Two - stages bloom of Margalefidinium cf. polykrikoides in a Mediterranean shallow bay (Ionian Sea, Italy)

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Two - stages bloom of Margalefidinium cf. polykrikoides in a Mediterranean shallow bay (Ionian Sea, Italy) ⁎

Leonilde Rosellia, , Maria Rosaria Vadruccia, Manuela Belmonteb, Pierangelo Cicirielloa, Fernando Rubinob, Nicola Ungaroa, Carmela Caroppob a b

Regional Agency for the Environmental Prevention and Protection (ARPA Puglia), Corso Trieste 27, Bari, Italy CNR-IRSA National Research Council-Water Research Institute, Unit of Taranto Via Roma, 3 – 74121 Taranto, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: Bloom Dinoflagellates Margalefidinium cf. polykrikoides Shallow bay Mediterranean Sea

The emergence of a red tide resulting in yellow-brownish discoloration of waters in Porto Cesareo bay (Italy) during July–August 2018 is reported. The species responsible for the bloom was the dinoflagellate Margalefidinium cf. polykrikoides. Cell densities reached 9.1 × 106 cells L−1 during the initial outbreak. A second peak was observed about three weeks later reaching 6.7 × 105 cells L−1. Study of live specimens showed great variation in cell size and shape. Different cyst morphotypes were found in the water samples and in the sediment. For the first time, we followed several stages of the life cycle of M. cf. polykrikoides in natural samples. Fish dieoffs in the bay were not observed, however this high-density bloom may have caused consequences on the ecosystem (amount of mucilage on the beach) and in turn, on tourism that is the main activity in the area during the summer season.

1. Introduction Harmful algal blooms (HABs) are natural phenomena, however some microalgal blooms can cause harm to humans and other organisms. These HABs have direct impacts on human health and negative influences on human wellbeing, mainly through their consequences to coastal ecosystem services (fisheries, tourism and recreation) and other marine organisms and environments (Davidson et al., 2011; Caroppo et al., 2016; Willis et al., 2018). These events can be favoured by anthropogenic pressures and eutrophication in coastal areas (Heisler et al., 2008; Sarkar, 2018). Global warming and associated changes in the oceans could affect HAB occurrences and toxicity as well, although forecasting the possible trends is still speculative and requires intensive multidisciplinary research (Wells et al., 2015). At the beginning of the 21st century, with expanding human populations, particularly in coastal and developing countries, mitigating HABs impacts on human health and wellbeing is becoming a more pressing public health need (Berdalet et al., 2016). Dynamics of HABs vary from one site to another, depending on the hydrographic and ecological conditions but also the complexity of the algal life cycle, which is composed of discrete life stages whose morphology, ecological niche (plankton/benthos), function, and lifespan vary (Wells et al., 2015; Figueroa et al., 2018; Glibert et al., 2018).

⁎

Margalefidinium polykrikoides (Margalef) Gómez et al., 2017 is a cosmopolitan dinoflagellate notorious for causing fish-killing HABs (Kim, 1998). Reactive oxygen species generated by M. polykrikoides are causative factors responsible for the oxidative damage of gill tissue leading to fish kills (Kim et al., 1999). This species has a wide distribution in the Asian and European waters, it is responsible for high mortalities of wild and farmed fish and causes large economic losses in the Pacific, Atlantic, and Indian oceans (Margalef, 1961; Matsuoka et al., 2008; Richlen et al., 2010; Kudela and Gobler, 2012). For example, it has been the cause of fisheries losses exceeding $100 million in Korea (Kim, 1998; Kim et al., 1999; Park et al., 2013). In the Mediterranean Sea, the presence of M. polykrikoides was detected in the Italian coast since the late 1990s and in the Black Sea since 2001 (Zingone et al., 2006; Iwataki et al., 2008; Kudela and Gobler, 2012), but it reached high concentrations in the Catalan Sea (Reñé et al., 2013). M. polykrikoides is a strong vertical migrator capable of utilizing both inorganic and organic nitrogen sources as well as mixotrophy and may be associated with moderate nutrient loading (Kudela and Gobler, 2012). These characteristics provide Margalefidinium with an adaptive capability conducive to rapid colonization of newly opened ecological niches, which may partially explain the apparent global expansion of its geographic range and bloom frequency (Kudela et al., 2008). While the

Corresponding author. E-mail address: [email protected] (L. Roselli).

https://doi.org/10.1016/j.marpolbul.2019.110825 Received 1 August 2019; Received in revised form 7 December 2019; Accepted 10 December 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Leonilde Roselli, et al., Marine Pollution Bulletin, https://doi.org/10.1016/j.marpolbul.2019.110825

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Fig. 1. (A) Water discoloration due to Margalefidinium cf. polykrikoides in Porto Cesareo Bay (Ionian Sea, Italy). (B) Copious amounts of viscous foam and mucus from M. cf. polykrikoides blooms in the beach.

2. Methods

production of resting cysts could contribute toward such an expansion, there are unanswered questions regarding the life stages of Margalefidinium, particularly regarding cyst formation and germination and how it contributes to the initiation and spread of blooms (Kim et al., 2007; Tang and Gobler, 2012; Jung et al., 2018). In this work, an exceptional yellow-brownish discoloration of the waters in the shallow Porto Cesareo Bay (Mediterranean, Ionian Sea, Italy) was observed in the July–August 2018 (Fig. 1A) and, it was found to be a bloom of M. cf. polykrikoides. We observed the event, that was notable for its intensity and time duration and, we followed it in order to highlight the bloom dynamics of M. cf. polykrikoides that is still not well-known. The high densities of M. cf. polykrikoides did not lead to fish die-offs in the bay but, a copious amount of viscous foam and mucus in the water column was documented in the days following the end of the blooms (Fig. 1B). The need for improving risk assessment to manage and prevent the occurrence of harmful blooms is globally enhancing. This is particularly true for those species that in the past persisted with low abundance in the phytoplankton assemblages and are currently responsible for high-biomass harmful events worldwide, as the case of Margalefidinium polykrikoides (Richlen et al., 2010).

