Physiological response of Prorocentrum lima (Dinophyceae) to varying light intensities

Physiological response of Prorocentrum lima (Dinophyceae) to varying light intensities Abstract The benthic dinoflagellate Prorocentrum lima is among the most common toxic morphospecies with a cosmopolitan distribution. This study explored if strains from different environments and different morphotypes, isolated from three locations in the Atlantic Iberian Peninsula and two from the Mediterranean Sea, showed different responses to varying light regimes, after confirming that all strains belonged to the same ribotype. Growth rates and photosynthetic parameters such as Fo, Fv/Fm, and rETRmax were analysed with a Coulter counter, a water-PAM and a fast repetition rate fluorometer. The photosynthetic properties were investigated in a high light stress experiment using strains acclimated to low light (LL) and high light (HL). The highest growth rate was 0.23 day−1 at 80 and 100 μmol photons m−2 s−1 for strains Dn150EHU and Dn60EHU, originated from different locations. Under control conditions (18°C and 90 μmol photons m−2 s−1), growth rate was on average 0.10 day−1. The HL stress exposure induced photodamage to all strains and the recovery period was not sufficiently long for full recovery of Fv/Fm. However, cells acclimated to HL showed a better recovery than the LL acclimated ones. Furthermore, some assumptions are discussed in relation to strains’ original location. dinoflagellates, growth rates, high light stress, microalgae, photophysiology INTRODUCTION During the last decades, the genus Prorocentrum Ehrenberg (Dinophyceae) has been the focus of several research works due to the fact that numerous species of dinoflagellates are known to produce toxins (Dodge 1975; Fukuyo 1981; Faust 1991; Morton and Tindall 1995; Pan, Cembella and Quilliam 1999; Yoo 2004; Hoppenrath et al.2013; López-Rosales et al.2014; Nascimento et al.2016). This genus has a worldwide distribution and contains about 60 species, from which 29 are known to be benthic (Hoppenrath et al.2013). Prorocentrum lima (Ehrenberg) Stein is the most commonly reported Prorocentrum benthic morphospecies, and latest studies, which included new geographic areas, have revealed a complex structure of genetically distant ribotypes (Nagahama et al.2011; Zhang et al.2015; Nascimento et al.2016). Moreover, variability within morphological features of P. lima is common (Nagahama et al.2011). Therefore, and for comparative purposes, it is crucial to know the precise molecular identity of the biological material employed, since physiological features can differ among lineages (e.g. Parsons et al.2012). Prorocentrum lima is widely present in the Mediterranean Sea (Aligizaki et al.2009; Vanucci et al.2010; Aissaoui et al.2014; Ben-Gharbia et al.2016) and in the Atlantic Iberian Peninsula (Vale, Veloso and Amorim 2009; Laza-Martínez et al.2011). Moreover, a broader study on the benthic Ostreopsis and Coolia (David et al.2013, 2014) revealed P. lima to be present in all 17 sites sampled along the Atlantic coast of the Iberian Peninsula. After a preliminary analysis (unpublished) revealed that all collected strains showed no phylogenetic differences, it was decided to investigate the response of this microalga to varying light irradiances. This was decided as the Iberian Peninsula coast presents contrasting temperatures and irradiances and also between its Atlantic and Mediterranean coasts, which may trigger local adaptations on benthic microalgae. Moreover, most of the recent work on Prorocentrum has been focused on the species taxonomy (Hoppenrath et al.2013; Zhang et al.2015; Nascimento et al.2016; Luo et al.2017) or chemical and toxin analyses (Huang et al.2014, 2015; Wang et al.2015; Prego-Faraldo et al.2017), with only a few related to P. lima ecophysiology (Gilbert, Burkholder and Kana 2012; Aissaoui et al.2014; Ben-Gharbia et al.2016). Light availability is one of the most important factors affecting marine environments, as light promotes photosynthetic activity, but can also inhibit many biological processes if radiation becomes excessive (Baek, Shimode and Kikuchi 2008; Celis-Plá et al.2014). Furthermore, changes in temperature, irradiation, salinity, nutrients and other environmental factors are known to influence cell proliferations (Heredia-Tapia et al.2002; Zhang and Hu 2011; Fraga et al.2012), as well as the amount of toxins they produce (Guerrini et al.2009). Although there is still much to uncover about the photophysiological properties of benthic species within the genus Prorocentrum, it is known that physiological and photoprotective mechanisms might differ among microalgae species according to their phylogeny and the environment where they grow (Stomp et al.2004). Epibenthic dinoflagellates are common in clear waters where high irradiances reach the benthos. As a mechanism of protection, it has been suggested that some of them can migrate to the macroalgae-shaded areas (Ballantine, Tosteson and Bardales 1988), and that microalgae possess an adaptive response system to highly variable light conditions (Ihnken, Eggert and Beardall 2010). Different levels of photoinhibition can be triggered by excessive irradiance (Franklin and Forster 1997) where dynamic photoinhibition can be defined as a short-term reversible, regulatory process for the controlled dissipation of excessive light energy, mediated by the xanthophyll cycle (e.g. Goss et al. 2006), and chronic photoinhibition as a slowly reversible process that may occur following prolonged exposure to excessive irradiances (Osmond and Grace 1995). These are based on the typical kinetics of relaxation (recovery of photosynthesis) of the biochemical and biophysical processes implicated in photoinhibition (Franklin and Forster 1997; Hanelt 1998) and can be assessed by changes in the chlorophyll fluorescence parameters (Franklin et al.1992; Jones and Hoegh-Guldberg 2001; Goss et al.2006). To gain insight into the photophysiology of these microalgae, several techniques have been developed. The fast repetition rate fluorometer (FRRF) and the water-PAM (pulse amplitude modulation) are highly sensitive apparatuses based on the variable in vivo chlorophyll fluorescence of phytoplankton that provide several photosynthetic parameters that have been widely used on phytoplankton research (Schreiber, Bilger and Neubauer 1994). The directly measured parameter F0 from the water-PAM and FRRF can be used as a proxy of algal biomass (Dijkman and Kromkamp 2006; Kromkamp et al.2008), and (Fm − F0)/Fm (Fv/Fm), the maximum quantum efficiency of photosystem II (PSII) photochemistry, can be used as a measure of stress (Baker 2008; Kromkamp et al.2008; Suggett et al.2012; Betancor et al.2015). In addition, rapid light curves (RLCs) or photosynthesis-irradiance (P-E) curves can be made under different controlled conditions (Lewis and Smith 1983; White and Critchley 1999; Johnson and Sheldon 2007) to estimate the relative electron transport rate (rETR) of PSII (Ralph and Gademann 2005). Other parameters derived from these curves, namely light-limited initial slope (α), saturation light intensity (Ek) and maximal relative electron transport rate (rETRmax), are good indicators of the light regime and photoacclimatory behaviour of the cells (Suggett et al.2012), and can provide a good estimate of the photosynthetic rate (Kromkamp et al.2008; Figueroa, Jerez and Korbee 2013). The present study aimed to know if P. lima strains isolated from different localities around the Atlantic Iberian Peninsula and the Mediterranean Sea showed local adaptations regarding their photophysiology. With this aim, localities representing different latitudes and from both coasts were selected. Strains from lower latitudes and from the Mediterranean were expected to be better adapted to high-light intensities. Additionally, one of the Mediterranean strains presented a roundish morphology, differing from the typical oval/ovoid P. lima strains, which allowed us to test if different cell shapes were related to adaptations leading to photophysiological differences. The cells’ photosynthetic performance was measured using an FRRF, a water-PAM and a Coulter counter. The strains’ identity was checked by molecular techniques prior to analyses. MATERIALS AND METHODS Study area The latitudinal range of the study was between 37°N and 44°N including five different locations from the Atlantic Iberian Peninsula and the Mediterranean Sea coasts (Fig. 1): San Sebastian in the north of the Iberian Peninsula (43.321397, –1.986706), Vigo in the northwest of the Iberian Peninsula (42.223871, –8.767948), Galé in the south of the Peninsula (37.078863, –8.314001), Villefranche-sur-Mer in the north Mediterranean Sea (43.703857, 7.320063) and Ibiza in the western Mediterranean Sea (38.967708, 1.241127). Although somewhat narrow, this latitudinal range presents a characteristic variability between the different localities: the north of the Atlantic Iberian Peninsula has higher annual mean cloud coverage compared to the south (Sanches-Lorenzo et al.2009). This is supported by the yearly sum of global solar irradiation with data from 1986 to 2005, generated by the Meteonorm software (www.meteronorm.com), which indicated that the location of San Sebastian, in the north of the Iberian Peninsula, received the smallest amount of ground solar radiance (1200–1300 Kwh/m2) from all five locations. Next, it was Vigo (1500–1600 Kwh/m2), Villefranche-sur-mer (1600–1700 Kwh/m2), Ibiza (1700–1800 Kwh/m2) and Galé (1900–2000 Kwh/m2). These values include a complete range of seasonal conditions and solar angles in each site (Paulescu et al.2013). Figure 1. View largeDownload slide Study area sampling sites. Figure 1. View largeDownload slide Study area sampling sites. Seasonal variability also differs between locations where the north (San Sebastian) is characterised by an oceanic climate with cool winters and warm summers where precipitation is dispersed throughout the year. Vigo has a warm-temperate climate characterised by warm dry summers and rainy winters. The other three locations of Galé, Ibiza and Villefranche-sur-Mer are characterised by a hot-temperate Mediterranean climate having mild winter temperatures and hot dry summers (Peel, Finlayson and McMahon 2007). The underwater light field also varies. In the Mediterranean, the water is very clear (e.g. Lüning 1990) with higher light intensity (about 10 m depth) reaching the benthos (Figueroa and Gómez 2001). Furthermore, the attenuation coefficient for PAR (KdPAR) is around 0.07–0.09 m−1 in the Mediterranean Sea (Wiencke et al.2000; Celis-Plá et al.2014), which is a great contrast with the Atlantic Iberian Peninsula coast locations where mean Kd varies from 0.6 to 0.9 m−1 (Gohin et al.2005; Brito et al.2013). Higher Kd values express