Expansion of invasive Ceratium furcoides ( Dinophyta ) toward north-central Brazil : new records in tropical environments

Aim: We record new occurrences of the invasive species Ceratium furcoides in reservoirs and their affluents in the Paraná River basin, State of Goiás (GO), central-western Brazil, and in some localities in the São Francisco River basin, northeastern region. Methods: Qualitative and quantitative phytoplankton samples were collected from Corumbá Reservoir and Cascatinha Falls, Caldas Novas, GO, and João Leite Reservoir, Goiânia, GO, both in the Paraná River basin, and samples from the São Franscisco River basin. Specimens of C. furcoides were observed with optical, epifluorescence and scanning electron microscopy. Results: The individuals of C. furcoides from these environments agreed morphologically with populations in other reservoirs in Brazil and other locations, especially concerning the tabulation and the shape of the fourth apical plate. These environments ranged from oligotrophic to eutrophic conditions. Physical and chemical variables of these waterbodies, compared to other environments where this species was found, demonstrate that C. furcoides is a highly eurytopic species. The dispersal pattern of C. furcoides seems to be more complex than upstream-downstream regulation, since the species occurs in high-altitude environments and systems upstream from previously recorded locations. Conclusion: An analysis based on the areas of occurrence and the chronology of the records demonstrated that C. furcoides has spread toward northern Brazil. Studies of the relationships among populations recorded in other parts of Brazil and South America are required in order to develop accurate models of dispersal for this invasive species, and will facilitate the development of management policies for aquatic systems in Brazil.


Introduction
An invasive species is defined as a "non-native species that, once established, can spread and rapidly dominate over native species" (Kernan, 2015).The establishment of an invasive species can be directly or indirectly associated with an anthropogenic event, and its dominance can generate negative effects on the biotic community and environment (Alpert et al., 2000;Kernan, 2015).These effects may result in profound ecological, evolutionary and economic impacts (Epanchin-Niell & Wilen, 2012;Kernan, 2015).Consequently, means of controlling biological invasions are important for ecosystem health.Rapid detection of invasive species and mapping areas with a potential risk of invasions are some of the main strategies for controlling invasions (Lennox et al., 2015;Mazzamuto et al., 2016).
Expansion of invasive Ceratium furcoides (Dinophyta)… Species of Ceratium were not observed in Brazilian freshwater systems before this century (Cavalcante et al., 2013).In 2007, S a n t o s -W i s n i e w s k i e t a l .( 2 0 0 7 ) a n d Silva et al. (2012) 2017) observed this species in aquatic systems from Paraná, and in 2012 this species was observed in several waterbodies in the states of Paraná and Rio Grande do Sul (Cavalcante et al., 2013(Cavalcante et al., , 2016;;Cassol et al., 2014Cassol et al., , 2017;;Jati et al., 2014).In 2013, Bressane et al. (2013) and Hackbart et al. (2015) recorded it in Barranco Alto, Minas Gerais, and in Jaguari and Jacareí reservoirs, São Paulo, respectively.In 2014, Oliveira et al. (2016)  The establishment and rapid development of invasive species such as C. furcoides in aquatic systems can affect the native communities and the water quality, producing various colors and unpleasant tastes and odors (Berthon, 2015;Meichtry de Zaburlín et al., 2016;Napiórkowska-Krzebietke et al., 2017).Species of this genus have been associated to fish-kill in temperate lakes because the oxygen depletion caused by blooms (Nicholls et al., 1980).Therefore, records of the occurrence of C. furcoides are important to provide information about its distribution, dispersion patterns, and contributing to the management of Brazilian aquatic systems.
We here record the occurrence of C. furcoides in reservoirs in the Paraná River basin and its sources in Goiás, central-western Brazil, and in some localities of the São Francisco River basin, northeastern Brazil.