2.1. Study area and environmental pressures Porto Cesareo Bay (Northern Ionian Sea, Apulia) (Fig. 2) is a shallow basin included in a marine protected area (MPA), which was formally established in 1997. The discoloration of waters occurred in the partial reserve of the MPA (C zone). In addition to fishing, the area provides recreational and aesthetic services including several beaches resorts, terrestrial and marine Sites of Community Importance with Posidonia oceanica meadows, coralligenous and submerged caves habitats (Guidetti et al., 2002; Terlizzi et al., 2002). The assessment of the anthropogenic pressures and expected impacts on the marine-coastal waters was carried out following the Italian SNPA-Guide Lines described in Fiorenza et al. (2018) and, summarized in Table 1. The discoloration water event was observed in a surface water body (SWB) which is already identified by Apulia Region according to the Water Framework Directive (WFD, 2000/60/CE) for monitoring program purposes and, it is codified as “ITR16184ACB3.s3_14”. It is a 1 km wide coastal area extending from the southern limit of the MPA of Porto Cesareo to Torre Colimena beach (Fig. 3A). The reference zones for the assessment of the pressure – impact relationships are: i) the afferent basin catchment area of the SWB and, ii) its relative buffer zone, as defined in the SNPA-Guide Lines (Table 1). In this study, the afferent basin catchment area corresponds Fig. 2. Location of water and sediment sampling sites in the Porto Cesareo bay, surveyed from July to September 2018. The dotted line indicates the area of discoloration waters phenomenon while the continuous line indicates the area of mucilage event. Water sample site DB (•); Surface sediment sampling sites D, C, M (Δ).

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Table 1 Method for the assessment of Pressure-Impact relationships from Fiorenza et al. (2018). In table are reported only the pressures considered as relevant for the study area. EI = Equivalent inhabitants; ha = hectare; N = nitrogen; Ptot = total phosphorous. *see the text for definition. PRESSURES Pressure indicators

Point source Presence of wastewater and their discharges (1.1)

Unitary EI = Sum of EI in the catchment area/km of shoreline

Diffuse source Discharge not connected to sewage network (2.6)

Number of EI not connected * 4.7 kgN/year/EI/ catchment area of the SWB (in ha)

Diffuse source Urban run-off (2.1)

Percentage of land coverage related to urban run-off in the buffer area

Diffuse source Agriculture (2.2)

Percentage of land coverage related to agriculture in the catchment area

Physical alteration (4.1)

Percentage ratio between the length of urbanized shoreline and the total length of buffer area

Threshold limits and reference area 2000 EI/km Catchment area* 100 kgN/ha/year Catchment area* 15% Buffer area*

50%

IMPACTS Type of Impact

Data sources

Impact indicators

Threshold limits and reference area

Water protection plan of Apulia Region (update 2015-2021)

Water protection plan of Apulia Region (update 2015-2021)

CORINE Land Cover maps (EEA, 2018)

CORINE Land Cover maps (EEA, 2018)

a) Annual average value of Ptot

a) > 0,3 µM

b) Number of algal blooms/peaks in one year

b) no bloom/peak

Catchment area* 50%

Buffer area*

Apulian Regional Technical Chart Italy (2011)

Data sources

Phosphorus concentration and list of phytoplankton taxa with cell density measured during the bloom event in the water body. Nutrient pollution

Type of pressure (WISE code)

Water body*

Data from monitoring program carried out by Apulia Region in the 2018 in the water body. Sampling was carried out bi-monthly. Water samples for nutrients analysis (including phosphorus) are collect in two stations localized at 500 m and 1000 m from the coast at two water depths (0.5 m and near the sea bottom).

“significant” related to a pressure means “a pressure that, on its own, or in combination with other pressures, would be liable to cause a failure to achieve the environmental objectives set out under Article 4 (good quality status)”. In this context, the impact is “significant” when the indicator values exceed the threshold limits. Hereafter, we refer to a “significant pressure or impact” sensu WFD.

to an extent area of 155 km2 including the territory of six municipalities (Avetrana, Porto Cesareo, Nardò, Leverano, Copertino and Veglie) (Fig. 3A). The buffer zone is defined as a 500 m wide area from the shoreline (Fig. 3A). The analysis of pressure-impact relationships was obtained using different data sources depending on the type of pressures in the study area and their indicators as well as on the type of expected impact (nutrient pollution) and its relative indicators (Table 1). The anthropogenic pressure types are those identified, classified and codified according to the WISE-WFD database (https://www.eea. europa.eu/data-and-maps/data/wise-wfd-3). We focused on the type of pressures that might cause nutrient enrichment and they were identified as among the most relevant for this study site (Table 1). In order to determine: i) point source pressure due to the presence of wastewater and their discharges (WISE 1.1) and diffuse source pressure due to the discharge not connected to sewerage network (WISE 2.6), the Water Protection Plan of Apulia Region (PTA Adozione proposta di Aggiornamento 2015–2021) was used; ii) diffuse source pressure due to the “urban run-off” and “agriculture” (WISE 2.1 and 2.2, respectively) pressures, the CORINE Land Cover maps (EEA, 2018) was used;; iii) physical alteration pressure (WISE code 4.1) the Apulian Regional Technical Chart Italy (2011) was used. ArchGIS ver 10.2 software was utilized to manage and display pressure data. Pressure indicators and the relative threshold limits in the reference areas related to each type of pressure are reported in Table 1. The assessment of expected impacts was made by using indicators of alterations of the ecosystem components, such as the variation of the water column chemical-physical properties or modification of biological communities (Fiorenza et al., 2018). According to this method, considering these types of pressure, the expected impact is nutrient pollution (Table 1). Impact indicators of nutrient pollution were applied on the data collected during the water discoloration event as well as during the marine-waters monitoring program routinely carried out by Apulia ARPA following the WFD in the above described SWB. Impact indicators and the relative threshold limits are also reported in Table 1. According to the WFD (2000/60/EC; CIS-WFD, 2003) the term