a higher influence of currents, resuspension of sediments and run-off. Tidal range also differs markedly between the Atlantic and the Mediterranean. Whereas the tidal regime in the Atlantic area is semidiurnal with a tidal range of ca. 2–4 m, tides are negligible in the Mediterranean Sea. The semidiurnal depth variation caused by the tidal regime means a parallel light-environment variation in the Atlantic area compared to the more stable environment in the Mediterranean. Moreover, tidal regime generates more turbidity in shallow shore waters due to sediment resuspension. Strains selection The strains used to study the photophysiological responses of cells from different locations (Fig. 1) representing differing light environments were as follows: Dn60EHU from San Sebastian (North of the Iberian Peninsula), Dn80EHU from Vigo (NW of the Iberian Peninsula), Dn116EHU from Galé (South of the Iberian Peninsula) and Dn150EHU from Villefranche-sur-Mer (Mediterranean Sea). For the high light (HL) stress experiment, strain Dn141EHU from Ibiza (Mediterranean Sea), representing a rounder morphotype, was also used (Fig. 2) for comparison with the elongated strains. Cells were collected at 1–2 m depth during low tides. Isolation and settlement of cultures were described in David et al. (2013). Cells were observed under light microscopy (Leica DMRB). Prior to the photophysiological analyses, strains’ identity was checked with molecular methods using the large ribosomal subunit (LSU) and the internal transcribed spacer (ITS) regions as markers. Results showed that all strains belonged to the lineage included in clade C of the LSU and clade D of the ITS tree from Zhang et al. (2015). This lineage includes all the existing Mediterranean and European sequences of P. lima. Additionally, all strains except Dn141EHU, which had a roundish morphology, showed the typical P. lima elongated morphology. The molecular and morphological characterisation of the strains will be reported elsewhere. Figure 2. View largeDownload slide Comparison of both P. lima morphotypes. (a) Typical cell of P. lima; (b) round morphotype. Scale bar = 10 μm. Figure 2. View largeDownload slide Comparison of both P. lima morphotypes. (a) Typical cell of P. lima; (b) round morphotype. Scale bar = 10 μm. Growth rates at different light intensities Strains were previously acclimated for a month in 50 mL Nuclon (ThermoFisher Scientific, Spain) culture flasks, containing 20 mL of F/2 Guillard's marine culture medium (Sigma), under 18°C, to a light intensity of 90 μmol photons m−2 s−1 (measured with a light meter (LI-250A, LI-COR, Fairborn, OH)) and a 12:12 light/dark hour cycle. Then two sets of experiments were performed. For the first one, an aliquot of 5 mL of the acclimated culture flasks was used to start fresh new cultures in new 50 mL Nuclon culture flasks, with a final volume of 20 mL to monitor their growth rate at this irradiance. Every other day, flasks were shaken and 400 μL of each culture was retrieved to an eppendorf tube, for cells count. This was measured in triplicate with a Coulter counter (Multisizer 3, Beckman coulter, Inc., Netherlands) during 20 days to compare later with the fluorescence values of the second experiment, given from the pulse amplitude modulated (PAM) fluorometer (water-PAM, Walz, Germany). From the exponential phase, growth rates were obtained by fitting the data in SigmaPlot 13.0 (Systat Software, Inc. GmbH, Germany) according to the following equation: Nt = N2exp(μ × t) where N2 is cell concentration at day 2 and Nt is the cell number at day t. During the same time, the second set was prepared. An aliquot of 1 mL was retrieved from each of the freshly renewed cultures made for Experiment 1, and placed in duplicate into a 24-multiwell culture plate that was left under cold fluorescent light, at a constant temperature of 18°C and seven different light intensities (30, 60, 80, 100, 150, 200 and 300 μmol photons m−2 s−1). Increases in the minimal fluorescence (F0) were measured in triplicate everyday, at the same hour, for 11 days. This was measured with a saturating light pulse from the fibre-optic version of the water-PAM (water-ED, Walz GmbH, Effeltrich, Germany). Culture plates were left in the dark for 10 min, and shaken before each measurement so cells could be detached from the bottom and be uniformly dispersed in the wells, and the fibre of the EDF unit of the water-PAM was placed in the middle of each well for the measurements. The residual fluorescence due to the empty well and culture medium without algae was subtracted before calculation of fluorescent yields. Growth rates, μ (day −1), were calculated from the whole exponential growth phase as μ = ln(Nt/N0)/t, where Nt and N0 are the dark acclimated fluorescence yields (F0) at time t and 0, respectively, and t = x days (Crow and Kimura 1970). Generation time (G) was calculated as G = ln2/μ (Guillard 1973). HL stress experiment Strains were previously acclimated in duplicate to a 12:12 light/dark hour cycle for 3 months at 18°C in 150 mL Nuclon culture flasks and diluted every 3 weeks by removing about 20 mL and adding new culture medium (Guillard F/2) until a final volume of 50 mL to maintain cultures in exponential phase. During that time, the two series of cultures were exposed to different irradiances: One to low light—LL (40 μmol photons m−2 s−1) and another to HL (150 μmol photons m−2 s−1) at 18°C before they were subjected to the HL stress. A short-term HL stress experiment was performed with all the given strains of P. lima, where 2.5 mL of each culture was transferred to a test tube and incubated for 10 min in the dark. The tube was then placed in front of a LED panel with a photon flux density of 500 μmol photons m−2 s−1 for 20 min to induce stress, and the cells’ recovery was recorded by following their Fv/Fm in the dark for the following 30 min (‘recovery phase’). This HL intensity was approximately three times their Ek, which is the irradiance value where photosynthesis switches from light-limited to light-saturated rates. Using values three times higher than their irradiance-onset saturation parameter was a way to ensure that cells would be in stress. This value was obtained from previous RLCs with the same cultures, by fitting data into a revised P-E model of Eilers and Peeters (1988) (Herlory et al.2007; Silsbe and Kromkamp 2012). During the entire experiment, RLCs on 2.5 mL subsamples were measured with an FRRF (FastTracka-II/FastAct) (Fast Repetition rate fluorometry, Chelsea Technologies Group Ltd) at crucial changing points in the experiment (point 0 in acclimated conditions, point 1 after 10 min of dark incubation, point 2 after 20 min of light stress (min 30), point 3 in the middle of recover (min 45), and point 4 at the end of the experiment (min 60)). Each RLC consisted of 10 irradiance steps between 0 and 1504 μmol photons m−2 s−1, and each light step lasted 30s. The values of the PSII quantum efficiency of the RLCs were then fitted in the Eilers and Peeters model as described above, to retrieve rETRmax values of each curve on each step. Furthermore, several acquisition points to monitor the Fv/Fm evolution were measured in between the RLCs. From RLC1 to RLC2 (light phase) measurements were taken every 3 min. From RLC2 to RLC3 (dark phase), measurements were taken every minute for the first 5 min and then every 2 min. From RLC3 to RLC4 (dark phase), these were taken every 5 min. Cells will be fully recovered if the final Fv/Fm value is similar to the initial ones. For a better comprehension, the RLC data were then separated into the photoinhibition HL period and the recovery period, where the first was from minute 10 to 30 and the latter from minute 30 until the end of the experiment (min 60). Hanelt (1998) revealed that during an HL stress, it was possible to describe the two kinetics of photoinhibition with two rate constants, associated with two fractions of PSII centres: a fraction (Pfast) associated with a fast rate constant (Kfast) and a ‘slow’ fraction (Pslow) with a slow rate constant (Kslow). Knowing these constants at any time (t) allows us to compare the different reactions of different strains to different irradiances. By fitting the changes of the PSII quantum efficiency during the inhibition and recovery phase we could calculate these data. Thus, the PSII quantum efficiency from the RLCs was fitted, with the solver added in Excel, using the following equations of Hanelt (1998):   \begin{equation*} {\rm{inhibition}}\,{\rm{kinetics}}:\frac{{\Delta F}}{{{{F\!'}_m}}} = {P_1} \cdot {e^{ - {k_1} \cdot t}} + {P_2} \cdot {e^{ - {k_2} \cdot t}}, \end{equation*}   \begin{equation*} {\rm{recovery}}:\frac{{\Delta F}}{{{{F\!'}_m}}} = {F_v}/{F_m} - \left( {{P_1} \cdot {e^{ - {k_1} \cdot t}} + {P_2} \cdot {e^{ - {k_2} \cdot t}}} \right). \end{equation*} So, P1 and P2 are the proportion of the centres with a fast and slow component, and k1 and k2 are the corresponding rate constants of fast and slow inhibition and recovery. It is thought that the RCII fraction with the components is related to the xanthophyll cycle, and P2 is probably related to a higher chronic damage of the PSII centres. Fv/Fm is the maximum PSII efficiency of the non-inhibited cells at t = 0 and P1 + P2 = Fv/Fm. Recovery becomes faster when k1 is larger or when k2 becomes smaller. Statistical analyses The statistical program SigmaPlot 13.0 (Systat Software, Inc. Germany) was used to analyse all data. Firstly, to find differences between strains and time on growth rate, the two-way analysis of variance (ANOVA) followed by the all pairwise multiple comparison Tukey's t-test was used to compare differences within groups. Then, the same method was employed to compare the estimated growth rates from the F0 values, between and among strains at different light intensities. To test if different light intensities presented relevant changes, a t-test was also performed between groups. Whenever equal variance failed, a Mann–Whitney Rank Sum test was performed. Furthermore, differences in the estimated growth rates from different methods were also tested. Tukey's t-test was also used to compare Fv/Fm values in the HL stress experiment. RESULTS Growth rates Under 90 μmol photons m−2 s−1, cell concentration measured with a Coulter counter increased exponentially in all cultures from day 2 until the end of the experiment, which lasted 20 days (Fig. 3), except for strains Dn80EHU and Dn60EHU, where cultures reached a stationary phase 2 days earlier, after the 18th day. A maximum cell density of 107 × 103 cells mL−1 was observed for strain Dn116EHU at the 20th day. Strains’ exponential growth rate averaged 0.098 ± 0.009 day−1. There were no significant differences among strains, except for Dn150EHU, which showed a significant lower growth rate (ANOVA, P = 0.0059; Table 