Invasion of Ceratium furcoides in subtropical lakes in Uruguay: environmental drivers and rst sh kill record

The invasive freshwater dinoagellate Ceratium furcoides is extending its distribution in South America with increasing environmental impacts associated with its blooms. We here report two events related to C. furcoides distribution expansion in Uruguay: 1) the rst appearance and main environmental drivers (physico-chemical variables, extreme wind events and zooplankton composition) of the bloom of C. furcoides in 2012 in a subtropical eutrophic shallow lake (Lake Blanca, Uruguay), and 2) the sh kill event of Prochilodus lineatus likely caused by C. furcoides in 2016 in a deep eutrophic lake (Puente de las Americas, Uruguay), which is the rst sh kill attributable to C. furcoides registered in the world. The bloom of C. furcoides in Lake Blanca started in spring 2012 (October) during a clear water period with high phytoplankton species replacement after a cyanobacterial bloom of Raphidiopsis raciborskii. Extreme wind events during this period resuspended cysts from the sediments, which likely started the C. furcoides bloom. High nutrient availability and low zooplankton grazing, allowed the bloom to expand and reach 96% of the phytoplankton biomass. Our results further indicate that the sh kill of Prochilodus lineatus in Lake Puente de las Americas was likely promoted by the high biomass of C. furcoides bloom, causing gill damage and clogging together with oxygen depletion in the benthic zone. Our study is the earliest record of C. furcoides in Uruguay and it shows the drastic consequences of C. furcoides bloom in freshwaters and its potential of inducing massive sh kills.


Introduction
Ceratium furcoides (Levander) Langhans 1925 is a freshwater dino agellate species which has recently expanded its geographic distribution range to southern South America (Meichtry de Zaburlín et al. 2016) and is currently considered invasive in this region (Boltovskoy et al. 2013).
Ceratium furcoides is a large-sized phytoplankton species (162-322 μm lineal dimension), K-selected, stress-tolerant, with a slow growth rate, low surface/volume ratio, high nutrient storage capacity and high resistance to herbivory (Bustamante Gil et al. 2012;Olrik 1994;Pollingher 1988;Reynolds 2006;Weithoff et al. 2001). C. furcoides shares morphological characteristics with Ceratium hirundinella (O.F. Müller 1773) Dujard, 1841 which is a common species in Uruguay (Fabre et al. 2010;Goyenola et al. 2014) and the distinction between the two species is di cult. The overall shape and size of these two species are similar, and both have an apical horn on the epitheca and a variable hypotheca with two or three antiapical horns (Huber-Pestalozzi 1950;Hickel 1988;Popovsky & P ester 1990). The main morphological differences between the two species occur in the epitheca, where the apical horn of C. furcoides is slightly thicker and cone-shaped than that of C. hirundinella, whose plate 4' is shorter and does not reach the apex of the apical horn ( Fig. 1) (Calado & Larsen 1997;Popovský & P ester 1990;Santos-Wisniewski et al. 2007). C. furcoides is often mistakenly identi ed as C. hirundinella (Bustamante Gil et al. 2012;Calado & Larsen 1997;Cavalcante et al. 2013) both because of the morphological similarity but also because of a high overlap in terms of ecological requirements. The genus Ceratium (Schrank) forms blooms in lakes and reservoirs during the warmer months in the pantropical region (Carty 2003;O'Sullivan & Reynolds 2004;Pollingher 1988). Although some species of the genus have been registered at oligotrophic conditions (Bucka & Zurek 1994;Padisàk 1985) they are typically found in eutrophic-hypereutrophic systems (Claps & Ardohain 2007;Hart 2007;Whittington et al. 2000) often co-occurring with cyanobacteria (Lund 1965). Species within the Ceratium genus are highly mobile and may control their position in the water column to perform diurnal vertical migration to areas with high availability of light and nutrients, particularly in deep or strati ed ecosystems (Heany & Talling 1980;Reynolds 2006). Consequently, both species may coexist in the same ecosystems (Gołdyn & Kowalczewska-Madura 2007;Hickel 1985;Stefaniac et al. 2007) but C. furcoides often become dominant (Boltovskoy et al. 2013;Meichtry de Zaburlín et al. 2016;Salusso & Moraña 2014) and eventually, this may lead to the exclusion of C. hirundinella (Salusso & Moraña 2014). The competitive ability C. furcoides and its potential to rapidly form cumulative blooms has been attributed to the ability to produce cysts. Under unfavourable environmental conditions or in response to density-dependent processes, C. furcoides can produce cysts for resistance and dispersion (Bustamante Gil et al. 2012). These cysts, deposited in surface sediments, can germinate during periods of mixing when environmental conditions are favourable, thereby constituting an important inoculum that may maintain high population densities throughout the year (Bustamante Gil et al. 2012;Cavalcante et al. 2016).
The expansion of C. furcoides has adverse consequences on water quality and ecosystem functioning. Accumulative blooms of C. furcoides often promote oxygen depletion, brown coloration, taste, odour, and may cause lter saturation in water treatment plants (Matsumura-Tundisi et al. 2010;Morales 2016;Taylor et al. 1995). While sh kill events due to C. hirundinella have been recorded (Bazán et al. 2007;Nicholls et al. 1980), there had been no clear evidence of a sh kill caused by C. furcoides, although some authors have mentioned its potential to cause sh kills (Campanelli et al. 2017;Matsumura-Tundisi et al. 2010;Morales 2016).
We report here two extreme events related to the expansion of C. furcoides distribution: 1) the earliest appearance and bloom of C. furcoides for Uruguay in 2012 in Lake Blanca, a subtropical eutrophic shallow lake, where we analyse in detail the environmental factors associated with its bloom. 2) As an extreme environmental impact derived from the bloom of C. furcoides, we register the sh kill event of Prochilodus lineatus (Valenciennes, 1837) associated with the bloom of C. furcoides in the deep eutrophic Lake Puente de las Americas in southern Uruguay in 2016, likely the rst sh kill attributable to C. furcoides registered in the world.