2.2. Field sampling and laboratory procedures For a period of two months, from the end of July to the end of September 2018, an emergency survey was performed due to the intense discolouration of the coastal waters in the northern part of Porto Cesareo Bay (40°16′10.7”N, 17°42′45.9″E) (Fig. 2). A phytoplanktonic bloom was observed starting from 30 July. Then, after a period of water transparency, another discolouration event occurred about 21 August. Investigation sampling was carried out at one station located in the area near the shore interested by the discolouration of waters (Station DB), collecting water at the surface using a telescopic rod (an extendable sampler for water sampling with interchangeable sample containers and holders). The sampling frequency was daily during the two events of discolouration and every three days during the period when the water was transparent, and in any case, until the end of September 2018. 2.2.1. Chemical-physical parameters Temperature, salinity and dissolved oxygen were recorded in situ with an Idromar 354 multiprobe. Nutrient concentrations nitrate (NO3−-N), phosphate (PO43−-P), total nitrogen (Ntot) and total phosphorus (Ptot) were measured according to segmented flow analysis (Aminot et al., 2009) by using Quaatro Seal Analytical Instruments. 2.2.2. Phytoplankton water samples At each sampling date, two phytoplankton samples were collected. One of them was kept in vivo while the other one was immediately fixed in Lugol's solution. Both of them were stored in a dark and cool place until the microscopic analysis in the laboratory. Each sample was 3

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was obtained from different samples characterising the different phases of the blooms. For scanning electron microscopy, 10 mL aliquots of fixed samples were filtered onto a 0.8 μm size Nucleopore membrane filter. The cells on the filter were rinsed a few times in distilled water, dehydrated with a graded ethanol series (50%, 70%, 80%, 90%, 95%, and 100%) and dried with hexamethyldisilazane (HMDS) (Botes et al., 2002). The filters were mounted on stub, sputter-coated with gold and viewed under a Leo 420 microscope. In order to remove mucilage on the cell surface several treatments were performed before the dehydration and drying (Honsell et al., 2010). 2.2.3. Cysts in the sediment To search for cysts, surface sediments were collected at three sampling stations at the end of the discolouration on 20 September 2018 when a mucilage event occurred in an adjacent bay (Fig. 2). The three sites were chosen on the basis of some environmental peculiarities: i) one was located in the area of the discolouration (Station D), one in the area interested by the mucilage event (Station M) and the last one in an area between the previous ones (Station C) (Fig. 2). At each site the sampling was carried out simply collecting the top 2 cm of the sandy bottom thanks to the low depth and two discrete samples were collected, one for the qualitative analysis, and the other for the quantitative one. Considering the sandy nature of the sediment, the qualitative analysis was carried out processing the whole sample to obtain additional information on rare species and to better analyse the morphology of M. cf. polykrikoides cyst types found during the quantitative analysis. Upon collection, to maintain the viability of the cysts, the samples were stored in an icebox approximately at 4 °C. All the samples were processed according to a sieving technique described in Montresor et al. (2010). For the qualitative analysis nearly 50 cm3 of sediment were utilized, while for the quantitative one an aliquot of nearly 2 cm3 was sub-sampled from each sample. The wet aliquots were weighed and screened trough a 20 μm mesh (Endecotts Limited steel sieves, ISO3310-1, London, England) using natural filtered (0.45 μm) sea water. The retained fraction was gently ultrasonicated for 1 min and screened again trough 75 and 20 μm stacked sieves. This allowed to obtain a fine-grained sediment fraction containing protistan cysts. The material retained on the 75 μm sieve was discarded. No chemicals were used to dissolve sediment particles in order to preserve calcareous and siliceous cyst walls. All the analyses were carried out under an LM (light microscopy) inverted microscope (Zeiss Axiovert 200 M) equipped with a Leica MC170-HD digital camera at 32 - 400× magnification. Both full (i.e. with cytoplasmic content) and empty (i.e. already germinated) cysts were counted. Considering the coarse nature of the sediment, at least 100 full cysts were counted per sample to obtain abundance values as homogeneous as possible. All resting stage morphotypes were identified based on published descriptions, the Modern Dinocyst Key website (https://www.marum.de/en/Modern_Dinocyst_Key.html). In order to estimate the water content in the sediments, a further set of subsamples was obtained, weighed and dried overnight at 70 °C. Quantitative data are reported as cysts g−1 of dry sediment (hereafter, cysts g−1). To clarify the topic concerning the different cyst types of Margalefidinium polykrikoides, particular attention was paid to the work of Kim et al. (2002), Tang and Gobler (2012), Li et al. (2015), Park et al. (2016) and Thoha et al. (2019).

Fig. 3. (A) Afferent basin catchment area (gray area) and buffer zone (white area) of the surface water body (SWB); (B) Agricultural land-use of the afferent basin of the SWB (green area); (C) Urban land-use of the buffer zone of the SWB (red area). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

examined under an inverted microscope (Nikon Eclipse Ti - S) equipped with a digital camera and an image analysis system (NIS elements). A quantitative analysis was performed on the fixed samples following the Utermöhl sedimentation method (Utermöhl, 1958; Zingone et al., 2010). The living samples were observed to obtain information on pigmentation, swimming behaviour, location of organelles, size, shape and location of chloroplasts, size and shape, and other features of the specimens, useful for their identification (Tomas, 1997). The evaluation of the size was based on the measurements of 200 cells at least. Population cell size structure, in terms of length and width of the cells,

3. Results 3.1. Water quality and anthropogenic pressures Salinity during the discoloration event ranged from 37.5–38.5, water temperature recorded ranged between 28 and 32 °C. Oxygen average concentration was around 10 mg L−1. The Ntot concentrations ranged from 54.21–63.38 μM during the first peak to 7.74–29.78 μM during the second peak, respectively. The NO3−- N concentrations 4

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Table 2 Pressure-Impact relationships in Porto Cesareo bay. In bold pressures and impact resulted significant sensu WFD. EI = Equivalent inhabitants; ha = hectare; N = nitrogen; Ptot = total phosphorous. *see the text for definition. IMPACTS

PRESSURES

Point source Presence of wastewater and their discharges (1.1)

Diffuse source Discharge not connected to sewerage network (2.6)

Threshold limits

2000 EI/km of shoreline

Pressure indicator values

Type of Impact

Impact indicators

Threshold limits

Impact indicator values

1615 EI/km of shoreline

100 kgN/ha/year

25 kgN/ha/year

Diffuse source Urban run-off (2.1)

15%

47%

Diffuse source Agriculture (2.2)

50%

85%

Physical alteration (4.1)