1) using this method. Figure 3. View largeDownload slide Growth curves of different strains of P. lima in the pre-acclimated culture flasks at 90 μmol photons m−2 s−1. Figure 3. View largeDownload slide Growth curves of different strains of P. lima in the pre-acclimated culture flasks at 90 μmol photons m−2 s−1. Table 1. Average growth rate values with their respective standard error values for each strain of stock cultures maintained at 90 μmol photons m−2 s−1. Strain   location  μ (day−1)  St. error  P. lima Dn60EHU  San Sebastian  0.1010  0.0130  P. lima Dn80EHU  Vigo  0.0946  0.0087  P. lima Dn116EHU  Galé  0.1087  0.0093  P. lima Dn150EHU  Villefranche-sur-mer  0.0875  0.0090  Strain   location  μ (day−1)  St. error  P. lima Dn60EHU  San Sebastian  0.1010  0.0130  P. lima Dn80EHU  Vigo  0.0946  0.0087  P. lima Dn116EHU  Galé  0.1087  0.0093  P. lima Dn150EHU  Villefranche-sur-mer  0.0875  0.0090  Average of all strains: 0.098 ± 0.009 day−1. View Large Growth rates of the strains measured daily with a water-PAM in the multiwell plates under different irradiances (from 30 and 300 μmol photons m−2 s−1) demonstrated that strains Dn150EHU and Dn60EHU were the ones which reached higher growth rates (0.23 day−1) (Fig. 4, Supplementary material), although these were not significantly different from the others (P > 0.5) and corresponded to different light intensities (80 and 100 μmol photons m−2 s−1). Strain Dn80EHU reached a maximum growth rate of 0.21 day−1 at 100 μmol photons m−2 s−1, and strain Dn116EHU had its maximum at 150 μmol photons m−2 s−1 with 0.17 day−1. Overall, strains grew better at moderate light intensities (80–150 μmol photons m−2 s−1) as at higher irradiances, growth rates were severely inhibited for most strains with the exception of Dn80EHU, which showed a growth rate of 0.13 day−1 at the highest irradiance (300 μmol photons m−2 s−1). At the lowest PAR tested (30 μmol photons m−2 s−1), growth rates were very low and similar (∼0.05 day−1). Figure 4. View largeDownload slide Growth rates of different strains of P. lima at different light intensities. Figure 4. View largeDownload slide Growth rates of different strains of P. lima at different light intensities. When comparing both methods, the Coulter counter and the water-PAM, slightly lower growth rates (although not statistical significant, P > 0.5) were estimated by the Coulter counter. HL stress During the HL stress experiment, aliquots of strains previously acclimated to HL and LL were exposed to a high-light stress and their recovery time was studied. Cells acclimated to LL showed higher initial values of the maximum quantum efficiency (Fv/Fm) than the cells acclimated to HL (Fig. 5). The photochemical efficiency of the different strains markedly decreased during the first minutes of exposure to 500 μmol photons m−2 s−1. Tukey's t-test, applied to compare final to initial acquisition points, showed that the cells acclimated to HL performed better than the same acclimated to LL. The difference of means within HL was less than the one in LL (0.090 < 0.115). Regarding the different strains, Dn150EHU from the Mediterranean Sea (Fig. 5B) presented nearly identical values of the initial and final quantum efficiency at HL and LL (Fv/Fm (i-f) = 0.09). The other strain (Dn141EHU) from the Mediterranean Sea (Fig. 5E; round morphotype) showed a similar result but performed better at HL (0.11). Strain Dn116EHU from the south of the Atlantic Iberian Peninsula was the one that better responded at HL (Fig. 5D), showing a difference of Fv/Fm(i-f) = 0.067, followed by Dn80EHU (0.08, Fig. 5A). However, strain Dn116EHU at LL, was the one with a higher difference (0.16) of Fv/Fm(i-f), meaning that it would take longer to reach the initial conditions. Regarding the north of the Iberian Peninsula, strain Dn60EHU (Fig. 5C) at HL was the one from all the HL-acclimated strains with a higher difference from the initial conditions (0.12). All data were fitted into the previously mentioned equations with the inhibition phase separated from the recovery phase and the fitted parameters can be found in Figs 6 and 7 and Table 2. Figure 5. View largeDownload slide Single acquisition points with the FRRF during HL stress experiment from strains acclimated at HL and LL. (A) Strain Dn80EHU, P. lima from Vigo; (B) strain Dn150EHU, P. lima from Villefranche-sur-mer; (C) strain Dn60EHU, P. lima from San Sebastian; (D) strain Dn116EHU, P. lima from Galé; (E) strain Dn141EHU, round morphotype of P. lima from Ibiza. At t = 0, a sample was collected from the culture and put in the dark for 10 min. Then, the sample was exposed to HL for 20 min. At 30 min, the sample was put back in the dark in order to measure the recovery. Figure 5. View largeDownload slide Single acquisition points with the FRRF during HL stress experiment from strains acclimated at HL and LL. (A) Strain Dn80EHU, P. lima from Vigo; (B) strain Dn150EHU, P. lima from Villefranche-sur-mer; (C) strain Dn60EHU, P. lima from San Sebastian; (D) strain Dn116EHU, P. lima from Galé; (E) strain Dn141EHU, round morphotype of P. lima from Ibiza. At t = 0, a sample was collected from the culture and put in the dark for 10 min. Then, the sample was exposed to HL for 20 min. At 30 min, the sample was put back in the dark in order to measure the recovery. Figure 6. View largeDownload slide Proportion of fast (P1) and slow (P2) reacting PSII centres during the 20 min inhibition phase in the HL experiment. Figure 6. View largeDownload slide Proportion of fast (P1) and slow (P2) reacting PSII centres during the 20 min inhibition phase in the HL experiment. Figure 7. View largeDownload slide Recovery parameters. Both left panels show the proportion (%) of the fast (top) and slowly (bottom) recovering PSII centres. The right panels show the rate constants (min−1) for recovery of the fast (top) and slow (bottom). Figure 7. View largeDownload slide Recovery parameters. Both left panels show the proportion (%) of the fast (top) and slowly (bottom) recovering PSII centres. The right panels show the rate constants (min−1) for recovery of the fast (top) and slow (bottom). Table 2. Fit parameters for the inhibition and recovery of the HL- and LL-acclimated strains in the HL stress experiment. Inhibition constant    Dn60EHU   Dn60EHU   Dn80EHU  Dn80EHU  Dn116EHU  Dn116EHU  Dn141EHU  Dn141EHU  Dn150EHU  Dn150EHU    HL  LL  HL  LL  HL  LL  HL  LL  HL  LL  P1  0.16  0.22  0.21  0.26  0.23  0.21  0.12  0.29  0.23  0.26  k1  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  P2  0.17  0.22  0.15  0.22  0.12  0.25  0.26  0.17  0.14  0.21  k2  0.00  0.00  0.00  0.00  0.00  0.03  0.03  0.00  0.00  0.00  Recovery constant  P1  0.21  0.17  0.20  0.22  0.26  0.29  0.28  0.18  0.26  0.22  k1  0.02  0.02  0.04  0.02  0.04  0.03  0.03  0.01  0.03  0.02  P2  0.12  0.27  0.16  0.26  0.09  0.17  0.11  0.28  0.11  0.25  k2  7.30  0.83  2.01  2.01  6.43  2.03  6.66  0.48  6.66  2.01  Inhibition constant    Dn60EHU   Dn60EHU   Dn80EHU  Dn80EHU  Dn116EHU  Dn116EHU  Dn141EHU  Dn141EHU  Dn150EHU  Dn150EHU    HL  LL  HL  LL  HL  LL  HL  LL  HL  LL  P1  0.16  0.22  0.21  0.26  0.23  0.21  0.12  0.29  0.23  0.26  k1  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  P2  0.17  0.22  0.15  0.22  0.12  0.25  0.26  0.17  0.14  0.21  k2  0.00  0.00  0.00  0.00  0.00  0.03  0.03  0.00  0.00  0.00  Recovery constant  P1  0.21  0.17  0.20  0.22  0.26  0.29  0.28  0.18  0.26  0.22  k1  0.02  0.02  0.04  0.02  0.04  0.03  0.03  0.01  0.03  0.02  P2  0.12  0.27  0.16  0.26  0.09  0.17  0.11  0.28  0.11  0.25  k2  7.30  0.83  2.01  2.01  6.43  2.03  6.66  0.48  6.66  2.01  View Large During the inhibition phase (Fig. 6), both the HL and the LL cells showed a very rapid decrease in the effective PSII quantum efficiency ΔF/Fm’, and the rate constant for fast inhibition (k1) equalled 9.05 min−1 for all cells acclimated to both HL and LL (Table 2). Most likely, the measuring frequency during the initial inhibition stage was too low to get a full resolution of k1. After 2 min, the minimal PSII quantum efficiency was reached for all the strains except for Dn116EHU-LL and Dn141EHU-HL which showed a biphasic decrease in the ΔF/Fm’ with a value for the slow rate constant of inhibition (k2) of 0.03 min−1. The proportion of fast reacting PSII centres varied between 46% and 66% for the elongated morphotypes and was generally higher in HL than in LL cells. This was the opposite for the round morphotype (Dn141EHU) where the P1-fraction varied from 32% to 63% and was higher in the LL cells. For the slow-responding PSII centres, this proportion was higher in the LL cells, except for the round morphotype where it was higher in the HL-acclimated cells. The elongated strain Dn60EHU had nearly identical proportions of P1 and P2 in LL and HL. However, the small differences between the strains and the acclimation state were not significantly different (P > 0.5). During the dark phase following the HL incubation, none of the strains fully recovered during the 30 min recovery period. In all strains, the recovery varied between 70%–80% and there were no differences in recovery percentage between the LL and HL strains. However, the kinetics of the recovery (Fig. 7) differed between the LL and HL strains: the LL-acclimated strains showed a more pronounced biphasic recovery than the HL ones, as the initial recovery was faster in the LL-acclimated strains. Interestingly, k1 for recovery was larger in the HL-acclimated strains, with the exception of the Dn60EHU strain where the k1 values in LL and HL were similar. Also, the k2 rate constants were larger in the HL-acclimated cells, strain Dn80EHU excepted, which showed equal k2 rate constants. No pattern was evident in discerning both morphotypes. The RLCs taken at critical points of the experiment are represented in Fig. 8. Unexpectedly, it seemed that the cells immediately deactivated themselves after 10 min of incubation in the dark. After 20 min of exposure to HL, the RLC parameters did not show any signs of photoinhibition: the RLC parameter rETRmax was either higher or similar to the growth condition (Fig. 8A, C, D and E, all LL). When the samples were transferred once more to the dark, in order to recover from the HL intensity, they downregulated their photosynthetic activity but never reached their initial conditions. In all the strains, LL-acclimated cells showed a higher rETRmax values than the HL-acclimated cells. Figure 8. View largeDownload slide rETRmax values of the RLCs taken with the FRRF during HL stress experiment from strains acclimated at HL and LL. (A) Strain Dn150EHU, P. lima from Villefranche-sur-mer; (B) strain Dn141EHU, round morphotype of P. lima from Ibiza; (C) strain Dn60EHU, P. lima from San sebastian; (D) strain Dn80EHU, P. lima from Vigo; (E) strain Dn116EHU, P. lima from Galé. All data were