the total (Figure 4), similar to what was presented by Villac et al. (2008) for the coast of São Paulo state (diatoms 62%, dinoflagellate 34%).

Discussion
Our results are analogous to those presented by Villac et al., (2008), who reported 193 distinct taxa over a longer extension of the São Paulo coast (between Cananéia and Ubatuba) from 2004 to 2006.Their inventory included 120 diatoms, 65 dinoflagellates, and 3 silicoflagellates.In the present study, however, we observed larger contributions of the diatom genera Pseudo-nitzschia, Thalassiosira, Chaetoceros, Hemiaulus, Cyclotella, Coscinodiscus, Guinardia, Rhizosolenia, Thalassionema, Cylindrotheca, and Leptocylindrus, and the dinoflagellate genera Prorocentrum, Scrippsiella, Tripos, Gymnodinium, Dinophysis, and Heterocapsa.One addition to Villac et al. (2008) inventory was the diazotrophic cyanobacteria Richelia intracellularis (unfrequent taxa), either free or in symbiosis with diatoms at both AB and SSC.Although this result can be partially related to our sampling design, differences in environmental conditions between the two studies cannot be discarded, reinforcing the importance of frequent assessments of phytoplankton genera or species.
The 5 to 6-week interval observations revealed some temporal distinctions in the taxonomic composition of the phytoplankton.For example, the diatom genera Thalassiosira and Pseudo-nitzschia were consistently frequent (Table 3, Figure 2).However, their abundances tended to alternate.In addition, Thalassiosira (class Mediophyceae) was frequent when taxa richness was high, while when Pseudonitzschia (class Bacillariophyceae) was predominant, the richness of taxa was low, and their highest abundances occurred synchronically to intrusions of South Atlantic Central Water in SSC.
In temperate marine ecosystems, the succession between dominant phytoplankton taxa tends to be seasonal, leading to blooms (e.g., Cui et al. 2018, Fragoso et al. 2021).The genus Pseudo-nitzschia, with about 55 species (Guiry & Guiry 2021), can form blooms in coastal regions globally (e.g., Trainer et al. 2012), and some species are known to be potentially harmful by producing the neurotoxin domoic acid (Hasle 2002).The genus Thalassiosira contains more than 100 species (Round et al. 1990), but as for Pseudo-nitzschia and other genera of the class Bacillariophyceae, such as Navicula, Pleurosigma, and Gyrosigma, species-level identification requires scanning electron microscopy.
Our results suggest not only the establishment of urgent monitoring programs for harmful algal blooms (HABs) given the frequent potential species year-round at both sites but also that these programs need to encompass proper techniques for distinguishing taxa, as species identification by optical microscopy alone is incomplete (e.g., Hoppenrath et al. 2007, Hamsher et al. 2011, Fernandes et al. 2014, Sterrenburg et al. 2015).
In our study area, the typical physical accumulations of phytoplankton cells nearshore can episodically include organisms that advect from the open ocean guided by winds (Lugomela et al. 2002), which may be the case for the diazotrophic cyanobacteria Trichodesmium spp.and Richelia intracellularis at both sampling sites.Slicks of the genus Trichodesmium are commonly observed in surface waters of the Brazilian Current (Detoni et al. 2016) or in inner shelf waters (< 50 m) during the summer (Brandini et al. 1989).The occurrences could be linked to the relatively low nitrogen input from the continent, as the observations took place during a dry period (Tocci 2016), favoring the growth of diazotrophic cyanobacteria.However, at least for Trichodesmium, the advection of waters from offshore by mesoscale winds (Castro Filho & Mirada 1998) could be a source of these organisms for the coast.Moreover, favorable upwelling winds will favor intrusions of the South Atlantic Central Water in the SSC, not only enhancing the local concentration of nutrients and primary production rates (Regaudie et al. 2017) but also transporting diatoms, such as Pseudo-nitzschia, that impacted the overall taxa richness.These results indicate the need for future phytoplankton monitoring programs assessing the offshore contribution of water masses to SSC.
The observation of unfrequent taxa of tycopelagic diatoms (Table 1) at AB and SSC included the predominance of Cylindrotheca closterium, Diploneis weissflogii, and Thalassionema nitzschioides.The Bacillariophyceae Cocconeis sp.1, D. didymus, D. cf.smithii, Licmophora tincta, Delphineis sp.1, Rhaphoneis sp.1, and Surirella sp.1 showed larger densities at AB than SSC, and some species only observed at AB, such as Diploneis didymus, Diploneis cf.smithii, Stenopterobia sp.1, Surirella sp.1, and Plagiogramma sp.1, probably a result from the bay hydrodynamics that due to its shallower depth (Siegle et al. 2018) allows organisms to resuspend to the water column during each tidal cycle.This continuous exchange of phytoplankton organisms between the sediments of the bay and SSC water needs further evaluation for a better description of this ecologically important system.
Note that some of the identified taxa are mentioned in the literature as non-toxin-producing bloom formers (Odebrecht et al. 2001, Hallegraeff et al. 2003, Moestrup 2004, Villac et al. 2008), such as the diatoms Asterionellopsis glacialis, Cerataulina pelagica, Cylindrotheca closterium, Guinardia delicatula, Leptocylindrus minimus, and Chaetoceros spp.; the dinoflagellates Tripos fusus and Tripus hircus; and the silicoflagellate Dictyocha fibula, may alternatively bloom.However, accumulations of these species may result in many other ecologic and economic impacts (Castro et al. 2016).Additionally, results also reveal the lower diatom diversity when the genus Pseudo-nitzschia was abundant, which occurred during SACW intrusions in the channel.
Our results stress the demand for the urgent implementation of monitoring programs that aid public policies for environmental safety.The occurrences of taxa are known to be potentially harmful, highlighting the dinoflagellates of the genera Alexandrium, cf.Gymnodinium, Dinophysis, Gonoyaulux, and Prorocentrum are unsettling.Although some initiatives are in place, our results demonstrate the need for a comprehensive monitoring program that includes modernized methodologies and hydrodynamical modeling.If conditions for blooming are favored with nutrients input by sewage and warming of seawater temperatures, they may cause fish death, mollusk poisoning, and several public health problems (Hallegraeff et al. 2003).

Figure 1 .
Figure 1.Location of the sampling sites at Araçá Bay (AB) and São Sebastião Channel (SSC) near the oceanographic buoy of SIMCosta Project.

Table 1 .
Environmental Variables measured during samplings at São