50%

66%

Nutrient pollution

Type of pressure (WISE Code)

a) Annual average value of Ptot

a) > 0,3 uM

a) 0,7 uM

b) Number of algal blooms in one year

b) no bloom/peak

b) two bloom/peak events

represent the 4% and the 92% of the Ntot during the first and the second peak, respectively. Ptot concentrations ranged from 0.57–1.33 μM to 0.32–0.85 μM during the first and the second peak, respectively. The PO43−- P concentrations represent the 40% and the 65% of the Ptot during the first and the second peak, respectively. The result of the analysis of pressure-impact relationships is reported in Table 2. In this study, the assessment of the impact nutrient pollution was performed only for the pressures that exceed the threshold limits and here, we refer to these pressures as “significant” sensu WFD (see Methods). In the study area, the type of pressure point source (WISE 1.1) and diffuse source (WISE 2.6) were determined. The first indicator value related to the study area was 1615 EI/km shoreline; the second indicator value, considering the total number of around 35,000 equivalent inhabitants estimated in the afferent basin catchment area, was 25 kg N / ha / year. In both cases the values of pressure indicators were lower than threshold limits, so these pressures are considered not significant (Table 2). Diffuse source pressures that are urban runoff (WISE 2.1) and agriculture (WISE 2.2), and physical alteration pressure (WISE 4.1) were also considered in the study area. The urban run-off covers the 47% land of the buffer area, while, the agricultural land use covers 85% of the catchment area. The physical alteration, estimated as percentage of urbanized shoreline, was 66%. All values resulted higher than the threshold limits (Table 2). Summarizing, these pressures resulted significant in the study area. Finally, according to the ItalianSNPA Guide Lines, the impact was estimated considering only the anthropogenic pressures that resulted significant. The impact typology that likely affects the study area is nutrient pollution and its relative indicators are: a) “Annual average value of Ptot” that showed a value of 0.7 μM L−1 higher than the threshold of 0.3 μM L−1 and, b) “number of microalgal blooms/peaks in one year”, that was two peaks observed in this study against no bloom/peak events which is the threshold (Table 2).

Resting stages produced by phytoplanktonic species were found at all the three sites examined in the bays. A total of 20 taxa of cyst producers were identified, both from quantitative and qualitative analysis, and all of them were produced by dinoflagellates (Table 4). For some species more than one cyst morphotype was identified, coming to a total number of 25 different morphotypes. Among them 4 were ascribed to M. cf. polykrikoides and three to Scrippsiella acuminata. The qualitative analysis revealed 8 species not observed in the quantitative samples. Among them the potentially toxic Alexandrium minutum and some species producing calcareous cysts like Calciodinellum operosum, Follisdinellum splendidum, Posoniella tricarinelloides and Scrippsiella kirschiae. The registered densities of full cysts varied from 103 cysts g−1 at Station D to 149 cysts g−1 at Station C, while the densities of empty ones were lower, ranging from 28 cysts g−1 at Station D to 59 cysts g−1 at Station C. Merging the registered abundance at the three sampling sites, the most abundant species was by far Margalefidinium cf. polykrikoides, with 230 full cysts g−1, representing 62% of the total assemblage, and 57 empty cysts g−1. Other abundant species were Scrippsiella acuminata and Scrippsiella sp.1.

3.2. Phytoplankton assemblages in the water column

3.4. Abundance and morphometrics of Margalefidinium cf. polykrikoides

A total of 31 microalgae taxa was identified from samples collected at the beginning of the bloom (30 July 2018), during the second bloom

Two distinct peaks in the abundance of M. cf. polykrikoides were observed. The first peak (9.08 × 106 cells L−1) was detected on 31 July

(28 August 2018) and, at the end of the bloom when Margalefidinium cf. polykrikoides was absent (24 September 2018). The micro-phytoplankton community was characterised by 20 taxa of diatoms, 9 dinoflagellates, 1 euglenophyta, and 1 coccolithophorid. The species list and abundance are summarized in Table 3. Dinoflagellates were numerically the most important group, followed by diatoms. However, in the former group M. cf. polykrikoides was by far the dominant species during the dense bloom events, reaching even the 99% of the total abundance. 3.3. Phytoplankton assemblages in the sediment

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Table 3 Abundance of micro-phytoplankton taxa in Porto Cesareo bay during proliferation of Margalefidinium cf. polykrikoides.

Dinoflagellates Ceratium furca Dinophysis sp. Gymnodinium spp. Gyrodinium spp. (> 20 μm) Margalefidinium cf. polykrikoides Prorocentrum micans Protoperidinium spp. Scrippsiella sp. Undetermined Dinophyceae thecate (> 20 μm) Total abundance of dinoflagellates Diatoms Amphora sp. Cerataulina pelagica Chaetoceros sp.p Chaetoceros wighamii Climacosphenia moniligera Cylindrotheca closterium complex Dactyliosolen blavyanus Dactyliosolen fragilissimus Guinardia striata Hemiaulus hauckii Leptocylindrus danicus Licmophora abbreviata Navicula sp. (< 20 μM) Pleurosigma spp. Proboscia alata Pseudo-nitzschia spp. del Nitzschia delicatissima complex Pseudo-nitzschia spp. del Nitzschia seriata complex Pseudo-nitzschia spp. Tabellaria fenestrata Thalassionema nitzschioides Total abundance of diatoms

Abundance

Abundance

Abundance

Taxon

Cell/L

Cell/L

Cell/L

30/07/2018

28/08/2018

24/09/2018

Dinoflagellates Alexandrium minutum Halim Calciodinellum operosum (Deflandre) Montresor Follisdinellum splendidum Versteegh Gonyaulax digitalis (Pouchet) Kofoid Gymnodinium impudicum (Fraga & Bravo) G. Hansen & Moestrup Gymnodinium nolleri Ellegaard & Moestrup Gymnodinium uncatenatum (Hulburt) Hallegraeff Margalefidinium polykrikoides (Margalef) Gómez, Richlen & Anderson Melodomuncula berlinensis Versteegh Pentapharsodinium dalei Indelicato & Loeblich Pentapharsodinium tyrrhenicum Montresor, Zingone & Marino Posoniella tricarinelloides (Versteegh) Streng et al. Scrippsiella acuminata (Ehrenb.) Kretschmann, Elbr., Soehner, Kirsch, Kusber & Gottischling Scrippsiella cf. erinaceus Kretschmann, Zinssmeister & Gottschling Scrippsiella kirschiae Zinssmeister, S. Soehner, S. Meier & Gottschling Scrippsiella lachrymosa Lewis Scrippsiella sp.1 Scrippsiella sp.4 Scrippsiella sp.5 Scrippsiella sp.6