normalised to the initial values. RLC 0 = min 0, RLC1 = min 10, RLC2 = min 30, RLC3 = min 45, RLC4 = min 60. Figure 8. View largeDownload slide rETRmax values of the RLCs taken with the FRRF during HL stress experiment from strains acclimated at HL and LL. (A) Strain Dn150EHU, P. lima from Villefranche-sur-mer; (B) strain Dn141EHU, round morphotype of P. lima from Ibiza; (C) strain Dn60EHU, P. lima from San sebastian; (D) strain Dn80EHU, P. lima from Vigo; (E) strain Dn116EHU, P. lima from Galé. All data were normalised to the initial values. RLC 0 = min 0, RLC1 = min 10, RLC2 = min 30, RLC3 = min 45, RLC4 = min 60. DISCUSSION Prorocentrum lima has generally been reported to be a slow-growing dinoflagellate. Several authors reported growth rates in a range from 0.06 to 0.24 d−1 with a prolonged exponential growth period (Pan, Cembella and Quilliam 1999; Bravo et al.2001; Heredia-Tapia et al.2002; Nascimento, Purdie and Morris 2005; Varkitzi et al.2010; Nascimento et al.2016). In concordance with Heredia-Tapia et al. (2002) and Varkitzi et al. (2010), our study revealed that under initial standard conditions (90 μmol photons m−2 s−1), cells showed an average growth rate of 0.10 d−1, which means a generation time of about 7 days. Considering the whole light-intensity range, maximum growth rates (0.23 d−1) were observed at 80 and 100 μmol photons m−2 s−1. Growth rates of 0.22–0.33 d−1 have previously been reported for Mediterranean strains (Vanucci et al.2010; Ben-Gharbia et al.2016). Although most of the Atlantic strains have been reported to grow slowly (Pan, Cembella and Quilliam 1999; Bravo et al.2001; Nascimento, Purdie and Morris 2005; Varkitzi et al.2010; Nascimento et al.2016), some authors have reported a faster growth at higher temperatures (0.47–0.75 d−1; 26°C–27°C; Morton and Norris 1990; Morton, Norris and Bomber 1992; Tomas and Baden 1993; Vale, Veloso and Amorim 2009). Studies on strains isolated from the Pacific region showed similar growth rates (0.11–0.35 d−1) to the Mediterranean strains, although these were performed at higher temperatures (25°C–29°C; Morton and Tindall 1995; Holmes et al.2001; Herradia-Tapia et al.2002). These results indicate that growth rates vary considerably depending on culturing conditions. When comparing the results obtained with the Coulter counter and the water-PAM, growth rates depicted from the former were slightly lower compared to the latter, although consistent between strains. While this method is valid for other benthic microalgae, as previously confirmed for Coolia monotis (David, Kromkamp and Orive 2017), Prorocentrum cells usually form clumps that might hamper the Coulter counter results. In our work, although the studied strains did not differ significantly in their growth rates at different light intensities, they seemed to grow better under moderate, but different, light intensities (80–150 μmol photons m−2 s−1) to reach their maximum growth rate. Strain Dn116EHU, from the south of the Atlantic Iberian Peninsula, was the one which seemed best adapted to proliferate under higher irradiances as opposed to the one collected in Villefranche-sur-mer, in the Mediterranean Sea (Dn150EHU), which seemed to prefer lower light intensities. This was interesting as waters in the Mediterranean Sea show water attenuation coefficients (Kd) much lower than in the Atlantic coast (Brito et al.2013; Celis-Plá et al. 2014). However, these are epiphytic dinoflagellates that might seek protection on macroalgae but waters in the Atlantic are more turbulent which makes epibenthic dinoflagellates more exposed to irradiances. Furthermore and according to our results, although the light field of epibenthic dinoflagellates is variable, it can be related to the strains’ sampling sites. Strains Dn80EHU and Dn60EHU seemed adapted to a higher range of light intensities, in agreement with the larger variability of environmental factors present in the NW and north of the Iberian Peninsula. However, intraspecific variability of physiological parameters is thought to exist but could not be confirmed at this phase. Other abiotic factors such as water turbulence, upwelling, salinity or temperatures might also influence the cells growth rate. Physiological differences from strains belonging to identical ribotypes but isolated from different environments have been described before. For example, Berden-Zrimec et al. (2008) found physiological differences (pigment composition and concentration, and in the delayed fluorescence decay kinetics and intensity) among P. minimum strains isolated from different areas in the same region and suggested that they could be a consequence of adaptations to specific conditions (e.g. salinity) in each area. However, P. minimum is a planktonic species and, whereas phytoplankton is associated with a particular water body, benthic dinoflagellates depend on a fixed substratum (Fraga et al.2012). In the present case, strains were isolated from macrophyte substrata. In studies performed with the also epiphytic and shade-adapted dinoflagellate genus Gambierdiscus, it was suggested that it might exploit the macroalgal host three-dimensional structure for protection from HL exposure (Villareal and Morton 2002). This behaviour allowed Gambierdiscus to thrive in a high-light environment. The same could happen in the present case with the P. lima strains. So, although P. lima strains are present in what is considered to be a high-light environment, the conditions derived from their epiphytic habitat, which is common for both Atlantic and Mediterranean strains, creates a moderate/LL field, that could have a larger impact in strains’ physiological responses. Regarding the HL stress experiment, cultures that were grown in HL showed lower maximum quantum efficiency than the ones grown in LL, suggesting that HL-exposed cells were suffering from chronic photodamage. Nevertheless, these had higher growth rates than the LL ones, denoting that the HL cultures apparently possessed a strategy to cope with this chronic D1-photodamage. This might be caused by the ‘excess PSII’ capacity, where damage to PSII does not necessarily results in impaired C-fixation (Bañares-España et al.2013). In addition, HL cultures showed a better recovery than the LL ones when comparing its final values with the initial ones. However, LL cultures showed higher rETRmax and higher Fv/Fm values at all stages, revealing a better performance of the cells. Still, our results indicated that none of the strains fully recovered during the half an hour they were monitored after the 20 min exposure to 500 μmol photons m−2 s−1. This showed that all strains suffered from photodamage, and that dynamic downregulation (qE) was not sufficient to prevent it. Other studies on benthic dinoflagellates also showed them to have more features of shade-adapted organisms rather than HL adapted ones (Fraga et al.2012; Tester et al.2013). This may happen since the D1 protein of PSII, which usually exhibits a rapid turnover in vivo at HL (Sundby, McCaffery and Anderson 1993) and provides a protective mechanism, exhibits a net loss at prolonged excess irradiances (Russel et al.1995). As stated previously, cultures grown in HL performed better in the recovery time than the same acclimated to LL as the algae near the sea surface are known to have a fast reaction to the photoinhibition and recovery (Hanelt 1998). A possible explanation is that strains acclimated to LL increase its pigment content to increase its light-harvesting capacity (Bañares-España et al.2013; Falkowski and Raven 2007), but a sudden exposure to a HL intensity can cause serious photodamage, while the ones acclimated to HL would have lower pigment contents. Although not statistically significant, some differences could be discerned between strain Dn141EHU (round morphotype) and the other strains (elongated morphotype), with respect to the proportion of the fast-responding (P1-fraction) and slow-responding (P2) PSII centres during the inhibition phase of the HL stress. During recovery, it was not possible to discern the round morphotype from the elongated ones indicating a similar capacity of regeneration. These could hypothetically represent different ecotypes adapted to different niches. All strains in the present study belong to the same ribotype. For further studies, it would be interesting to study the physiological variability among cryptic phylogenetic groups of the P. lima complex. Many of the studies on the morphospecies P. lima do not include phylogenetic analyses of the analysed strains, which hampers the comparison among them. Furthermore, as depicted from Ben-Gharbia et al. (2016) more studies need to originate from different geographical areas, especially from the Indo-Pacific region where data are scarce. Also, since both round and elongated morphotypes can be found in the same ribotype, it is suggested that further studies should explore if these shapes are adaptive features of different ecotypes. Overall, our results are in agreement with Pan, Cembella and Quilliam (1999) when referring to P. lima as a shade-adapted epibenthic species with long generation times. The comparison of both roundish and elongate morphotypes and strains from the Atlantic Iberian Peninsula and the Mediterranean Sea (differing environments: higher vs. lower latitude; more turbulent and turbid vs. more calm and transparent waters) did not reveal significant differences in their photophysiology. However, different growth rates and small differences found in the photosynthetic properties of the elongated morphotypes, suggest that strains were acclimated to the local light environment, which differed at different locations. Further studies are necessary to investigate this pattern in a broader scale including tropical and cold temperate environments. SUPPLEMENTARY DATA Supplementary data are available at FEMSEC online. FUNDING Financial support for this research was provided by the Department for environment of Bizkaiko Foru Aldundia, the Bilbao-Bizkaia Water Consortium, and the Basque Government (projects IT-699-13 and UPV/EHU-PPG17/67). A grant from the Portuguese funding institution FCT—Fundação para a Ciência e a Tecnologia awarded to H. David (SFRH/BPD/121365/2016) is also acknowledged. Conflict of interest. None declared. REFERENCES Aissaoui A, Armi Z, Akrout F et al.   Environmental factors and seasonal dynamics of Prorocentrum lima population in coastal waters of the Gulf of Tunis, south Mediterranean. Water Environ Res  2014, 86: 2256– 70. Google Scholar CrossRef Search ADS PubMed  Aligizaki K, Nikolaidis G, Katikou P et al.   Potentially toxic epiphytic Prorocentrum (Dinophyceae) species in Greek coastal waters. Harmful Algae  2009, 8: 299– 311. Google Scholar CrossRef Search ADS   Baek SH, Shimode S, Kikuchi T. 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Physiological response of Prorocentrum lima (Dinophyceae) to varying light intensities