Study area
Lake Blanca is a subtropical shallow lake (Zmax= 3.2 m, fetch=1.3 km) located at the south-eastern coast of Uruguay (34°89' S; 54°83' W, Area: 5.4 km 2 ), used for tap water provision (Kruk et al. 2009;Pacheco et al. 2010). It is polymictic, eutrophic lake with low plant coverage during the study period, although it is often covered by submerged plants (Mazzeo et al. 2003). The lake has experienced several episodes of persistent cyanobacterial blooms of Microcystis aeruginosa and Raphidiopsis raciborskii (formerly Cylindrospermopsis raciborskii), limiting its exploitation as a tap water source (Pacheco et al. 2010).
Lake Puente de las Americas (34°87' S; 56°03' W, Area: 0.13 km 2 ), is a relatively deep mine pit lake with a short fetch (Zmax= 7.5, fetch= 0.66km) located in a periurban area in south Uruguay. It originated from sand mining and is described as a eutrophic to hypereutrophic lake with recurrent strati cation and anoxia in the hypolimnion, especially during summer. The lake has recently undergone drastic ecosystem alterations due to the relocation of sediments at the lakeshore (Goyenola, et al. 2014;Goyenola et al. 2018). Lake Puente de las Americas is located nearby the strongly eutrophicated Carrasco stream, having sporadic water connection depending on the hydrological conditions (Goyenola et al. 2014). At least since 2010, the lake has had intensive phytoplankton blooms, with high turbidity and frequent presence of cyanobacterial blooms of Planktothrix agardhii and Raphidiopsis raciborskii. The previous presence of Ceratium in this lake was restricted to C. hirundinella registered in June 2013 (Goyenola et al. 2014) while C. furcoides had not previously been recorded.