119 19 190 114 4,764,601 79

237,085 58 38

1704

136

4,764,799

237,143

361

39 2298

674

119

4635 8180 39 857 97 39 136 818

79

78 78

95 285 76 76 152 133 247 38 152 38 57

3170

15,171

77

58 252,372

• • • • • • • • • • •

• • • • • • • • • • • •

•

The bloom observed at the study area was caused by an unarmoured dinoflagellate identified as Margalefidinium cf. polykrikoides. Our observations from light and scanning electron microscopy can be summarized as follows. M. cf. polykrikoides appeared as single cells (Fig. 5A, see also Supplementary Video 1) and, especially during the second peak, as twocelled chains (Fig. 5B). Single cells were ellipsoidal, with the epicone widely conical and the hypocone subspherical and bilobed (Fig. 5C). The cingulum encircled the cell about 1.8–2.0 turns and was highly concave (Fig. 5D). The apical groove or the anterior extension of the sulcus was not visible in the observations by light microscopy. The sulcus runs closely down just below the cingulum (Fig. 6A and B). A spherical nucleus was large and positioned at the anterior of the epicone (Fig. 7A). A reddish orange pigmented body (eyespot or stigma) was visible on the anterior dorsal side (Fig. 7A). Chloroplasts were rodlike shaped and aligned longitudinally in parallel (Fig. 7B, C). The transversal flagellum was curling-like (Fig. 7D, see also Supplementary Video 2). In many cases, individual specimens and two-celled chains were surrounded by a transparent and thin hyaline membrane (Fig. 5B, C and D). However, cells lacking extracellular mucilage were not observed under scanning electron microscope (Fig. 8) making it impossible to look at the apical groove that is an important diagnostic feature for the identification of M. polykrikoides (Matsuoka et al., 2008; Iwataki et al., 2010). In the water samples, we found a variety of forms that compared with the description published by Tang and Gobler (2012) and Shin et al. (2017), seems to represent different life stages of M. cf. polykrikoides (Fig. 9). However, Lugol's preservation did not allow us to give such a clear conclusion.

19 133 2207

58

0 4,767,969

• • • •

qt

3.5. Morphological description of the bloom forming species in the water column

324 136 39

ql

length of 17.96 ± 1.89 μm and width of 14.08 ± 1.40 μm. Other morphological features were the same as for the single cell stage.

305

Euglenophytes Eutreptiella sp. Coccolithophorids Syracosphaera pulchra Total abundance of other groups Phytoplankton total abundance

Table 4 List of the cyst taxa identified from surface sediments in Porto Cesareo bay. The results of the qualitative (ql) and quantitative (qt) analyses are reported.

57 57 2625

2018 while the second one (6.70 × 105 cells L−1) on 23 August 2018, about three weeks later (Fig. 4). In both cases, M. cf. polykrikoides dominated the phytoplankton assemblages contributing > 99% of the overall. After the first peak, the cell concentrations showed their lowest density for sixteen days, ranging from 8 to 4 × 102 cells L−1. Then abundances started to grow until the achievement of the second peak of 6 × 105 cells L−1 (Fig. 4). For ten days, cell densities were around 105 cells L−1, starting to decrease from the beginning of September until the disappearance of M. cf. polykrikoides cells at the end of the month. During the first peak, M. cf. polykrikoides was mainly observed as single cells. Also, temporary cysts were counted in the water samples reaching a concentration of 6 × 104 cysts L−1 (around 4% of the total counting). The second peak was characterised by new germling cells consisting of two (see figures in the following sections). During the first peak, single cells ranged in size with a length of 24.36 ± 5.44 μm and a width of 18.40 ± 4.26 μm (n = 100). Cells during the second phase of the event were on average smaller than the beginning of the bloom, with lengths of 14.08 ± 4.27 μm and widths of 13.82 ± 1.30 μm (n = 100). In this last case, cells ranged in dimensions from twisted cells with length of 10.20 ± 1.40 μm and width of 13.57 ± 1.18 μm to single cells with

3.6. Cyst morphology in the sediment In the surface sediment samples four different types of encysted stages of Margalefidinium cf. polykrikoides were discovered, presumably 6

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Fig. 4. Abundance of Margalefidinium cf. polykrikoides during the sampling period at the monitoring station DB.

and Gobler (2012) as a mature resting cyst (Fig. 1I).

representing different phases of the encystment process (Fig. 10). They were named as type 1 to type 4. i) Cyst type 1 – Pre-cyst intermediate stage. This cyst type is quite large, with the diameter measuring up to 52 μm. The hyaline double-layered wall is smooth, without mucus and inside, together with a central red accumulation body, both the sulcus and cingulum are visible. ii) Cyst type 2 – Pre-cyst, late stage: this morphotype has a spherical shape with a diameter of approximately 28 μm. The hyaline wall is double-layered, enwrapped in a variable amount of mucus. Inside the (para-) sulcus is visible. This stage is very similar to that reported by Tang and Gobler (2012) in Fig. 5 – day 10, as an intermediate stage in the germination process of cysts produced in laboratory cultures. iii) Cyst type 3 – Temporary cyst: the cyst is graygreenish in colour with a double-layered hyaline wall, always surrounded by mucus. An orange accumulation body is visible in the centre of the cyst, together with an intense microgranulation. The diameter ranged between 28 and 37 μm. This cyst is very similar to that identified by Thoha et al. (2019) in surface sediments from Lampung Bay (Indonesia) and illustrated in Fig. 2i. iv) Cyst type 4 – Mature cyst: this cyst type has an irregular spherical shape, with a double-layered smooth wall without mucus. It is dark brown in colour with a well visible orange accumulation body and microgranulations. The diameter ranged from 28 to 34.7 μm. This cyst is similar to that reported by Tang