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Abstract

Abstract The benthic dinoflagellate Prorocentrum lima is among the most common toxic morphospecies with a cosmopolitan distribution. This study explored if strains from different environments and different morphotypes, isolated from three locations in the Atlantic Iberian Peninsula and two from the Mediterranean Sea, showed different responses to varying light regimes, after confirming that all strains belonged to the same ribotype. Growth rates and photosynthetic parameters such as Fo, Fv/Fm, and rETRmax were analysed with a Coulter counter, a water-PAM and a fast repetition rate fluorometer. The photosynthetic properties were investigated in a high light stress experiment using strains acclimated to low light (LL) and high light (HL). The highest growth rate was 0.23 day−1 at 80 and 100 μmol photons m−2 s−1 for strains Dn150EHU and Dn60EHU, originated from different locations. Under control conditions (18°C and 90 μmol photons m−2 s−1), growth rate was on average 0.10 day−1. The HL stress exposure induced photodamage to all strains and the recovery period was not sufficiently long for full recovery of Fv/Fm. However, cells acclimated to HL showed a better recovery than the LL acclimated ones. Furthermore, some assumptions are discussed in relation to strains’ original location. dinoflagellates, growth rates, high light stress, microalgae, photophysiology INTRODUCTION During the last decades, the genus Prorocentrum Ehrenberg (Dinophyceae) has been the focus of several research works due to the fact that numerous species of dinoflagellates are known to produce toxins (Dodge 1975; Fukuyo 1981; Faust 1991; Morton and Tindall 1995; Pan, Cembella and Quilliam 1999; Yoo 2004; Hoppenrath et al.2013; López-Rosales et al.2014; Nascimento et al.2016). This genus has a worldwide distribution and contains about 60 species, from which 29 are known to be benthic (Hoppenrath et al.2013). Prorocentrum lima (Ehrenberg) Stein is the most commonly reported Prorocentrum benthic morphospecies, and latest studies, which included new geographic areas, have revealed a complex structure of genetically distant ribotypes (Nagahama et al.2011; Zhang et al.2015; Nascimento et al.2016). Moreover, variability within morphological features of P. lima is common (Nagahama et al.2011). Therefore, and for comparative purposes, it is crucial to know the precise molecular identity of the biological material employed, since physiological features can differ among lineages (e.g. Parsons et al.2012). Prorocentrum lima is widely present in the Mediterranean Sea (Aligizaki et al.2009; Vanucci et al.2010; Aissaoui et al.2014; Ben-Gharbia et al.2016) and in the Atlantic Iberian Peninsula (Vale, Veloso and Amorim 2009; Laza-Martínez et al.2011). Moreover, a broader study on the benthic Ostreopsis and Coolia (David et al.2013, 2014) revealed P. lima to be present in all 17 sites sampled along the Atlantic coast of the Iberian Peninsula. After a preliminary analysis (unpublished) revealed that all collected strains showed no phylogenetic differences, it was decided to investigate the response of this microalga to varying light irradiances. This was decided as the Iberian Peninsula coast presents contrasting temperatures and irradiances and also between its Atlantic and Mediterranean coasts, which may trigger local adaptations on benthic microalgae. Moreover, most of the recent work on Prorocentrum has been focused on the species taxonomy (Hoppenrath et al.2013; Zhang et al.2015; Nascimento et al.2016; Luo et al.2017) or chemical and toxin analyses (Huang et al.2014, 2015; Wang et al.2015; Prego-Faraldo et al.2017), with only a few related to P. lima ecophysiology (Gilbert, Burkholder and Kana 2012; Aissaoui et al.2014; Ben-Gharbia et al.2016). Light availability is one of the most important factors affecting marine environments, as light promotes photosynthetic activity, but can also inhibit many biological processes if radiation becomes excessive (Baek, Shimode and Kikuchi 2008; Celis-Plá et al.2014). Furthermore, changes in temperature, irradiation, salinity, nutrients and other environmental factors are known to influence cell proliferations (Heredia-Tapia et al.2002; Zhang and Hu 2011; Fraga et al.2012), as well as the amount of toxins they produce (Guerrini et al.2009). Although there is still much to uncover about the photophysiological properties of benthic species within the genus Prorocentrum, it is known that physiological and photoprotective mechanisms might differ among microalgae species according to their phylogeny and the environment where they grow (Stomp et al.2004). Epibenthic dinoflagellates are common in clear waters where high irradiances reach the benthos. As a mechanism of protection, it has been suggested that some of them can migrate to the macroalgae-shaded areas (Ballantine, Tosteson and Bardales 1988), and that microalgae possess an adaptive response system to highly variable light conditions (Ihnken, Eggert and Beardall 2010). Different levels of photoinhibition can be triggered by excessive irradiance (Franklin and Forster 1997) where dynamic photoinhibition can be defined as a short-term reversible, regulatory process for the controlled dissipation of excessive light energy, mediated by the xanthophyll cycle (e.g. Goss et al. 2006), and chronic photoinhibition as a slowly reversible process that may occur following prolonged exposure to excessive irradiances (Osmond and Grace 1995). These are based on the typical kinetics of relaxation (recovery of photosynthesis) of the biochemical and biophysical processes implicated in photoinhibition (Franklin and Forster 1997; Hanelt 1998) and can be assessed by changes in the chlorophyll fluorescence parameters (Franklin et al.1992; Jones and Hoegh-Guldberg 2001; Goss et al.2006). To gain insight into the photophysiology of these microalgae, several techniques have been developed. The fast repetition rate fluorometer (FRRF) and the water-PAM (pulse amplitude modulation) are highly sensitive apparatuses based on the variable in vivo chlorophyll fluorescence of phytoplankton that provide several photosynthetic parameters that have been widely used on phytoplankton research (Schreiber, Bilger and Neubauer 1994). The directly measured parameter F0 from the water-PAM and FRRF can be used as a proxy of algal biomass (Dijkman and Kromkamp 2006; Kromkamp et al.2008), and (Fm − F0)/Fm (Fv/Fm), the maximum quantum efficiency of photosystem II (PSII) photochemistry, can be used as a measure of stress (Baker 2008; Kromkamp et al.2008; Suggett et al.2012; Betancor et al.2015). In addition, rapid light curves (RLCs) or photosynthesis-irradiance (P-E) curves can be made under different controlled conditions (Lewis and Smith 1983; White and Critchley 1999; Johnson and Sheldon 2007) to estimate the relative electron transport rate (rETR) of PSII (Ralph and Gademann 2005). Other parameters derived from these curves, namely light-limited initial slope (α), saturation light intensity (Ek) and maximal relative electron transport rate (rETRmax), are good indicators of the light regime and photoacclimatory behaviour of the cells (Suggett et al.2012), and can provide a good estimate of the photosynthetic rate (Kromkamp et al.2008; Figueroa, Jerez and Korbee 2013). The present study aimed to know if P. lima strains isolated from different localities around the Atlantic Iberian Peninsula and the Mediterranean Sea showed local adaptations regarding their photophysiology. With this aim, localities representing different latitudes and from both coasts were selected. Strains from lower latitudes and from the Mediterranean were expected to be better adapted to high-light intensities. Additionally, one of the Mediterranean strains presented a roundish morphology, differing from the typical oval/ovoid P. lima strains, which allowed us to test if different cell shapes were related to adaptations leading to photophysiological differences. The cells’ photosynthetic performance was measured using an FRRF, a water-PAM and a Coulter counter. The strains’ identity was checked by molecular techniques prior to analyses. MATERIALS AND METHODS Study area The latitudinal range of the study was between 37°N and 44°N including five different locations from the Atlantic Iberian Peninsula and the Mediterranean Sea coasts (Fig. 1): San Sebastian in the north of the Iberian Peninsula (43.321397, –1.986706), Vigo in the northwest of the Iberian Peninsula (42.223871, –8.767948), Galé in the south of the Peninsula (37.078863, –8.314001), Villefranche-sur-Mer in the north Mediterranean Sea (43.703857, 7.320063) and Ibiza in the western Mediterranean Sea (38.967708, 1.241127). Although somewhat narrow, this latitudinal range presents a characteristic variability between the different localities: the north of the Atlantic Iberian Peninsula has higher annual mean cloud coverage compared to the south (Sanches-Lorenzo et al.2009). This is supported by the yearly sum of global solar irradiation with data from 1986 to 2005, generated by the Meteonorm software (www.meteronorm.com), which indicated that the location of San Sebastian, in the north of the Iberian Peninsula, received the smallest amount of ground solar radiance (1200–1300 Kwh/m2) from all five locations. Next, it was Vigo (1500–1600 Kwh/m2), Villefranche-sur-mer (1600–1700 Kwh/m2), Ibiza (1700–1800 Kwh/m2) and Galé (1900–2000 Kwh/m2). These values include a complete range of seasonal conditions and solar angles in each site (Paulescu et al.2013). Figure 1. View largeDownload slide Study area sampling sites. Figure 1. View largeDownload slide Study area sampling sites. Seasonal variability also differs between locations where the north (San Sebastian) is characterised by an oceanic climate with cool winters and warm summers where precipitation is dispersed throughout the year. Vigo has a warm-temperate climate characterised by warm dry summers and rainy winters. The other three locations of Galé, Ibiza and Villefranche-sur-Mer are characterised by a hot-temperate Mediterranean climate having mild winter temperatures and hot dry summers (Peel, Finlayson and McMahon 2007). The underwater light field also varies. In the Mediterranean, the water is very clear (e.g. Lüning 1990) with higher light intensity (about 10 m depth) reaching the benthos (Figueroa and Gómez 2001). Furthermore, the attenuation coefficient for PAR (KdPAR) is around 0.07–0.09 m−1 in the Mediterranean Sea (Wiencke et al.2000; Celis-Plá et al.2014), which is a great contrast with the Atlantic Iberian Peninsula coast locations where mean Kd varies from 0.6 to 0.9 m−1 (Gohin et al.2005; Brito et al.2013). Higher Kd values express a higher influence