Physico-chemical and biological methods
Within the framework of different research programs, we monthly sampled phytoplankton, zooplankton and environmental variables in Lake Blanca from May 2011 (July 2010 for phytoplankton) to October 2013, with a fortnightly sampling frequency during the warmest months (November to May). The samples were taken at three sampling points along a pelagic mid-lake transect. At each sampling point, we measured: temperature, pH, conductivity, turbidity and dissolved oxygen at mid-depth using a YSI 650 MDS multiprobe. We collected depthintegrated samples with a tube sampler (10 cm diameter) integrating the entire water column. From the integrated water samples, we collected sub-samples for physico-chemical, phytoplankton and zooplankton analyses. Zooplankton samples were collected by ltering 10 L of the column-integrated water sample through a 50-µm mesh net. Zooplankton and phytoplankton samples were preserved with acidi ed lugol's iodine solution to 5%. Pelagic chlorophyll-a (Chl_a) samples were collected and ltered in situ through GF/C glass ber lters, followed by extraction in 95% cold ethanol and spectrophotometrical measures (absorbance 665-750 nm) in the laboratory, following Nusch (1980) andISO10260 (1992). Total nitrogen (TN) and phosphorus (TP) concentrations were determined according to Valderrama (1981), and nitrate (NO 3 ), ammonia (NH 4 ) and orthophosphate (PO 4 ) were determined according to APHA (2005).
We examined the potential role of extreme wind events as trigger promoting C. furcoides bloom in Lake Blanca. Historical data series on the maximum wind speed (sustained at least for 3 hours) were obtained from a nearby meteorological station for the period 2010-2013 (Laguna del Sauce WMO N°86586 INUMET). Furthermore, due to the use of Lake Blanca for drinking water provision, we analysed the presence of C. furcoides in tap water after the potabilization process in the water treatment plant and in a distribution tank using qualitative samples taken with a 20-µm mesh plankton net.

Plankton identi cation and counting
Phytoplankton was identi ed to species level and counted in random elds at 100 to 400X magni cation using an Olympus CKX 41 inverted microscope (Utermöhl 1958). Counting lasted until at least 100 individuals of the most frequent phytoplankton species (Lund et al. 1958), considering the organism (cell, colony or lament) as the unit and calculating the biovolume according to Hillebrand et al. (1999). We did not consider picoplankton (smaller than 2 μm) or tychoplankton (periphytic resuspended organisms). We treated the cells with NaClO 20% to separate cell wall plates and perform dino agellates identi cation based on Popovský & P ester (1990) and Steidinger & Tanger (1997). Ceratium furcoides individuals were rst differentiated from the almost similar C. hirundinella by its shape and then identi ed based on the position of the apical plate 4' that does not reach the apex (Fig. 1).
Zooplankton was identi ed and counted in the laboratory according to Paggi & de Paggi (1974). We calculated the ratio mesozooplankton (calanoid copepods and cladocerans) to microzooplankton (copepod nauplii and rotifers) (Meso:micro) from abundance data (ind.L -1 ) as a proxy of zooplankton grazing potential on phytoplankton (Pacheco et al. 2010).

Data analysis
The environmental and biological variables explaining the phytoplankton composition in Lake Blanca were analysed by canonical correspondence analysis (CCA) to constrain the composition variance. We rst performed a detrended correspondence analysis to determine which constrained method to use, and as the longest axis was longer than three standard deviations we selected the unimodal-based method (CCA) (ter Braak & Verdonschot 1995). We selected the environmental and biological explanatory variables to incorporate in the CCA model by stepwise selection, comparing R 2 with the full model and checking the signi cance of each variable and model by Monte-Carlo permutation test. We inspected the variance in ation factor (VIF) of the variables to identify collinearity, excluding variables with VIF>10 or VIF=0 (ter Braak & Verdonschot 1995).
We analysed the environmental and biological variables associated with the bloom of C. furcoides in Lake Blanca by partial least-squares regressions (PLS), cross-validating the model by permutation test and selecting the variables best explaining the high abundances of C. furcoides.
We z-scored standardised environmental data and log (x+1) transformed the phytoplankton biovolume data, excluding rare species with a lower contribution than 1% to the total phytoplankton biomass from the analysis, to avoid bias in the sensitive methods. All the statistical analyses were performed in R 3.6.2 (R Core Team, 2019) using the 'vegan' package (Oksanen et al. 2013).