4. Discussion The bloom event in Porto Cesareo bay (Southern Italy) examined during this study was caused by Margalefidinium cf. polykrikoides. Given the striking visual attributes of Margalefidinium blooms (large, dense, surface patches of colored water), the occurrence of these events in new regions or areas is more likely to be a real phenomenon than a function of improved methods of detection compared to other HABs that form less distinguishable blooms (Kudela and Gobler, 2012). The bloom here described was characterised by relatively high water temperature (28–32 °C) and salinity (37.8–38.5) values. These conditions were apparently causative to the growth of M. cf. polykrikoides, with maximum cell concentrations reaching 9.1 × 106 cells L−1 during the initial outbreak (30 July – 1 August 2018) and 6.7 × 105 cells L−1 during the second peak observed about three weeks later (23–24 August 2018). Based on laboratory experiments, temperature has the greatest influence on M. polykrikoides growth rate, followed by salinity, and then the interaction between temperature and salinity (Kim et al., 2004). However, even though rising ocean temperatures may intensify M. polykrikoides blooms in temperate zones, it could render this species less 7

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Fig. 5. Light micrographs of Margalefidinium cf. polykrikoides from the Ionian Sea. Cells are oriented apically. (A) Single cell (B) Two-celled chain showing the hyaline membrane (hm). (C) Cell showing the rounded epitheca and bilobed hypotheca. (D) Cingulum encircled the cell. Scale bars: 10 μm.

Particularly, from the pressure-impact analysis, Porto Cesareo bay resulted potentially subjected to nutrient pollution impact deriving from the land-use anthropogenic activities (see Results Section). In addition, we found in the area subjected to the discoloration of waters, total phosphorus concentrations higher than those detected during the routinely monitoring samplings for the nearly marine-coastal waters (0.7 μM and annual average concentration 0.26 ± 0.06 μM, respectively) (Annual report of SWBs monitoring program). Several authors

toxic (Griffith and Gobler, 2019). Additional factors that likely promotes the expansion of M. polykrikoides across the globe are supposed to be enhanced nutrient loading (Heisler et al., 2008), ballast water transport (Smayda, 2007), or climate change (Hallegraeff, 2010). However, in most cases the experimental evidences partly support each of these hypotheses prohibiting firm conclusions regarding the causes of these blooms and their expansion (Kudela and Gobler, 2012).

Fig. 6. Deeper focus of single cells of Margalefidinium cf. polykrikoides showing the cingulum (A) and the sulcus running just below the cingulum (B) Scale bars: 10 μm. 8

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Fig. 7. Light micrographs of Margalefidinium cf. polykrikoides showing (A) the nucleus (n) and the position of the eyespot (e) visible in the anterior of the cell; (B, C) rod-shaped chloroplasts (ch) and (B) the longitudinal flagellum (lf); (B) the curling-like transversal flagellum (tf) are visible. Scale bars: 10 μm.

(e.g. Viques and Hargraves, 1995; Lee and Lee, 2006; Anton et al., 2008; Mulholland et al., 2009; Morse et al., 2011) have also associated increases in Margalefidinium densities with rainfall, attributed to changes in both the physical environment and the influx of dischargeassociated nutrients. We observed heavy and short rainfall events few days before the peaks occurred. On one side, we suggest that the shallow waters and semi-enclosed morphology of the bay together with rainfall during the summer season, when the population can increase by 50 times compared to the resident one (ISTAT, 2018), could amplify the nutrients impact and trig the initial outbreak of the bloom. On the other side, adopting M. polykrikoides mixotrophy (Lee and Kim, 2007; Tang and Gobler, 2012), the intensity and time duration of the bloom here described might be due to the own self ecology of this species. We observed in the natural samples, different stages of the same bloom that seem those reported in the life cycle obtained in laboratory experiments by Tang and Gobler (2012). The adaptive capability to rapid colonization of newly opened ecological niches and the production of resting cysts could contribute to the apparent global expansion of the geographic range and bloom frequency of this species (Kudela et al., 2008). Nevertheless, there are unanswered questions regarding its life cycle, particularly about cyst formation and germination and how they contribute to the initiation and spread of blooms (Kim et al., 2007; Tang and Gobler, 2012; Jung et al., 2018). Although M. polykrikoides is primarily recorded from tropical to warm-temperate areas, during the last decades the species expanded its distribution in temperate coastal waters of the

Fig. 8. Scanning electron micrographs of Margalefidinium cf. polykrikoides, showing the cell covered by mucilaginous envelopment. Scale bar: 2 μm.

9

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Fig. 9. Putative life stages of Margalefidinium cf. polykrikoides collected in water natural samples: (A) pellicles (temporary cyst) showing the red pigmented body; (B) gymnodinium-like cell with heavy pigmentation; (C, D) temporary cysts showing the hyaline membrane (hm); (E, F) temporary and immature cysts with red accumulation body; (G-I) other forms. Scale bars: 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

reasonably just been produced due to their presence also in the water samples before their settlement. For their identification many factors were considered, namely the unusual abundance of temporary cyst stages in sandy sediments, the presence of the same stages in the water column during and immediately after the peaks, the morphological similarity to analogous stages reported in literature. Usually sandy bottoms contain very low densities of cysts, both as resting and temporary ones. In this study the exceptional abundance of M. cf. polykrikoides in the water allowed us to discover cysts in the sediments with densities comparable to those found in muddy or sandy-muddy sediments (e.g. Rubino et al., 2017). Besides this, temporary cysts of M. polykrikoides are not commonly observed in natural sediment samples. In fact, due to their physiological features and ecological role, normally they germinate in very short times after their production, and often they do not even have the time to reach the sea bottom (Bravo and Figueroa, 2014;

Mediterranean Sea (Reñé et al., 2013). The potential for cysts to overwinter in sediments and initiate blooms when environmental conditions are suitable could play a major role in its expansion, given the seasonal occurrence of the species in temperate areas. Nevertheless, reports of M. polykrikoides in Mediterranean Sea, both in the pelagos as active stages and in the sediment as cysts, are relatively rare, with low abundance and never in samples from beaches (Reñé et al., 2013; Rubino et al., 2016; Ferraro et al., 2017; Rogelja et al., 2018). In our knowledge, the occurrence of a two-stages bloom in a shallow bay near to the beach during the summer season can be considered as exceptional taking in to account the concentrations of this species detected in other costal sites around the globe (Moreira et al., 2016; Curtiss et al., 2008; Richlen et al., 2010; Gárate-Lizárraga, 2013; Jeong et al., 2017). In our sediment samples we were able to identify four different cyst morphotypes ascribable to M. cf. polykrikoides, all of them were had 10