of currents, resuspension of sediments and run-off. Tidal range also differs markedly between the Atlantic and the Mediterranean. Whereas the tidal regime in the Atlantic area is semidiurnal with a tidal range of ca. 2–4 m, tides are negligible in the Mediterranean Sea. The semidiurnal depth variation caused by the tidal regime means a parallel light-environment variation in the Atlantic area compared to the more stable environment in the Mediterranean. Moreover, tidal regime generates more turbidity in shallow shore waters due to sediment resuspension. Strains selection The strains used to study the photophysiological responses of cells from different locations (Fig. 1) representing differing light environments were as follows: Dn60EHU from San Sebastian (North of the Iberian Peninsula), Dn80EHU from Vigo (NW of the Iberian Peninsula), Dn116EHU from Galé (South of the Iberian Peninsula) and Dn150EHU from Villefranche-sur-Mer (Mediterranean Sea). For the high light (HL) stress experiment, strain Dn141EHU from Ibiza (Mediterranean Sea), representing a rounder morphotype, was also used (Fig. 2) for comparison with the elongated strains. Cells were collected at 1–2 m depth during low tides. Isolation and settlement of cultures were described in David et al. (2013). Cells were observed under light microscopy (Leica DMRB). Prior to the photophysiological analyses, strains’ identity was checked with molecular methods using the large ribosomal subunit (LSU) and the internal transcribed spacer (ITS) regions as markers. Results showed that all strains belonged to the lineage included in clade C of the LSU and clade D of the ITS tree from Zhang et al. (2015). This lineage includes all the existing Mediterranean and European sequences of P. lima. Additionally, all strains except Dn141EHU, which had a roundish morphology, showed the typical P. lima elongated morphology. The molecular and morphological characterisation of the strains will be reported elsewhere. Figure 2. View largeDownload slide Comparison of both P. lima morphotypes. (a) Typical cell of P. lima; (b) round morphotype. Scale bar = 10 μm. Figure 2. View largeDownload slide Comparison of both P. lima morphotypes. (a) Typical cell of P. lima; (b) round morphotype. Scale bar = 10 μm. Growth rates at different light intensities Strains were previously acclimated for a month in 50 mL Nuclon (ThermoFisher Scientific, Spain) culture flasks, containing 20 mL of F/2 Guillard's marine culture medium (Sigma), under 18°C, to a light intensity of 90 μmol photons m−2 s−1 (measured with a light meter (LI-250A, LI-COR, Fairborn, OH)) and a 12:12 light/dark hour cycle. Then two sets of experiments were performed. For the first one, an aliquot of 5 mL of the acclimated culture flasks was used to start fresh new cultures in new 50 mL Nuclon culture flasks, with a final volume of 20 mL to monitor their growth rate at this irradiance. Every other day, flasks were shaken and 400 μL of each culture was retrieved to an eppendorf tube, for cells count. This was measured in triplicate with a Coulter counter (Multisizer 3, Beckman coulter, Inc., Netherlands) during 20 days to compare later with the fluorescence values of the second experiment, given from the pulse amplitude modulated (PAM) fluorometer (water-PAM, Walz, Germany). From the exponential phase, growth rates were obtained by fitting the data in SigmaPlot 13.0 (Systat Software, Inc. GmbH, Germany) according to the following equation: Nt = N2exp(μ × t) where N2 is cell concentration at day 2 and Nt is the cell number at day t. During the same time, the second set was prepared. An aliquot of 1 mL was retrieved from each of the freshly renewed cultures made for Experiment 1, and placed in duplicate into a 24-multiwell culture plate that was left under cold fluorescent light, at a constant temperature of 18°C and seven different light intensities (30, 60, 80, 100, 150, 200 and 300 μmol photons m−2 s−1). Increases in the minimal fluorescence (F0) were measured in triplicate everyday, at the same hour, for 11 days. This was measured with a saturating light pulse from the fibre-optic version of the water-PAM (water-ED, Walz GmbH, Effeltrich, Germany). Culture plates were left in the dark for 10 min, and shaken before each measurement so cells could be detached from the bottom and be uniformly dispersed in the wells, and the fibre of the EDF unit of the water-PAM was placed in the middle of each well for the measurements. The residual fluorescence due to the empty well and culture medium without algae was subtracted before calculation of fluorescent yields. Growth rates, μ (day −1), were calculated from the whole exponential growth phase as μ = ln(Nt/N0)/t, where Nt and N0 are the dark acclimated fluorescence yields (F0) at time t and 0, respectively, and t = x days (Crow and Kimura 1970). Generation time (G) was calculated as G = ln2/μ (Guillard 1973). HL stress experiment Strains were previously acclimated in duplicate to a 12:12 light/dark hour cycle for 3 months at 18°C in 150 mL Nuclon culture flasks and diluted every 3 weeks by removing about 20 mL and adding new culture medium (Guillard F/2) until a final volume of 50 mL to maintain cultures in exponential phase. During that time, the two series of cultures were exposed to different irradiances: One to low light—LL (40 μmol photons m−2 s−1) and another to HL (150 μmol photons m−2 s−1) at 18°C before they were subjected to the HL stress. A short-term HL stress experiment was performed with all the given strains of P. lima, where 2.5 mL of each culture was transferred to a test tube and incubated for 10 min in the dark. The tube was then placed in front of a LED panel with a photon flux density of 500 μmol photons m−2 s−1 for 20 min to induce stress, and the cells’ recovery was recorded by following their Fv/Fm in the dark for the following 30 min (‘recovery phase’). This HL intensity was approximately three times their Ek, which is the irradiance value where photosynthesis switches from light-limited to light-saturated rates. Using values three times higher than their irradiance-onset saturation parameter was a way to ensure that cells would be in stress. This value was obtained from previous RLCs with the same cultures, by fitting data into a revised P-E model of Eilers and Peeters (1988) (Herlory et al.2007; Silsbe and Kromkamp 2012). During the entire experiment, RLCs on 2.5 mL subsamples were measured with an FRRF (FastTracka-II/FastAct) (Fast Repetition rate fluorometry, Chelsea Technologies Group Ltd) at crucial changing points in the experiment (point 0 in acclimated conditions, point 1 after 10 min of dark incubation, point 2 after 20 min of light stress (min 30), point 3 in the middle of recover (min 45), and point 4 at the end of the experiment (min 60)). Each RLC consisted of 10 irradiance steps between 0 and 1504 μmol photons m−2 s−1, and each light step lasted 30s. The values of the PSII quantum efficiency of the RLCs were then fitted in the Eilers and Peeters model as described above, to retrieve rETRmax values of each curve on each step. Furthermore, several acquisition points to monitor the Fv/Fm evolution were measured in between the RLCs. From RLC1 to RLC2 (light phase) measurements were taken every 3 min. From RLC2 to RLC3 (dark phase), measurements were taken every minute for the first 5 min and then every 2 min. From RLC3 to RLC4 (dark phase), these were taken every 5 min. Cells will be fully recovered if the final Fv/Fm value is similar to the initial ones. For a better comprehension, the RLC data were then separated into the photoinhibition HL period and the recovery period, where the first was from minute 10 to 30 and the latter from minute 30 until the end of the experiment (min 60). Hanelt (1998) revealed that during an HL stress, it was possible to describe the two kinetics of photoinhibition with two rate constants, associated with two fractions of PSII centres: a fraction (Pfast) associated with a fast rate constant (Kfast) and a ‘slow’ fraction (Pslow) with a slow rate constant (Kslow). Knowing these constants at any time (t) allows us to compare the different reactions of different strains to different irradiances. By fitting the changes of the PSII quantum efficiency during the inhibition and recovery phase we could calculate these data. Thus, the PSII quantum efficiency from the RLCs was fitted, with the solver added in Excel, using the following equations of Hanelt (1998):   \begin{equation*} {\rm{inhibition}}\,{\rm{kinetics}}:\frac{{\Delta F}}{{{{F\!'}_m}}} = {P_1} \cdot {e^{ - {k_1} \cdot t}} + {P_2} \cdot {e^{ - {k_2} \cdot t}}, \end{equation*}   \begin{equation*} {\rm{recovery}}:\frac{{\Delta F}}{{{{F\!'}_m}}} = {F_v}/{F_m} - \left( {{P_1} \cdot {e^{ - {k_1} \cdot t}} + {P_2} \cdot {e^{ - {k_2} \cdot t}}} \right). \end{equation*} So, P1 and P2 are the proportion of the centres with a fast and slow component, and k1 and k2 are the corresponding rate constants of fast and slow inhibition and recovery. It is thought that the RCII fraction with the components is related to the xanthophyll cycle, and P2 is probably related to a higher chronic damage of the PSII centres. Fv/Fm is the maximum PSII efficiency of the non-inhibited cells at t = 0 and P1 + P2 = Fv/Fm. Recovery becomes faster when k1 is larger or when k2 becomes smaller. Statistical analyses The statistical program SigmaPlot 13.0 (Systat Software, Inc. Germany) was used to analyse all data. Firstly, to find differences between strains and time on growth rate, the two-way analysis of variance (ANOVA) followed by the all pairwise multiple comparison Tukey's t-test was used to compare differences within groups. Then, the same method was employed to compare the estimated growth rates from the F0 values, between and among strains at different light intensities. To test if different light intensities presented relevant changes, a t-test was also performed between groups. Whenever equal variance failed, a Mann–Whitney Rank Sum test was performed. Furthermore, differences in the estimated growth rates from different methods were also tested. Tukey's t-test was also used to compare Fv/Fm values in the HL stress experiment. RESULTS Growth rates Under 90 μmol photons m−2 s−1, cell concentration measured with a Coulter counter increased exponentially in all cultures from day 2 until the end of the experiment, which lasted 20 days (Fig. 3), except for strains Dn80EHU and Dn60EHU, where cultures reached a stationary phase 2 days earlier, after the 18th day. A maximum cell density of 107 × 103 cells mL−1 was observed for strain Dn116EHU at the 20th day. Strains’ exponential growth rate averaged 0.098 ± 0.009 day−1. There were no significant differences among strains, except for Dn150EHU, which showed a significant lower growth rate (ANOVA, P = 0.0059; Table 1) using this