Many dinoflagellates produce benthic cysts as a part of their life cycle (reviewed in Belmonte and Rubino, 2019). The resting cysts accumulate in the sediments and serve as potential seed banks that secure the survival of the species over time (Lundholm et al., 2011; Ribeiro et al., 2013). They also play a crucial role in bloom initiation, especially important in the case of harmful algal bloom species (HABs) (Anderson et al., 2003; Garcés et al., 2004). Encystment is considered as an adaptive strategy the species play out to respond to environmental stresses driven by variations of environmental conditions, such as temperature, salinity, nutrient concentration or daylength. These variations can also influence the patterns of spatial distribution of dinocysts (Anderson et al., 1984; Devillers and de Vernal, 2000; Sgrosso et al., 2001; Grigorszky et al., 2006; Richter et al., 2007; Zonneveld and Susek, 2007; Kremp et al., 2009; Lundgren and Granéli, 2011; Shin et al., 2018). Furthermore, intraspecific morphological variability among cysts produced by some species, including dinoflagellates like Scrippsiella spp., has been associated to variations in environmental conditions, both in cultures (Kokinos and Anderson, 1995) and in the field (Olli and Anderson, 2002; Zonneveld and Susek, 2007; Mertens et al., 2009) and this intraspecific variability can be read as a quantitative measure of environmental variations (Mertens et al., 2009; Mertens et al., 2012a; Persson et al., 2013; Shin et al., 2013; Hoyle et al., 2019).

The cosmopolitan dinoflagellate Scrippsiella acuminata (Ehrenberg, 1836) Kretschmann, Elbrächter, Zinssmeister, S. Soehner, Kirsch, Kusber & Gottschling, 2015 (the currently accepted name of Scrippsiella trochoidea (Stein, 1883) Loeblich III, 1976), is one of the dominant species in the phytoplankton community in the Black Sea, responsible for bloom events in the coastal areas of this basin (Nesterova et al., 2008; Terenko and Terenko, 2009; Moncheva et al., 2019 and the references therein). Even the cysts of this species are widespread in the basin, being dominant in the western region off the Bulgarian coast (Rubino et al., 2010). Their presence is reported also from many sites in the South (Mudie et al., 2017) and in the Northwest off the Ukrainian coast where the abundance increases with the depth in the shelf zone (Nikonova, 2010; Nikonova, 2015; Mudie et al., 2021).

Many studies, aimed to clarify the taxonomic characters both of thecae and cysts of S. acuminata and the cyst-theca relationships, showed that actually this species should be considered as a member of a complex (Montresor et al., 2003; Gottschling et al., 2005) with cryptic species genetically differentiated but morphologically indistinguishable so far (Zinssmeister et al., 2011; Kretschmann et al., 2014).

Based on the high morphological variability of the S. acuminata cysts already observed in the Black Sea (Dzhembekova et al., 2020), and in the Mediterranean (Ferraro et al., 2017; Rubino et al., 2017) and reported also from the East China Sea (Gu et al., 2008), the aim of the present study was to clarify whether the geographic distribution of the different cyst morphotypes of S. acuminata in the surface sediments of many coastal and shelf sites of the Black Sea, could be related to other environmental variables, besides hypoxia and pH, as reported by Shin et al. (2013) and Ishikawa et al. (2019) from the Pacific Ocean. Accordingly, sea surface temperature (SST), sea surface salinity (SSS), currents speed (CS), station depth, nitrates (NO₃) and phosphates (PO₄) were analyzed as plausible drivers of the spatial distribution and abundance of S. acuminata cyst morphotypes.