Fish kill register methods
As part of a research program in the Lake Puente de las Americas, we performed three monthly sampling campaigns during June to September 2016. We measured Secchi disk depth, dissolved oxygen and turbidity and collected depthintegrated chlorophyll a and phytoplankton samples following the same analytical methods as for the Lake Blanca samples. The sampling campaign included the day of the sh kill, 29 August 2016. Prochilodus lineatus (Valenciennes, 1837) is a large-sized (aprox. 80 cm in total length and 7 kg of fresh biomass) migratory sh from the Río de la Plata basin (Castro & Vari 2003;Zaniboni et al. 2004). It feeds mainly on organic matter from the sediment (detritivore) also consuming periphyton and phytoplankton, and therefore has a fundamental role in freshwater food webs and ecosystem functioning (Flecker 1996, Benedito et al. 2018. Dead sh were manually collected at the lakeshore and frozen until laboratory processing. In the laboratory, we analysed in microscope the material accumulated in the gills. We compared the general conditions of the gills, the amount of material accumulated on the laments, and the phytoplankton from this accumulated material with the gills and material from healthy sh obtained in the subsequent sampling campaign (September 2016).

Results
The environmental characteristics were highly variable in Lake Blanca during the period 2011-2013 (Table I, Fig. 2). Turbidity was at its historical maximum during the C. furcoides bloom, coinciding with the lowest transparency (Secchi Disk depth) (Fig. 2). The levels of nutrients, chlorophyll-a and transparency corresponded to mesotrophic-eutrophic conditions (Carlson 1977). Nitrogen concentrations varied widely, with a TN peak during the initial cyanobacterial bloom in 2011, while NH 4 was low throughout the period except for a peak during the second half of 2012; NO 3 had the highest concentrations in 2013 during the C. furcoides bloom (Fig. 2, Table I). Phosphorus varied widely as well, with low PO 4 concentrations during 2013 (Fig. 2), and similarly to TN, TP rapidly declined at the end of the study period (Oct 2013, Fig. 2).
The phytoplankton community showed substantial variations during 2010-2013, with alternating periods of high and low biomass and marked taxa replacements (Fig. 3). From July 2010 to July 2011, cyanobacteria dominated the phytoplankton community In Lake Blanca, with extremely high biomasses (c.a. 60 mm 3 .L -1 ) and a persistent bloom of Raphidiopsis raciborskii (formerly Cylindrospermopsis raciborskii). This was followed by a period of relatively low phytoplankton biomass and taxa replacement from desmids (mostly Closterium acicularis and Staurastrum leptocladum) to chlorophytes (Coelastrum sp., Pediastrum spp. and Botryococcus sp.), followed by the bloom of C.
furcoides. An abrupt change in phytoplankton composition occurred just before C. furcoides bloom. This period was characterised by low phytoplankton biomass, mainly represented by tychoplanktonic diatoms and euglenoids, while also dino aggelate cysts and resuspended sediment appeared in the samples. The period coincided with the most extreme wind events, with wind speeds above 100 km. h -1 for more than 3 hours (Fig. 3).
The C. furcoides bloom was characterised by rapid growth (Fig. 3) thus, it constituted more than 96% of the total phytoplankton biovolume, and led to collapse of other groups, particularly dino agellates (Fig. 4). Before the consolidation of the C. furcoides bloom, dino agellates were mainly represented by Peridinium spp. and low biomasses of C. hirundinella (Fig. 4). After the C. furcoides bloom, both the Peridinium spp. and the C. hirundinella populations reached low biomasses (Peridinium spp. maximum= 1.97 mm 3 .L -1 ) relative to C. furcoides (25.76 mm 3 .L -1 ) (Fig 4). The zooplankton in Lake Blanca was largely dominated by small-sized rotifers such as Keratella spp.,