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Fig. 10. Different types of encysted stages of Margalefidinium cf. polykrikoides collected in the sediment: (A) cyst type 1 – pre-cyst intermediate stage; (B) cyst type 2 – pre-cyst, late stage; (C) cyst type 3 – temporary cyst; (D) cyst type 4 – mature cyst. Scale bars: 10 μm.

collected in the sediments. The presence of a hyaline membrane seems to be an important feature detected in the water samples life stages both for vegetative and cyst morphotypes. Another feature of M. cf. polykrikoides (mainly in the vegetative forms) is the presence of mucus (Figs. 5, 9). Moreover, at the end of this severe bloom, a huge amount of viscous foam and mucus in the water column of a basin adjacent to the study area was observed (Fig. 1B). Functions and benefits that mucilage might provide have been proposed mainly for freshwater microalgae even though mechanisms are not always clear and no compelling evidence is available to verify some of the following hypothesis (see reference in Reynolds, 2006). Mucilage-bound algae are usually more common in nutrient-poor waters than in enriched systems and the organisms that produce variable amounts of mucilage produce more when nutrients (especially phosphorous) are depleted (Margalef, 1997). In such nutrient-limited environments, it is possible that a mucilaginous coat provides cheap mechanisms for increasing the size of the individuals whilst simultaneously providing a microenvironment wherefrom the rapid uptake of the nutrient across the cell wall is eased (Wolf-Gladrow and Riebesell, 1997). In fact, according to another hypothesis, the mucilage could provide a repository for the concentration and storage of essential nutrients (e. g. Lange, 1976). It is supposed also that the mucilage could minimize unnecessary metabolic activity (Margalef, 1997): nutrientdeficient cells may be prevented from completing their division cycle even though they do not stop photosynthesis (Vaulot, 1995). The mucilage could improve the self-regulated buoyancy of organisms, making controlled migrations in natural environment feasible (Reynolds, 1997). Another important possibility is that the mucilage helps to

Belmonte and Rubino, 2019). We did not observe resting cysts in the sediments, at least with the morphologies described by Matsuoka and Fukuyo (2003) or Li et al. (2015) and reported by Thoha et al. (2019), even considering that the sampling was carried out some weeks after the bloom and that, normally resting cysts are hypnozygotes and probably they need more time to complete their development (Belmonte and Rubino, 2019). However, for A. minutum, for example, Figueroa et al. (2007) reported a time after the sexual cross in mating and resting cyst formation up to 15 days during laboratory experiments. We did not find also the hyaline cysts described by Kim et al. (2002, 2007) from Korean waters and reported by Rubino et al. (2010) in the southern Mediterranean. Certainly, M. polykrikoides exhibits a complex life cycle, with many different morphological and functional stages both in the water column and in the sediments and, all of them are object of deepening knowledge worldwide. Thanks to the observations in natural samples and comparing findings in literature, we hypothesised different phases of the bloom event. During the initial outbreak, M. cf. polykrikoides appeared as single vegetative cells, no colonial organisms were detected. Also, different morphotypes of temporary cysts (pellicles) were found in the water samples. During the second peak, on average, cells were smaller than those at the beginning of the bloom. Moreover, two-celled chains were abundant. These new germlings cells were surrounded by a thin hyaline membrane. Toward the end of the second peak, different forms showing the largest size of the specimens were also found. Unfortunately, Lugol's preservation did not allow us to give more than a suggestion about the nature of those forms. However, to confirm these suggestions we are running germination experiments on the cysts 11

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F.R and M.B. performed the analysis on cysts in the sediment. L.R. wrote the manuscript with contribution from C.C, M.R.V., F.R and N.U.

protect against oxidative processes and leads to tolerance of high external concentrations of oxygen; in fact, it has been demonstrated that a low-redox microenvironment is maintained within the mucilage, apparently through the production of sulphydryl radicals (Sirenko, 1972). Moreover, the mucilaginous envelopes could provide a defense against the uptake of toxic cations in the acidic environments tolerated by some algae (Coesel, 1994; Freire-Nordi et al., 1998). Finally, mucilage reduces the edibility of algae by making them too large to be ingested (Komárková and Šimek, 2003) and prevents digestion during the passage through the guts of some consumers (Porter, 1976; Canter-Lund and Lund, 1995). Unfortunately, we do not have data that permit to study the above cited functions fulfilled by mucilage for M. cf. polykrikoides life stages. However, we highlight the need to deepen the knowledge of this ecological aspect that might have a role in the over-sustainment of the Margalefidinium bloom. The production of the huge amounts of mucilage has been already documented in the eastern Pacific where these substances caused suffocation of scleractinian corals indicating that the copious production of polysaccharides may be an important indirect mechanism of mortality (Guzmán et al., 1990). Based on our observational evidence during the monitoring period, the high densities of Margalefidinium did not lead to fish die-offs in the bay such as reported in other sites (Gárate-Lizárraga, 2013). The unusual event for the Porto Cesareo bay has been followed daily by local media news and newspapers, beyond the local stakeholders and tourists because of its feature of exceptionality and media impact during the tourism season. Although HABs, and M. cf. polykrikoides blooms are important in other parts of the world for their consequences on ecosystems good and services, it is interesting to point out the implications in the present study site. Tourism industry in Apulia region has an important socio-economic value, especially during the summer season, that is about 20 billion US$ annually (Report of the Regional Tourism Promotion Agency, 2016). Mucilage and discoloration could become a big menace to tourism and recreational activities that for example, in the Mediterranean Sea cover the 92% of the Gross Marine Product of 450 billion US$ annually (Randone, 2017).