The present study represents one of the largest basin-scale surveys (depth 13.1–2100 m) of Scrippsiella acuminata (the currently accepted name of S. trochoidea) cyst distribution in the Black Sea conducted to this date. As a consequence, a great variability in cysts abundance throughout the study area was observed, both for the whole dinoflagellate encysted community and the S. acuminata complex. Even considering the natural variability linked to the different seasons of sampling, the huge amplitude of densities observed (ranging from 5 to 5,981 cysts g^-1 dry weight of sediment for viable forms), most likely could be associated with the different abiotic and biotic conditions occurring within the Black Sea study region, as demonstrated in the recent literature. In a comprehensive study of cysts distribution from 43 samples at water depths of 71–905 m on the outer Ukrainian Shelf and upper continental slope, Mudie et al. (2021) reported the presence of Scrippsiella spp. cysts at almost all the investigated sites with densities ranging between 2-89 cysts g^-1 dry weight of sediment. The cysts of S. acuminata (as S. trochoidea) were recorded at 6 sites only with very low abundance (1–10 cysts g^-1 dry weight of sediment) ( Figure 2 ). Nikonova (2010; 2015) found much higher densities (190–1,690 cysts g^-1 dry weight of sediment), at three stations in the same area close to those observed in our study (Stations 1, 5 and 6) ( Table 2 ; Figure 2 ). Over the investigated 34 sites, our samples recorded a presence of the S. acuminata cysts widespread throughout the entire area and particularly higher relative abundance (46 to 100% of Scrippsiella viable forms) as compared to the data (less than 5%) documented by Mudie et al. (2017). The study of Mudie et al. (2017) was based on information from different datasets considering data from top 2 cm of sediment, similar to the current study. However, the use of hydrochloric acid impose difficulties in the differentiation of the cyst morphotypes of species with organic walls such as S. trochoidea (= S. acuminata), as highlighted in the paper (Mudie et al., 2017). Even if the highest densities were detected at the deep open sea stations (up to 4,275 cysts g^-1), high values were also observed at the coastal stations (up to 2,556 cysts g^-1) and at some shelf stations, suggesting no distinct pattern of spatial distribution related to depth and/or distance from the coast. Similarly, Mudie et al. (2021) exploring the relationship to sediment grain size, abiotic and biotic oceanographic parameters, including methanogenesis, found that Scrippsiella cysts (mostly cysts of S. acuminata) tend to be more abundant at higher depths on the continental slope, but this species could be also dominant on the continental shelf-break around 200 m depth.

Cysts identified in surface sediment samples are usually assumed as recently produced in relation to the sedimentary regime of the area. Nevertheless, the natural encysted community represents an integration in time and space of the species encystment/excystment dynamics and consists of different “dormancy aged” assemblages (Kim and Han, 2000) expression of different time intervals with a succession of highly variable environmental conditions. They reflect the dominant ecological processes for plankton over several years rather than a seasonal signature (Dale, 1976), that imposes a further complication to explain dinocyst distribution patterns with environmental specific conditions at the time of sampling (Devillers and de Vernal, 2000). Taking into account various factors such as sedimentation, sampling method, and amount of bioturbation, Mudie et al. (2017) estimated that the time span represented by the Black Sea surface sediment samples is between less than five years to a few decades. Therefore, in the present study, environmental multiyear data were used in the statistical analysis, to account for the disparity between the time scale of the cyst record and the time scale of the environmental factors (Ribeiro and Amorim, 2008; García-Moreiras et al., 2021) and to ensure better approximation of the model linking environmental gradients with cysts abundance data.

In this study, Redundant discriminant analysis (RDA) helped to explore the relationships between the environmental variables and the S. acuminata cyst assemblages. All the cysts morphotypes were found to be associated to a single or a combination of environmental variables, confirming that cysts beds are repositories of ecophysiological diversity for the planktonic communities (Ribeiro et al., 2013). The links revealed by the applied multivariate analyses between temperature, salinity, phosphates, and nitrates and the spatial distribution of cyst morphotypes conform to findings in other studies (e.g., Sala-Pérez et al., 2020); salinity was generally reported to induce primarily variation in cyst process morphology of some Gonyaulacacean dinoflagellate species (e.g., Ellegaard, 2000; Mertens et al., 2009). In particular, process length variation has been observed to mostly change with water density (as a function of salinity and temperature) (Mertens et al., 2011; Mertens et al., 2012a; Mertens et al., 2012b). However, other variables have been mentioned as drivers of intraspecific variation, such as water column deoxygenation (Pross, 2001; Mudie et al., 2021), temperature and nutrients, associated with intraspecific cyst body size variation (Mertens, 2013; Mousing et al., 2017). In our study, the diagnostic of morphological variability of the cyst produced by S. acuminata was particularly related to the cyst wall surface structure (calcification and length of the processes), cyst body shape and size. The literature provides contradictory links between nutrients levels and Scrippsiella cyst morphotypes. Accordingly, the naked-type (i.e. non-calcified) cysts of S. acuminata were perceived as a morphological response to pH in the sediments and it has been suggested that they could be an indicator for eutrophication or acidification (Shin et al., 2013). On the contrary, Wang et al. (2007) reported high rates of formation of the naked cyst-type of S. acuminata during the bloom and shortly after the bloom, perceived to be related to the cellular nutrient limitation, while Gu et al. (2008) did not find any effect of nutrients on calcification in laboratory conditions.

Our results suggest that the formation of different morphotypes of cysts of S. acuminata complex within the water depths studied is regulated by specific environmental conditions (salinity, temperature, nitrates and phosphates) and even if some morphotypes appeared to have more confined geographical distribution, our data did not explain the existence of explicit ecological niches. The ecological niche theory (Pocheville, 2015) assumes that differences in species composition among communities are caused by heterogeneity in the environment or by limiting resources and environmental filtering of species according to their environmental requirements, such as climate, oceanographic conditions, both in pelagic and coastal areas, and competition for resources such as nutrients for marine algae, as a result of biotic interactions (Cadotte and Tucker, 2017).