Filinia spp. (microzooplankton) and the small-bodied cladoceran Bosmina spp. (mesozooplankton).
According to the CCA, phytoplankton composition in Lake Blanca was largely explained by turbidity (Turb), the ratio of mesozooplankton to microzooplankton (Meso:micro), total phosphorus (TP) and ammonium (NH 4 ) concentrations (CCA: F= 11.78, p=0.008; Fig. 5), with the rst two axes explaining 59.8% over 69.9% of the total constrained variance. C. furcoides biomass was positively correlated with turbidity and the Meso:micro ratio and negatively with the NH 4 concentration. Similarly, the PLS analysis also identi ed turbidity and the Meso:micro ratio as positive factors and zooplankton biomass, NH 4 and TP as factors negatively correlated with C. furcoides based on the eight selected components, explaining 86.9% of the total variance.
At the end of the study period, during the biomass peak of C. furcoides, we observed cells with cyst formation (Fig. 6) and the presence of cysts in the water column. These cysts passed the water puri cation process and were found in the tap water. Furthermore, we observed both cysts and adult organisms of C. furcoides in one of the tap water distribution tanks, located 2 km away from the potabilization plant (El Chorro, Maldonado 34°54'02" S; 54°48'54" W); the adults with deformities in the apical and antiapical horns (Fig. 6).
Although in this study we did not analyze the sh community in Lake Blanca, we did not record sh kills in Laguna Blanca during the study period.

Fish kill event
On 29 August 2016 a massive sh kill of Prochilodus lineatus (Characiformes) was registered in Lake Puente de las Americas during a C. furcoides bloom. Before the bloom (19 June), the lake exhibited intermediate phytoplankton biomass (Chl_a = 50µg.L -1 ) in the surface water, the transparency (SD = 1.0 m) was intermediate and the turbidity (Turb = 5 NTU) low, while intermediate levels of dissolved oxygen in the surface water prevailed (OD = 73-86%). The phytoplankton was dominated by cyanobacteria (mostly Dolichospermum circinalis and Aphanocapsa elachista up to 5000 org.mL -1 ). On the sampling event on 30 August 2016, we registered the bloom of C. furcoides in Lake Puente de las Americas. As in Blanca Lake, the bloom of C. furcoides in this lake was characterised by a drastic increase in phytoplankton biomass (Chl_a = 170.4 µg.L -1 ) with 99% consisting of C. furcoides, high turbidity (Turb = 18.2 NTU) and low transparency (SD = 0.6m). Furthermore, the bloom increased the surface oxygen to oversaturation (81.6-115.9%). On the subsequent sampling event (22 September 2016), the phytoplankton biomass was markedly lower (16.8-19.5µg.L -1 ) the bloom of C. furcoides had collapsed and high amounts of C. furcoides cysts but not adult organisms were recorded. Dissolved oxygen and saturation depth pro les showed hypoxia or anoxia at depths of more than 2m, coinciding with the beginning of the bloom of C. furcoides in late August (Fig. 8). Turbidity also decreased (Turb = 13 NTU), while surface saturation of dissolved oxygen remained high, 104-107%. At this time, the phytoplankton included various groups such as diatoms (mainly Aulacoseira distans and Cyclotella meneghiniana), euglenoids (Trachelomonas spp.), Chlorophyta (Scenedesmus spp.), Charophyta (Staurastrum spp.) and Plagioselmis nannoplanctica.
In the initial visual inspection of the dead sh, the cause of the sh kill could not be determined.
Although it was not possible to record the number of dead sh in the shoreline, the high density of dead sh observed were clearly much higher than the limit to de ne a sh kill for lakes of 25 ind.km 2 (La & Cook 2011). This indicated that the event was not an incidental mortality restricted to few organisms, but a massive mortality caused by extendedenvironmental stress in the lake. In the microscopic analysis, we found dense accumulations of C. furcoides in all gill laments, clogging the laments and even stuck into the gill tissue (Fig. 8). Contrary, the specimens of P. lineatus collected after the bloom on 22 September 2016 had normal gill tissue (Fig. 9), without phytoplankton accumulations and the presence of only a few Aulacoseira granulata, Cyclotella meneghiniana and Staurastrum sp.individuals.