method. Figure 3. View largeDownload slide Growth curves of different strains of P. lima in the pre-acclimated culture flasks at 90 μmol photons m−2 s−1. Figure 3. View largeDownload slide Growth curves of different strains of P. lima in the pre-acclimated culture flasks at 90 μmol photons m−2 s−1. Table 1. Average growth rate values with their respective standard error values for each strain of stock cultures maintained at 90 μmol photons m−2 s−1. Strain   location  μ (day−1)  St. error  P. lima Dn60EHU  San Sebastian  0.1010  0.0130  P. lima Dn80EHU  Vigo  0.0946  0.0087  P. lima Dn116EHU  Galé  0.1087  0.0093  P. lima Dn150EHU  Villefranche-sur-mer  0.0875  0.0090  Strain   location  μ (day−1)  St. error  P. lima Dn60EHU  San Sebastian  0.1010  0.0130  P. lima Dn80EHU  Vigo  0.0946  0.0087  P. lima Dn116EHU  Galé  0.1087  0.0093  P. lima Dn150EHU  Villefranche-sur-mer  0.0875  0.0090  Average of all strains: 0.098 ± 0.009 day−1. View Large Growth rates of the strains measured daily with a water-PAM in the multiwell plates under different irradiances (from 30 and 300 μmol photons m−2 s−1) demonstrated that strains Dn150EHU and Dn60EHU were the ones which reached higher growth rates (0.23 day−1) (Fig. 4, Supplementary material), although these were not significantly different from the others (P > 0.5) and corresponded to different light intensities (80 and 100 μmol photons m−2 s−1). Strain Dn80EHU reached a maximum growth rate of 0.21 day−1 at 100 μmol photons m−2 s−1, and strain Dn116EHU had its maximum at 150 μmol photons m−2 s−1 with 0.17 day−1. Overall, strains grew better at moderate light intensities (80–150 μmol photons m−2 s−1) as at higher irradiances, growth rates were severely inhibited for most strains with the exception of Dn80EHU, which showed a growth rate of 0.13 day−1 at the highest irradiance (300 μmol photons m−2 s−1). At the lowest PAR tested (30 μmol photons m−2 s−1), growth rates were very low and similar (∼0.05 day−1). Figure 4. View largeDownload slide Growth rates of different strains of P. lima at different light intensities. Figure 4. View largeDownload slide Growth rates of different strains of P. lima at different light intensities. When comparing both methods, the Coulter counter and the water-PAM, slightly lower growth rates (although not statistical significant, P > 0.5) were estimated by the Coulter counter. HL stress During the HL stress experiment, aliquots of strains previously acclimated to HL and LL were exposed to a high-light stress and their recovery time was studied. Cells acclimated to LL showed higher initial values of the maximum quantum efficiency (Fv/Fm) than the cells acclimated to HL (Fig. 5). The photochemical efficiency of the different strains markedly decreased during the first minutes of exposure to 500 μmol photons m−2 s−1. Tukey's t-test, applied to compare final to initial acquisition points, showed that the cells acclimated to HL performed better than the same acclimated to LL. The difference of means within HL was less than the one in LL (0.090 < 0.115). Regarding the different strains, Dn150EHU from the Mediterranean Sea (Fig. 5B) presented nearly identical values of the initial and final quantum efficiency at HL and LL (Fv/Fm (i-f) = 0.09). The other strain (Dn141EHU) from the Mediterranean Sea (Fig. 5E; round morphotype) showed a similar result but performed better at HL (0.11). Strain Dn116EHU from the south of the Atlantic Iberian Peninsula was the one that better responded at HL (Fig. 5D), showing a difference of Fv/Fm(i-f) = 0.067, followed by Dn80EHU (0.08, Fig. 5A). However, strain Dn116EHU at LL, was the one with a higher difference (0.16) of Fv/Fm(i-f), meaning that it would take longer to reach the initial conditions. Regarding the north of the Iberian Peninsula, strain Dn60EHU (Fig. 5C) at HL was the one from all the HL-acclimated strains with a higher difference from the initial conditions (0.12). All data were fitted into the previously mentioned equations with the inhibition phase separated from the recovery phase and the fitted parameters can be found in Figs 6 and 7 and Table 2. Figure 5. View largeDownload slide Single acquisition points with the FRRF during HL stress experiment from strains acclimated at HL and LL. (A) Strain Dn80EHU, P. lima from Vigo; (B) strain Dn150EHU, P. lima from Villefranche-sur-mer; (C) strain Dn60EHU, P. lima from San Sebastian; (D) strain Dn116EHU, P. lima from Galé; (E) strain Dn141EHU, round morphotype of P. lima from Ibiza. At t = 0, a sample was collected from the culture and put in the dark for 10 min. Then, the sample was exposed to HL for 20 min. At 30 min, the sample was put back in the dark in order to measure the recovery. Figure 5. View largeDownload slide Single acquisition points with the FRRF during HL stress experiment from strains acclimated at HL and LL. (A) Strain Dn80EHU, P. lima from Vigo; (B) strain Dn150EHU, P. lima from Villefranche-sur-mer; (C) strain Dn60EHU, P. lima from San Sebastian; (D) strain Dn116EHU, P. lima from Galé; (E) strain Dn141EHU, round morphotype of P. lima from Ibiza. At t = 0, a sample was collected from the culture and put in the dark for 10 min. Then, the sample was exposed to HL for 20 min. At 30 min, the sample was put back in the dark in order to measure the recovery. Figure 6. View largeDownload slide Proportion of fast (P1) and slow (P2) reacting PSII centres during the 20 min inhibition phase in the HL experiment. Figure 6. View largeDownload slide Proportion of fast (P1) and slow (P2) reacting PSII centres during the 20 min inhibition phase in the HL experiment. Figure 7. View largeDownload slide Recovery parameters. Both left panels show the proportion (%) of the fast (top) and slowly (bottom) recovering PSII centres. The right panels show the rate constants (min−1) for recovery of the fast (top) and slow (bottom). Figure 7. View largeDownload slide Recovery parameters. Both left panels show the proportion (%) of the fast (top) and slowly (bottom) recovering PSII centres. The right panels show the rate constants (min−1) for recovery of the fast (top) and slow (bottom). Table 2. Fit parameters for the inhibition and recovery of the HL- and LL-acclimated strains in the HL stress experiment. Inhibition constant    Dn60EHU   Dn60EHU   Dn80EHU  Dn80EHU  Dn116EHU  Dn116EHU  Dn141EHU  Dn141EHU  Dn150EHU  Dn150EHU    HL  LL  HL  LL  HL  LL  HL  LL  HL  LL  P1  0.16  0.22  0.21  0.26  0.23  0.21  0.12  0.29  0.23  0.26  k1  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  P2  0.17  0.22  0.15  0.22  0.12  0.25  0.26  0.17  0.14  0.21  k2  0.00  0.00  0.00  0.00  0.00  0.03  0.03  0.00  0.00  0.00  Recovery constant  P1  0.21  0.17  0.20  0.22  0.26  0.29  0.28  0.18  0.26  0.22  k1  0.02  0.02  0.04  0.02  0.04  0.03  0.03  0.01  0.03  0.02  P2  0.12  0.27  0.16  0.26  0.09  0.17  0.11  0.28  0.11  0.25  k2  7.30  0.83  2.01  2.01  6.43  2.03  6.66  0.48  6.66  2.01  Inhibition constant    Dn60EHU   Dn60EHU   Dn80EHU  Dn80EHU  Dn116EHU  Dn116EHU  Dn141EHU  Dn141EHU  Dn150EHU  Dn150EHU    HL  LL  HL  LL  HL  LL  HL  LL  HL  LL  P1  0.16  0.22  0.21  0.26  0.23  0.21  0.12  0.29  0.23  0.26  k1  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  9.05  P2  0.17  0.22  0.15  0.22  0.12  0.25  0.26  0.17  0.14  0.21  k2  0.00  0.00  0.00  0.00  0.00  0.03  0.03  0.00  0.00  0.00  Recovery constant  P1  0.21  0.17  0.20  0.22  0.26  0.29  0.28  0.18  0.26  0.22  k1  0.02  0.02  0.04  0.02  0.04  0.03  0.03  0.01  0.03  0.02  P2  0.12  0.27  0.16  0.26  0.09  0.17  0.11  0.28  0.11  0.25  k2  7.30  0.83  2.01  2.01  6.43  2.03  6.66  0.48  6.66  2.01  View Large During the inhibition phase (Fig. 6), both the HL and the LL cells showed a very rapid decrease in the effective PSII quantum efficiency ΔF/Fm’, and the rate constant for fast inhibition (k1) equalled 9.05 min−1 for all cells acclimated to both HL and LL (Table 2). Most likely, the measuring frequency during the initial inhibition stage was too low to get a full resolution of k1. After 2 min, the minimal PSII quantum efficiency was reached for all the strains except for Dn116EHU-LL and Dn141EHU-HL which showed a biphasic decrease in the ΔF/Fm’ with a value for the slow rate constant of inhibition (k2) of 0.03 min−1. The proportion of fast reacting PSII centres varied between 46% and 66% for the elongated morphotypes and was generally higher in HL than in LL cells. This was the opposite for the round morphotype (Dn141EHU) where the P1-fraction varied from 32% to 63% and was higher in the LL cells. For the slow-responding PSII centres, this proportion was higher in the LL cells, except for the round morphotype where it was higher in the HL-acclimated cells. The elongated strain Dn60EHU had nearly identical proportions of P1 and P2 in LL and HL. However, the small differences between the strains and the acclimation state were not significantly different (P > 0.5). During the dark phase following the HL incubation, none of the strains fully recovered during the 30 min recovery period. In all strains, the recovery varied between 70%–80% and there were no differences in recovery percentage between the LL and HL strains. However, the kinetics of the recovery (Fig. 7) differed between the LL and HL strains: the LL-acclimated strains showed a more pronounced biphasic recovery than the HL ones, as the initial recovery was faster in the LL-acclimated strains. Interestingly, k1 for recovery was larger in the HL-acclimated strains, with the exception of the Dn60EHU strain where the k1 values in LL and HL were similar. Also, the k2 rate constants were larger in the HL-acclimated cells, strain Dn80EHU excepted, which showed equal k2 rate constants. No pattern was evident in discerning both morphotypes. The RLCs taken at critical points of the experiment are represented in Fig. 8. Unexpectedly, it seemed that the cells immediately deactivated themselves after 10 min of incubation in the dark. After 20 min of exposure to HL, the RLC parameters did not show any signs of photoinhibition: the RLC parameter rETRmax was either higher or similar to the growth condition (Fig. 8A, C, D and E, all LL). When the samples were transferred once more to the dark, in order to recover from the HL intensity, they downregulated their photosynthetic activity but never reached their initial conditions. In all the strains, LL-acclimated cells showed a higher rETRmax values than the HL-acclimated cells. Figure 8. View largeDownload slide rETRmax values of the RLCs taken with the FRRF during HL stress experiment from strains acclimated at HL and LL. (A) Strain Dn150EHU, P. lima from Villefranche-sur-mer; (B) strain Dn141EHU, round morphotype of P. lima from Ibiza; (C) strain Dn60EHU, P. lima from San sebastian; (D) strain Dn80EHU, P. lima from Vigo; (E) strain Dn116EHU, P. lima from Galé. All data were normalised to