In order to add new information about the relation between environmental drivers and S. acuminata morphotypes a very cautious approach should be used to interpret the data, considering the high cryptic diversity within the Scrippsiella species complex (Montresor et al., 2003). Essentially, the taxonomy of species within S. acuminata complex is still quite challenging and problematic. DNA-based results showed the presence of two S. acuminata ribotypes in the Romanian Black Sea assigned ambiguously as S. cf. erinaceus and S. aff. acuminata (Žerdoner Čalasan et al., 2019). Thus, additional molecular analyses would be vital (Žerdoner Čalasan et al., 2019) to clarify if the morphotypes are ecotypes, produced under distinctive environmental conditions, indicating different survival strategies of a single species (Sgrosso et al., 2001), or they represent a variety of cryptic species (Zinssmeister et al., 2011).

We found out that the Black Sea sediments harbor phenotypically diverse seed banks of S. acuminata complex as described also in other geographic areas (Montresor et al., 2003; Gu et al., 2008; Rubino et al., 2016; Rubino et al., 2017). Noteworthy, S. acuminata morphotypes dominated in the sediment trap sample, collected at 1,000 depth, confirming the high efficiency of the species in cyst production (Montresor et al., 1994; Wang et al., 2004; Morozova et al., 2016). The environmental and hydrodynamic conditions in the water column can influence the resting stage assemblages as species composition and abundance, and the resting stage encystment/excystment dynamics, by coupling the bentho-pelagic compartments (Casabianca et al., 2020). The conditions leading to bloom seeding and initiation are still not fully understood, although seeding from planktonic populations is considered the most plausible hypothesis (García-Moreiras et al., 2021 and the references therein). The high S. acuminata complex cyst densities documented in our study suggest the build-up and growth in time of significant cyst beds. They play a crucial role in the dinoflagellate bloom dynamics, seeding the planktonic blooms and refueling the communities in the water column (Kremp, 2001; Rubino and Belmonte, 2021). Moreover, they ensure the persistence of the species in the environment, serving as a benthic reservoir of biodiversity (Belmonte and Rubino, 2019). The geographical spread of Scrippsiella blooms in the Black Sea indicates that most likely the interplay between the planktonic and benthic habitat is advantageous and common for S. acuminata, allowing it to dominate in the plankton communities and proliferating to bloom densities (Nesterova et al., 2008; Moncheva et al., 2019). Outbursts of S. acuminata have been observed basin-wide since the ‘70s in abundance ranging between 7.8 x 10⁵–125.4 x 10⁶ cells/L – in Odessa Bay (Ukraine) in 2005 (8.0 x 10⁶ cells/L, Terenko and Terenko, 2009) and Crimea in 2011 (4.4 x 10⁶ cells/L, Bryantseva and Gorbunov, 2012), in Romanian waters (25.8–125.4 x 10⁶ cells/L in the period 1973–2005, Bodeanu, 2002; Nesterova et al., 2008), in Varna Bay (Bulgaria) (1.9 x 10⁶ cells/L, Moncheva et al., 1995) in Turkish waters – Bay of Sinop (7.8 x 10⁵ cells/L during 1995–1996, Turkoglu and Koray, 2002) and Trabson (1.34 x 10⁶ cells/L in 2000, Feyzioğlu and Oğut, 2006), and in Russian waters (Novorosiysk) (8.6 x 10⁶ cells/L in 2005–2006, Yasakova, 2010).

Although the new finding of different cyst morphotypes and ecological affinity to the environmental conditions and regional distribution advances our understanding of S. acuminata complex, many uncertainties remain about their ecological role in the benthic seed banks and plankton dynamics above all. Moreover, there are unanswered questions about the persistence and expansion of homogenous/heterogeneous vegetative populations in the water column in relation to the dynamics and distribution of cyst beds in the sediment. Further research focusing on the pelagic/benthic coupling however seems promising for large-scale monitoring and tracking phytoplankton community alterations and climate change effects.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

SM, ND, and IZ conceived the study. ND, SM, FR, and IZ wrote the manuscript. FR and MB analysed the cysts. SM and NS carried out the samplings and organized the research cruises. VS extracted and prepared the environmental data and maps. IZ carried out statistical analyses. All authors commented on the manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by the National Science Fund, Ministry of Education and Science (MES), Bulgaria under project “Phytoplankton cysts – an intricacy between a “memory” or a “potential” for Black sea biodiversity and algal blooms” (Grant number DN01/8, 16.12.2016) and the National Science Program “Environmental Protection and Reduction of Risks of Adverse Events and Natural Disasters”, approved by the Resolution of the Council of Ministers № 577/17.08.2018 and supported by the Ministry of Education and Science (MES) of Bulgaria (Agreement № D01-230/06.12.2018).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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