Discussion
This study constitutes the earliest record of Ceratium furcoides in Uruguay (October 2012) and likely the rst record worldwide of a sh kill attributable to its bloom. Invasive C. furcoides has expanded its distribution to Uruguay, an area previously indicated as having high invasion potential (Meichtry de Zaburlín et al. 2016). Lake Blanca basin is not connected to the geographically closest record of this species, the Salto Grande reservoir, 500 km north in the large river Uruguay. Therefore, although C. furcoides probably colonized Salto Grande from northern Argentina or Brasil, where it has been extensively recorded ( The recording of cysts and adult organisms in the drinking water distribution tank further evidences the dispersal potential of C. furcoides. Even though the adult organisms of C. furcoides were retained during the potabilization because of their large size (160 µm average linear dimension) and their sensitivity to chlorination, the small-sized (22 µm linear dimension) and more resistant cysts can go through the water puri cation process and germinate somewhere else. However, all these adult organisms developed from cysts presented deformities, possibly because of their sensitivity to chlorination.
The occurrence of C. furcoides bloom after a cyanobacteria dominance, as seen in Lake Blanca (with R. raciborskii and Microcystis aeruginosa) has been described previously (Bustamante Gil et al. 2012;Cavalcante et al. 2016;Matsumura-Tundisi et al. 2010). C. furcoides require high nutrient availability and resuspension of its cysts from the sediments to start the bloom (Almanza et al. 2016;Bustamante Gil et al. 2012;Claps & Ardohain 2007;Hart 2007;Matsumura-Tundisi et al. 2010;Whittington et al. 2000). Consequently, a high nutrient uptake-and storage capacity, as well as the ability to swim, which give access both to light on the surface and benthic nutrients (Reynolds et al. 2006) may provide this species with a high competitive ability under eutrophic conditions. This further indicates that eutrophication promotes suitable conditions to facilitate the invasion of C. furcoides in freshwater ecosystems. Lake Blanca experienced a period of high transparency with low phytoplankton biomass just before the bloom of C. furcoides started. The rapid bloom and dominance of C. furcoides can be explained by the colonization from the cyst bank in the sediments (Bustamante Gil et al. 2012;Hansson et al. 1994;Reynolds & Walby 1975), indicating that C. furcoides was already present in Lake Blanca before this study although not been registered in pelagic samples. The extreme wind events before the bloom, likely facilitated resuspension of the cysts from the sediments, as described elsewhere (Bustamante Gil et al. 2012;Matsumura-Tundisi et al. 2010). Other cyst-forming species, such as Peridinium spp. and C. hirundinella, also increased their biomass in this period but rapidly disappeared, likely due to competitive exclusion by C. furcoides (Moreira et al. 2015;Rengefors et al. 2004;Reynolds 2006). The high biomass of C. furcoides bloom led to high turbidity and a decline in the concentration of dissolved nutrients due to the rapid nutrient uptake and storage in the cells, coinciding with previous ndings (Matsumura-Tundisi et al. 2010;Bustamante Gil et al. 2012).
We found that the bloom of C. furcoides in Lake Blanca began just before summer with warm temperatures and lasted throughout the following year, continuing even during cold months, indicating that high temperatures may have triggered the bloom but low temperatures did not affect its persistence. This nding coincides with previous studies reporting that high temperatures are important during the initial stages to trigger the bloom but are not necessary to maintain the bloom (Meichtry de Zaburlín et al. 2016, Cavalcante et al. 2016. Once established, the bloom of C. furcoides may persist due to its large size, providing the species with a low grazing susceptibility (Pollingher 1988, Olrik 1994 which is typically even lower in subtropical freshwaters due to the dominance of small-sized zooplankton (Bustamante Gil et al. 2012;Cavalcante et al. 2016;Crossetti et al. 2019;Meerhoff et al. 2007;Morales 2016). In accordance, the zooplankton in Lake Blanca consisted mostly of small taxa, such as small cladocerans and rotifers (Bosmina, Keratella, and Filinia) as also reported in previous studies Pacheco et al. 2010) and consequently, they did not control C. furcoides by grazing (Bustamante Gil et al. 2012;Reynolds 2006).