Material and Methods
Cascatinha Falls is located in the eastern area of the Serra de Caldas State Park (PESCaN), a preserved area near Caldas Novas, Goiás, at 750 m a.s.l.Its waters flow along Saia Velha Stream, Caldas Stream and Pirapitinga River until they enter the north end of the Corumbá Reservoir (17°44'53.23"S;48°33'38.77"W)(Ramos & Carneiro, 2010).Corumbá Reservoir is located 30 km from Caldas Novas, at 668 m a.s.l.The reservoir has an area of 65 km 2 and a total volume of 1.5 km 3 , and was formed by damming the Corumbá River in the Paraná Hydrographic Region (Furnas, 2015).
The São Francisco River is the main aquatic system of the São Francisco Hydrographic Region.It is the longest Brazilian river, reaching 2,914 km from its sourse in the Canastra Mountain Range, State of Minas Gerais, to its mouth in the Atlantic Ocean on the border of the states of Alagoas and Sergipe.Along its course, the river is dammed five times, forming the reservoirs Três Marias, Sobradinho, Luiz Gonzaga, Paulo Afonso and Xingó (Brasil, 2006).
Qualitative samples were taken at Cascatinha Falls, Corumbá Reservoir, and at ten points in the São Francisco River, using a plankton net of 25 µm mesh (Table 1).The samples were fixed with Transeau's solution (Bicudo & Menezes, 2006) and deposited in the Herbarium of the Federal University of Goiás and the Herbarium of the State University of Londrina, respectively (Table 1).Quantitative samples were composed of 100 mL of water collected from the subsurface at each site described in Table 1, except Cascatinha Falls (GO-Casc) and the margin of the Corumbá Reservoir (GO-Cor-MARG).These samples were da Silva, W.J. et al.
For scanning electron microscopy (SEM) analyses, small probes of each material were fixed with glutaraldehyde for 1 h and dehydrated in an acetyl alcohol series series of 20, 40, 60, 80 and 100%, for 150 min.Part of the dehydrated material was deposited under cover slips and dried in desiccators using silica.The cover slips were fixed on aluminum stubs, using carbon-conductive ink.The material was sputtered with a layer of gold in a Desk V sputter coater (Denton Vacuum, LLC).The preparations were analyzed in a Jeol JSM-6610 scanning electron microscope (Jeol, USA), with an electrical potential of 6 kV, spotsize 25-40.
In the Corumbá and João Leite river basins, physical and chemical variables including water temperature, pH, oxidation-reduction potential (ORP), electrical conductivity, turbidity, dissolved oxygen (DO), and total dissolved solids (TDS) were obtained using a Horiba U21 multiparameter water quality meter.In the João Leite River basin, concentrations of nitrate and total phosphorus (TP) were measured according to APHA (2005).The data for these variables from the São Francisco River Basin were obtained from Technical Report DILAB 003/2015 (http://cbhsaofrancisco.org.br/?wpfb_dl=1980).
The physical and chemical characteristics of the environments in which C. furcoides was recorded were variable (Table 3).

