First detection of the bloom forming Unruhdinium penardii (Dinophyceae) in a Mediterranean reservoir: insights on its ecology, morphology and genetics

The freshwater genus Unruhdinium includes dinoflagellates hosting a tertiary diatom endosymbiont. Some of the species belonging to this genus form high-biomass blooms. In this study, data on the ecology, morphology and molecular identity of Unruhdinium penardii were reported for the first time from a Mediterranean reservoir (Cedrino Lake, Sardinia, Italy). The ecology of the species and its bloom events were examined along a multiannual series of data (2010-2017). Cell morphology was investigated using field samples and six cultures established by cell isolation. A molecular identification of the six strains was performed. Wild and cultured cells shared the same morphology, showing a prominent apical pore complex and two/three more or less prominent hypothecal spines as distinctive characters in light microscopy. Molecularly, the six cultured strains corresponded to the same taxonomic entity with sequences only differing in a few polymorphic positions for the studied markers SSU rDNA, LSU rDNA, ITS and endosymbiont SSU rDNA. All markers showed 99.5%−100% similarity with the available U. penardii sequences. Seasonality of U. penardii revealed its preference for the colder semester (from December to June) with bloom events restricted to late winter/early spring months. Three blooms resulting in reddish water discolorations were observed along the study period (2011, 2012 and 2017). GLMs revealed a significant role of water depth, temperature, and reactive phosphorous in determining the highest cell densities (>5 x 104 cells L–1). The results obtained contribute to the increase of field ecology knowledge on this species, demonstrating it is well established in the Mediterranean area, and being able to produce recurrent high biomass blooms in the studied reservoir. No n-c om me rci al us e o nly C.T. Satta et al. 72 Padedda et al., 2017). Cedrino Lake belongs to the Italian Network of Long Term Ecological Research (LTER-Italy; deims.org/9010f9db-3d6b-4253-9604-4e10f6714000). The Regional Sardinian Agency ‘Ente Acque della Sardegna’ (ENAS) is the manager of the Cedrino Lake since 2006. Samplings were conducted from July 2010 to May 2018 at one station close to the deepest part of the lake (Fig. 1) following different patterns (Supplementary Tab. 1). In fact, the data collection activities were carried out under various projects with different objectives. Consequently, samplings were conducted monthly in 2010 and 2011 and bi-monthly from 2012 to 2018 (Supplementary Tab. 1). During the sampling period several interruptions occurred, mainly due to adverse weather conditions or sampling difficulties. Water samples were collected from selected water depth layers using a Niskin bottle. Samples for phytoplankton and chlorophyll a (Chla) analyses were collected from 0, 1, 2.5, 5, 7.5 and 10 m water depth layers. Phytoplankton samples were immediately fixed in Lugol’s iodine solution (1% final concentration) for the cell density estimate, determined following Utermoḧl (1958) under an inverted microscope Axiovert 25 (Carl Zeiss, Oberkochen, Germany) at 200x magnification. Further non-fixed samples were taken for the observation of live cells, for cell culturing and for formalin-fixation. Cell counts were made for each of the six depths from 2010 to 2013, whereas only one sample corresponding to the depth with the highest Chla was counted from 2014 to 2018. Chla was determined as described by Goltermann et al. (1978). Water temperature (Temp), conductivity (Con), dissolved oxygen (DO) and pH were measured in situ with multi-parameter probes (YSI 6600 V2 and Hydrolab DS5). Transparency was measured with a Secchi disk (SD). Euphotic zone depth was calculated using SD measures (Zeu: 2.5 times the SD depth; Poikane, 2009). Water samples for nutrient analyses were collected from Fig. 1. Geographical location of the Cedrino Lake (Sardinia, Italy) and sampling station placement. No n-c om me rci al us e o ly Unruhdinium penardii in a Mediterranean reservoir 73 each of the selected water depth layers (0, 1, 2.5, 5, 7.5, and 10 m) plus further depth layers along the entire water column (15, 20, and 30 m). Concentrations of nutrients such as reactive (P-PO4) and total (TP) phosphorus, ammonium (N-NH4), nitrate (N-NO3), (N-NO2) and total nitrogen (TN), were determined according to the methods of Strickland and Parsons (1972). Total dissolved inorganic nitrogen (DIN) was calculated as the sum of NNH4, N-NO3, and N-NO2. Surface sediment samples were taken with a grab at Tab. 1. Unruhdinium penardii cell densities in the samples corresponding to the highest chlorophyll a values at each data sampling in the photic zone and U. penardii cell densities and chlorophyll a values calculated as weighted average in the photic zone. Data corresponding to U. penardii cell densities higher than 5 x 104 cells L–1 are reported in bold. U. pen Depth Chla U. pen WA Chla WA Disc (cells x 103 L–1) (m) (mg m–3) (cells x 103 L–1) (mg m–3) (m) 2010 Jul 0 2.5 17.5 0 19.6 1.0 Aug 0 1 18.5 0 17.3 1.5 Sep 0 0 56.9 0 51.1 1.5 Oct 0 0 14.6 0 11.5 1.2 Dec 5.8 1 0.6 3.5 0.3 2.5 2011 Jan 0 1 0.6 0 0.3 3.0 Feb 0 7.5 1.1 2.1 0.7 1.1 Mar 758.2 2.5 21.9 710.9 18.3 2.0 Apr 5.9 5 24.1 16.6 18.8 1.7 May 0 0 0.9 1.6 0.6 3.0 Jun 0 1 4.9 0 4.2 2.3 Jul 0 1 8.2 0 7.0 1.7 Aug 0 1 29.2 0 27.4 1.5 Sep 0 0 58.8 2.6 50.9 1.0 Dec 4.0 2.5 0.1 2.3 0.1 1.6 2012 Feb 25,632 0 359.7 9380.9 151.92 0.7 Apr 1.9 1 1.0 0.8 0.4 7 Jun 10.1 0 101.8 10.5 99.0 0.6 Aug 0 2.5 78.2 0 70.2 0.5 Oct 0 1 29.1 0 24.7 1.8 Dec 0 0 14.4 0.5 13.0 3.2 2013 Feb 0 5 13.5 29.2 10.1 1.5 May 6.0 0 39.0 6.0 37.5 1 Jul 0 2 14.6 0 9.9 1.4 Sep 0 0 8.6 0 7.4 2.6 2014 Jan 88 2.5 8.5 7.9 1.7 Mar 32 2.5 29.7 27.0 1.7 May 0 0 9.4 9.1 2.5 2015 Jan 3 1 1.9 1.6 1.7