The bloom of C. furcoides can cause different environmental impacts, and sh kills is one of the most drastic consequences. Although we did not evidence sh kills at Lake Blanca, where we recorded the process of emergence and bloom of C. furcoides, we observed the sh kill of P. lineatus in Lake Puente de las Americas, during the bloom of C. furcoides. We found that the gills of P. lineatus were affected both by clogging and histopathological lesions due to accumulations of C. furcoides. The affectation of the gills may have accentuated the sensitivity of P. lineatus to anoxia in the benthic zone promoted by the bloom of C. furcoides likely caused the mass mortality by asphyxia of P. lineatus. Furthermore, sh death by asphyxia is facilitated by clogged gills with excess mucus production and edema in secondary lamellae caused by Ceratium (Onoue 1990) and add to explain mass mortality of aquatic organisms due to anoxic conditions promoted by the genus Ceratium (Bazán et al. 2007;Mahoney & Steimle 1979;Nicholls et al. 1980;Onoue 1990).
The expansion of C. furcoides in the subtropical region of South America has accelerated in recent years (Meichtry de Zaburlín et al. 2016) and in Uruguay, besides the record in Lake Blanca and Lake Puente de las Americas reported here, C. furcoides was recently found in shallow lakes Lake Escondida in 2014 (34°82' S; 54°62' W), Lake Sauce in 2016 (34°82' S; 55°06' W), in the river Santa Lucia in 2015 (34°30' S; 56°23' W) (JP Pacheco unpubl. Data) and the reservoir San Francisco in 2015 (34°39' S; 55°21' W) (Meerhoff et al. 2017). The rapid spread of C. furcoides, facilitated by eutrophic conditions, may entail sh kill as reported for Lake Puente de las Americas in this study. The risk of sh kills is higher in deep strati ed lakes where the phytoplankton blooms may cause anoxia in the hypolimnion, particularly at night. Oxygen depletion is not common in shallow lakes exposed to the mixing by the wind and this could explain why we did not register sh kills in the shallow Lake Blanca.
Given the rapid expansion and impacts of C. furcoides in South American freshwater ecosystems, it is necessary to increase the surveillance capacity in phytoplankton biomonitoring programs, beyond the more widespread focus on cyanobacteria. This is particularly needed for genera holding invasive species as these may otherwise spread unnoticed. The spread of C. furcoides in Uruguay may have been a silent invasion due to di culties in distinguishing this species from C. hirundinella. Improving phytoplankton biomonitoring programs allows to act proactively and avoid environmental consequences of the blooms as the massive sh kill reported in this study.
Declarations Table   Table I Figure 1 Ceratium furcoides from Lake Blanca, Uruguay in 2012. Ventral views before (left) and after (right) being treated with NaClO 20% to separate the cell wall plates. Arrows indicate the 4' plate not reaching the apex of the apical horn.  Temporal dynamics of phytoplankton groups as biovolume, total chlorophyll-a (blue area) (upper panel) and maximum maintained wind speed (> 3 hours) (lower panel) in Lake Blanca for the period July 2010 -October 2013. Note that dino agellates (Dinophyceae) in the upper plot, correspond almost exclusively to C. furcoides. The blue horizontal line in the lower panel represents the average maximum wind speed and the dashed red line represents the 99% upper quartile of extreme wind events.

Figure 4
Temporal variation of dino agellates C. furcoides, C. hirundinella and Peridinium sp. biovolume before and during the bloom of C. furcoides in Lake Blanca. Note the biovolume data in the x-axis is expressed in Log of biomass (µm3.L-1) to represent together the extremely different values among species.

Figure 6
Ceratium furcoides during the bloom in Lake Blanca in 2012-2013. Top panel: normal appearance of C. furcoides (left) and one organism with the cyst formed inside the cell wall (black arrow). Lower panel: Adult organisms of C. furcoides from one of the tap water distribution tanks in El Chorro, Maldonado. All organisms presented deformities in their apical horns (a, b) or lack of one of their antiapicals (c, d).

Figure 7
Dissolved oxygen concentration and oxygen saturation in-depth in Puente de las Americas Lake, Uruguay from June to September 2016.  Gills of P. lineatus after the collapse of the bloom of C. furcoides in Puente de las Americas Lake, Uruguay, on September 22th, 2016. Upper panels: general view of the gills at 3 and 8 X. Lower panels: sediments and phytoplankton found in the gills at 40 X.