the initial values. RLC 0 = min 0, RLC1 = min 10, RLC2 = min 30, RLC3 = min 45, RLC4 = min 60. Figure 8. View largeDownload slide rETRmax values of the RLCs taken with the FRRF during HL stress experiment from strains acclimated at HL and LL. (A) Strain Dn150EHU, P. lima from Villefranche-sur-mer; (B) strain Dn141EHU, round morphotype of P. lima from Ibiza; (C) strain Dn60EHU, P. lima from San sebastian; (D) strain Dn80EHU, P. lima from Vigo; (E) strain Dn116EHU, P. lima from Galé. All data were normalised to the initial values. RLC 0 = min 0, RLC1 = min 10, RLC2 = min 30, RLC3 = min 45, RLC4 = min 60. DISCUSSION Prorocentrum lima has generally been reported to be a slow-growing dinoflagellate. Several authors reported growth rates in a range from 0.06 to 0.24 d−1 with a prolonged exponential growth period (Pan, Cembella and Quilliam 1999; Bravo et al.2001; Heredia-Tapia et al.2002; Nascimento, Purdie and Morris 2005; Varkitzi et al.2010; Nascimento et al.2016). In concordance with Heredia-Tapia et al. (2002) and Varkitzi et al. (2010), our study revealed that under initial standard conditions (90 μmol photons m−2 s−1), cells showed an average growth rate of 0.10 d−1, which means a generation time of about 7 days. Considering the whole light-intensity range, maximum growth rates (0.23 d−1) were observed at 80 and 100 μmol photons m−2 s−1. Growth rates of 0.22–0.33 d−1 have previously been reported for Mediterranean strains (Vanucci et al.2010; Ben-Gharbia et al.2016). Although most of the Atlantic strains have been reported to grow slowly (Pan, Cembella and Quilliam 1999; Bravo et al.2001; Nascimento, Purdie and Morris 2005; Varkitzi et al.2010; Nascimento et al.2016), some authors have reported a faster growth at higher temperatures (0.47–0.75 d−1; 26°C–27°C; Morton and Norris 1990; Morton, Norris and Bomber 1992; Tomas and Baden 1993; Vale, Veloso and Amorim 2009). Studies on strains isolated from the Pacific region showed similar growth rates (0.11–0.35 d−1) to the Mediterranean strains, although these were performed at higher temperatures (25°C–29°C; Morton and Tindall 1995; Holmes et al.2001; Herradia-Tapia et al.2002). These results indicate that growth rates vary considerably depending on culturing conditions. When comparing the results obtained with the Coulter counter and the water-PAM, growth rates depicted from the former were slightly lower compared to the latter, although consistent between strains. While this method is valid for other benthic microalgae, as previously confirmed for Coolia monotis (David, Kromkamp and Orive 2017), Prorocentrum cells usually form clumps that might hamper the Coulter counter results. In our work, although the studied strains did not differ significantly in their growth rates at different light intensities, they seemed to grow better under moderate, but different, light intensities (80–150 μmol photons m−2 s−1) to reach their maximum growth rate. Strain Dn116EHU, from the south of the Atlantic Iberian Peninsula, was the one which seemed best adapted to proliferate under higher irradiances as opposed to the one collected in Villefranche-sur-mer, in the Mediterranean Sea (Dn150EHU), which seemed to prefer lower light intensities. This was interesting as waters in the Mediterranean Sea show water attenuation coefficients (Kd) much lower than in the Atlantic coast (Brito et al.2013; Celis-Plá et al. 2014). However, these are epiphytic dinoflagellates that might seek protection on macroalgae but waters in the Atlantic are more turbulent which makes epibenthic dinoflagellates more exposed to irradiances. Furthermore and according to our results, although the light field of epibenthic dinoflagellates is variable, it can be related to the strains’ sampling sites. Strains Dn80EHU and Dn60EHU seemed adapted to a higher range of light intensities, in agreement with the larger variability of environmental factors present in the NW and north of the Iberian Peninsula. However, intraspecific variability of physiological parameters is thought to exist but could not be confirmed at this phase. Other abiotic factors such as water turbulence, upwelling, salinity or temperatures might also influence the cells growth rate. Physiological differences from strains belonging to identical ribotypes but isolated from different environments have been described before. For example, Berden-Zrimec et al. (2008) found physiological differences (pigment composition and concentration, and in the delayed fluorescence decay kinetics and intensity) among P. minimum strains isolated from different areas in the same region and suggested that they could be a consequence of adaptations to specific conditions (e.g. salinity) in each area. However, P. minimum is a planktonic species and, whereas phytoplankton is associated with a particular water body, benthic dinoflagellates depend on a fixed substratum (Fraga et al.2012). In the present case, strains were isolated from macrophyte substrata. In studies performed with the also epiphytic and shade-adapted dinoflagellate genus Gambierdiscus, it was suggested that it might exploit the macroalgal host three-dimensional structure for protection from HL exposure (Villareal and Morton 2002). This behaviour allowed Gambierdiscus to thrive in a high-light environment. The same could happen in the present case with the P. lima strains. So, although P. lima strains are present in what is considered to be a high-light environment, the conditions derived from their epiphytic habitat, which is common for both Atlantic and Mediterranean strains, creates a moderate/LL field, that could have a larger impact in strains’ physiological responses. Regarding the HL stress experiment, cultures that were grown in HL showed lower maximum quantum efficiency than the ones grown in LL, suggesting that HL-exposed cells were suffering from chronic photodamage. Nevertheless, these had higher growth rates than the LL ones, denoting that the HL cultures apparently possessed a strategy to cope with this chronic D1-photodamage. This might be caused by the ‘excess PSII’ capacity, where damage to PSII does not necessarily results in impaired C-fixation (Bañares-España et al.2013). In addition, HL cultures showed a better recovery than the LL ones when comparing its final values with the initial ones. However, LL cultures showed higher rETRmax and higher Fv/Fm values at all stages, revealing a better performance of the cells. Still, our results indicated that none of the strains fully recovered during the half an hour they were monitored after the 20 min exposure to 500 μmol photons m−2 s−1. This showed that all strains suffered from photodamage, and that dynamic downregulation (qE) was not sufficient to prevent it. Other studies on benthic dinoflagellates also showed them to have more features of shade-adapted organisms rather than HL adapted ones (Fraga et al.2012; Tester et al.2013). This may happen since the D1 protein of PSII, which usually exhibits a rapid turnover in vivo at HL (Sundby, McCaffery and Anderson 1993) and provides a protective mechanism, exhibits a net loss at prolonged excess irradiances (Russel et al.1995). As stated previously, cultures grown in HL performed better in the recovery time than the same acclimated to LL as the algae near the sea surface are known to have a fast reaction to the photoinhibition and recovery (Hanelt 1998). A possible explanation is that strains acclimated to LL increase its pigment content to increase its light-harvesting capacity (Bañares-España et al.2013; Falkowski and Raven 2007), but a sudden exposure to a HL intensity can cause serious photodamage, while the ones acclimated to HL would have lower pigment contents. Although not statistically significant, some differences could be discerned between strain Dn141EHU (round morphotype) and the other strains (elongated morphotype), with respect to the proportion of the fast-responding (P1-fraction) and slow-responding (P2) PSII centres during the inhibition phase of the HL stress. During recovery, it was not possible to discern the round morphotype from the elongated ones indicating a similar capacity of regeneration. These could hypothetically represent different ecotypes adapted to different niches. All strains in the present study belong to the same ribotype. For further studies, it would be interesting to study the physiological variability among cryptic phylogenetic groups of the P. lima complex. Many of the studies on the morphospecies P. lima do not include phylogenetic analyses of the analysed strains, which hampers the comparison among them. Furthermore, as depicted from Ben-Gharbia et al. (2016) more studies need to originate from different geographical areas, especially from the Indo-Pacific region where data are scarce. Also, since both round and elongated morphotypes can be found in the same ribotype, it is suggested that further studies should explore if these shapes are adaptive features of different ecotypes. Overall, our results are in agreement with Pan, Cembella and Quilliam (1999) when referring to P. lima as a shade-adapted epibenthic species with long generation times. The comparison of both roundish and elongate morphotypes and strains from the Atlantic Iberian Peninsula and the Mediterranean Sea (differing environments: higher vs. lower latitude; more turbulent and turbid vs. more calm and transparent waters) did not reveal significant differences in their photophysiology. However, different growth rates and small differences found in the photosynthetic properties of the elongated morphotypes, suggest that strains were acclimated to the local light environment, which differed at different locations. Further studies are necessary to investigate this pattern in a broader scale including tropical and cold temperate environments. SUPPLEMENTARY DATA Supplementary data are available at FEMSEC online. FUNDING Financial support for this research was provided by the Department for environment of Bizkaiko Foru Aldundia, the Bilbao-Bizkaia Water Consortium, and the Basque Government (projects IT-699-13 and UPV/EHU-PPG17/67). A grant from the Portuguese funding institution FCT—Fundação para a Ciência e a Tecnologia awarded to H. David (SFRH/BPD/121365/2016) is also acknowledged. Conflict of interest. None declared. REFERENCES Aissaoui A, Armi Z, Akrout F et al.   Environmental factors and seasonal dynamics of Prorocentrum lima population in coastal waters of the Gulf of Tunis, south Mediterranean. Water Environ Res  2014, 86: 2256– 70. Google Scholar CrossRef Search ADS PubMed  Aligizaki K, Nikolaidis G, Katikou P et al.   Potentially toxic epiphytic Prorocentrum (Dinophyceae) species in Greek coastal waters. Harmful Algae  2009, 8: 299– 311. Google Scholar CrossRef Search ADS   Baek SH, Shimode S, Kikuchi T. 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