Discussion
Two species of Ceratium have been recorded in Brazilian aquatic systems: C. furcoides and C. hirundinella (Cavalcante et al., 2013).The main differences in these species are the shape and size of the plate 4", which does not reach the apex in C. furcoides (Matsumura-Tundisi et al., 2010).This characteristic was observed in specimens found in the São Francisco River, João Leite and Corumbá Reservoirs, confirming the identity of this taxon as C. furcoides (Figures 4,6,9,10).
There is no consensus about the environmental conditions that contribute to the establishment of C. furcoides.Species of Ceratium have been observed in deep and shallow environments, oligo-to eutrophic, and medium to large sizes (Padisák et al., 2009).More specifically, C. furcoides has been positively related to ammonium concentrations and negatively related to alkalinity, nitrate concentration,   (Nishimura et al., 2015), or in mesotrophic environments, with warmer temperatures and higher electrical conductivity (Silva et al., 2012).Cavalcante et al. (2016) found that high organic-matter content, nutrient availability, well-oxygenated water and temperatures from 15 to 25 °C are the main environmental factors that contribute to the establishment of C. furcoides in reservoirs in southern Brazil.In this study, the João Leite, Corumbá and São Francisco systems were acid to slightly alkaline, and the water temperatures were above 25 °C.Moreover, it is not clear if well-oxygenated water is a favorable condition for the development of this species or is a result of its development, since oxygen is a product of photosynthesis Thus, this species has occurred in environments with different DO concentrations; da Silva, W.J. et al.
Acta Limnologica Brasiliensia, 2018, vol.30, e210 e.g., in João Leite DO concentrations were low, in contrast to the Corumbá and São Francisco systems.Organic matter can also be influenced by the high biovolumes of this species.Despite the values of nutrient concentrations are sparse in the environments studied here, these resources seem facilitate the establishment and development of the species.Crossetti et al. (2018) observed that the recurrence of C. furcoides in Garças reservoir was associated with higher transparency, lower water stability, higher dissolved inorganic nitrogen and lower TP values.C. furcoides is hitherto known in Brazil in aquatic environments from the Paraná River, São Francisco River, Atlantic (East/South regions), and Uruguay River basins.No records have been reported from the Amazon River, Tocantins-Araguaia River or Atlantic (North/Northeast) basins, nor in some other parts of the world (Figures 11 and 12).The establishment of C. furcoides in Brazilian aquatic systems is a recent event (Santos-Wisniewski et al., 2007;Cavalcante et al., 2013).Based on the dates of occurrence, Cavalcante et al. (2013) hypothesized that the dispersal of C. furcoides seems to be radial, i.e., northward and southward from the initial appearance in the southeastern region.However, the relationships among the populations in the different Brazilian aquatic systems have not been studied.
In several cases, C. furcoides was observed in very low densities and did not appear in quantitative samples, such as at Cascatinha Falls.This suggests that the species may be present in some environments but not in sufficient numbers to be recorded.The failure to find it in some samples may also indicate methodological problems during sampling, since species of Ceratium can migrate vertically.Almost all samples taken in Brazilian waterbodies are from the subsurface.However, in some cases, high densities of species of Ceratium can be found down to 7 m, where light conditions are more suitable or due to stratification (Harris et al., 1979).In some aquatic systems in Brazil where C. furcoides was found, no temporal records exist, which makes the time of its arrival uncertain.These problems add to the uncertainty regarding the actual localities where this species occurs and its distribution patterns.
For some environments in Brazil, it is possible to infer an approximate date of the invasion because of long-term historical floristic studies, such as in Billings Reservoir (Matsumura-Tundisi et al., 2010; Nishimura et al., 2015).Because the João  showing the apical (2'), precingular (1" and 5"), postcingular (4"' and 3"') and antapical (2"") plates.Figure 9: Cell in ventral view showing the apical (4 '), precingular (1", 5" and 6"), postcingular (1"' and 5"') and antapical (1"") plates; note the fourth apical plate (4') not reaching the apex.Leite River basin is the water source for the metropolitan region of Goiânia, the biotic and abiotic conditions of this system have been long exhaustively monitored, mainly after the creation of the reservoir in 2009 (Cunha & Borges, 2015).Thus, C. furcoides certainly did not occur in this environment before March 2016.Historical studies of the phytoplankton composition in Corumbá Reservoir are scarce, but the existing studies support the hypothesis that C. furcoides did not occur there before 2011 (Silva et al., 2001;I. S. Nogueira, pers. observ.).
While the answer for "when" C. furcoides arrives in Brazilian waterbodies seems to be easier to propose, "how" it occur and is occurring is still not possible to verify.Some researchers suggest by boats or migratory birds as some of the main dispersive ways for phytoplanktonic species (Padisák et al., 2016).From that, the establishment of this species in Brazilian waters and its spread can be associated to adequate environmental conditions and drivers upstream-downstream or downstream-upstream.Until 2011, C. furcoides was observed in only two hydrographic regions, Paraná and São Francisco.The existence of cascades of reservoirs in large rivers is presumed to contribute considerably to its spread (Padisák et al., 2016).This seems to be a reaseonable explanation for invasions at the upstream-downstream direction.Corumbá and João Leite reservoirs are in the Paraná Hydrographic Region, upstream of the systems where C. furcoides was previously recorded.
This sugest the progression of the invasion of this species in aquatic systems toward the central-northern region.
The continuous progression of this species, reaching aquatic systems in the state of Goiás, should be considered and studied.This state contains a watershed divide between tributaries of three of the main river basins of the country.Although these river basins are mostly separated by high-altitude formations, these seem not to limit the movement of C. furcoides, since the species occurs in high-altitude systems (Moreira et al., 2015).This suggests that the distribution pattern is more complex than can be explained by the connectivity of aquatic systems.However, this factor may facilate its spread, as in the case of reservoir cascades in large rivers, which are presumed to contribute considerably to the establishment of C. furcoides (Padisák et al., 2016).
The harmful of C. furcoides is few known.Despite this species is not toxic, it reach great density during some phases because the low preference by grazers and (Santer, 1996) and good enviromental conditions (Crossetti et al., 2018).Similar blooms have contributed to high depletion of oxygen in water column and consequently fish-kill in some lakes and problems in water treatment (Nicholls et al., 1980).Understanding the occurrences and distribution patterns of C. furcoides is essential to the development of control policies, which should be used to guarantee the future water quality of Brazilian aquatic systems for different uses in a period when drinking water is becoming scarce.Promptly detecting the occurrence of an invasive species such as C. furcoides is essential to understand the ecological factors that can facilitate the establishment of this species and to develop models to prevent its spread (Lennox et al., 2015;Mazzamuto et al., 2016).Future studies should analyze the relationships among populations of C. furcoides in Brazil, and make efforts to discover the mechanisms related to the expansion success of this species.

Figure 12 .
Figure 12.Occurrence of Ceratium furcoides in the world.
recorded C. furcoides in the Rio Grande basin, State of Minas Gerais, in March and December, respectively.In 2008, C. furcoides was also found in Billings Reservoir, State of São Paulo (Matsumura-Tundisi et al., 2010).Oliveira et al. (2011) recorded C. furcoides in reservoirs in the states of Alagoas, Bahia, Pernambuco and Sergipe in April 2009, and Nishimura et al. (2015) found this species in Billings Reservoir and Guarapiranga Reservoir, São Paulo, in September 2009.In 2010, Moreira et al. (2015) observed C. furcoides in a temporary lagoon at high altitude in Minas Gerais.In 2011, Rosini et al. (2016) recorded C. furcoides in Ilha Solteira Reservoir, São Paulo and Cavalcante et al. (

Table 1 .
Sites of occurrence of Ceratium furcoides and sample codes.

Table 2 .
Density and biovolume of Ceratium furcoides in Corumbá Reservoir and São Francisco River (for sites codes, see Table1).

Table 3 .
Mean values of physical and chemical variables analyzed in reservoirs and rivers in the Corumbá River basin, Goiás, and the São Francisco River basin, Alagoas (ORP: oxidation-reduction potential; DO: dissolved oxygen; TDS: total dissolved solids).