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors thank to the executives of the Department of Lecce of Regional Agency for the Environmental Prevention and Protection, Dr. A. D'Angela, Dr. A. Romano, Dr. R. Bucci for their availability in supporting this study. The authors also thank Dr. P. D'Ambrosio and Mr. Ilario Peluso, staff of the Marine Protected Area of Porto Cesareo and Prof. A. Terlizzi for their support during sampling. Thanks to Prof. Annalisa Falace for her support in the identification of the mucilage samples, Dr. Maria Grazia Grasso for providing nutrient analysis, Dr. Francesca Fanelli for providing SEM picture and Dr. Vito La Ghezza for providing the shapefiles for the catchment area. References Aminot, A., Kerouel, R., Coverly, S., 2009. Chapter 8 in practical guidelines for the analysis of seawater. In: Wurl, O. (Ed.), Nutrients in Seawater Using Segmented Flow Analysis. CRC, 978-1420073065, pp. 143–178 2009 408 pp ISBN-13. Annual Reports of SWBs Monitoring Program. Available online at. www.arpa.puglia.it/ web/guest/monitoraggio_CIS. Anton, A., Teoh, P.L., Mohd-Shaleh, S.R., Mohammad-Noor, N., 2008. First occurrence of Cochlodinium blooms in Sabah, Malaysia. Harmful Algae 7, 331–336. doi.org/https:// doi.org/10.1016/j.hal.2007.12.013. Belmonte, G., Rubino, F., 2019. Resting cysts from coastal marine plankton. Oceanogr. Mar. Biol. Annu. Rev. 57, 1–88. https://doi.org/10.1007/978-3-030-21213-1_5. Berdalet, E., Fleming, L.E., Gowen, R., Davidson, K., Hess, P., Backer, L.C., Moore, S.K., Hoagland, P., Enevoldsen, H., 2016. Marine harmful algal blooms, human health and wellbeing: challenges and opportunities in the 21st century. J. Mar. Biol. Assoc. U. K. 96 (1), 61–91. https://doi.org/10.1017/S0025315415001733. Botes, L., Price, B., Waldron, M., Pitcher, G.C., 2002. A simple and rapid scanning electron microscope preparative technique for delicate “Gymnodinioid” Dinoflagellates. Microsc. Res. Tech. 59, 128–130. https://doi.org/10.1002/jemt.10184. Bravo, I., Figueroa, R., 2014. Towards an ecological understanding of dinoflagellate cyst functions. Microorganisms 2, 11–32. https://doi.org/10.3390/ microorganisms2010011. Canter-Lund, H.M., Lund, J.W.G., 1995. Freshwater Algae: Their Microscopic World Explored. Biopress, Bristol. Caroppo, C., Cerino, F., Auriemma, R., Cibic, T., 2016. Phytoplankton dynamics with a special emphasis on harmful algal blooms in the Mar Piccolo of Taranto (Ionian Sea, Italy). Environ. Sci. Pollut. Res. 23, 12691–12706. https://doi.org/10.1007/s11356015-5000-y. CIS – WFD – EC, 2003. Common implementation strategy for the water framework directive (2000/60/EC), guidance document n°3. In: Analysis of Pressures and Impacts, (150 pp). Coesel, P.F.M., 1994. On the ecological significance of a mucilaginous envelope in planktic desmids. Algol. Stud. 73, 65–74. Curtiss, C.C., Langlois, G.W., Busse, L.B., Mazzillo, F., Silver, M.L., 2008. The emergence of Cochlodinium along the California Coast (USA). Harmful Algae 7 (2008), 337–346. https://doi.org/10.1016/j.hal.2007.12.012. Davidson, K., Tett, P., Gowen, R., 2011. Harmful algal blooms. In: Hester, R.E., Harrison, R.M. (Eds.), Marine Pollution and Human Health. Issues in Environmental Science and Technology. 33. Royal Society of Chemistry, Cambridge, pp. 95–127. EEA European Environmental Agency, 2018. Coordination of Information of the Environment Land Cover 2011 Seamless Vector Data (CORINE Land Covermap). Available online at. https://land.copernicus.eu/pan-european/corine-land-cover/ clc2018. EU Water Framework Directive, 2000. European Commission directive 2000/60/EC of the European Parliament and of the council of 23 October 2000 establishing a framework for community action in the field of water policy. Off. J. Eur. Communities 327, 1–72 (Brussels). Ferraro, L., Rubino, F., Belmonte, M., Da Prato, S., Greco, M., Frontalini, F., 2017. A multidisciplinary approach to study confined marine basins: the holobenthic and merobenthic assemblages in the Mar Piccolo of Taranto (Ionian Sea, Mediterranean). Mar. Biodivers. 47, 887–911. Figueroa, R.I., Garcés, E., Bravo, I., 2007. Comparative study of the life cycles of Alexandrium tamutum and Alexandrium minutum (Gonyaulacales, Dinophyceae) in culture. J. Phycol. 43, 1039–1053. https://doi.org/10.1111/j.1529-8817.2007. 00393.x. Figueroa, R.I., Estrada, M., Garcés, E., 2018. Life histories of microalgal species causing

5. Conclusions We highlight the fundamental importance to implement both surveillance monitoring and seasonally oriented or ad hoc alerts during unusual events. Advanced techniques need to be implemented that permit sampling phytoplankton communities at ecologically relevant scales and to perform rapid and automated microscopic analysis of natural waters (e. g. laboratory or in situ cytofluorimetry) (Roselli et al., 2019). In fact, in the meanwhile we conclude this study an even more severe bloom of M. cf. polykrikoides was recorded in the first week of July 2019 (2.4 × 107 cell L−1) in the same bay of the 2018 summer causing again heavy consequences on the tourism industry. In addition, the same morphotypes of cysts here described were found in the sediment weeks later the bloom. And, high concentrations of M. cf. polykrikoides were observed along the Ionian coast that seems as a spreading of this species (unpublished data). The need for improving risk assessment to manage and prevent the occurrence of harmful blooms is globally enhancing. This is particularly true for those species that in the past persisted with low abundance in the phytoplankton assemblages and are currently responsible for high-biomass harmful events worldwide provoking environmental and socio-economic damages. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marpolbul.2019.110825. Author contributions L.R. designed the research and performed the analysis on phytoplankton. M.R.V., N.U. and P.C. performed the analysis on pressureimpact relationship. L.R. and C.C. wrote the description of the species. 12

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