Species composition of testate amoebae in Lake Monte Alegre , Ribeirão Preto , SP , Brazil

We aimed to perform the first survey of testate amoebae community composition in Lake Monte Alegre at São Paulo state, and contribute to mitigate the taxonomic impediment regarding the diversity of testate amoebae in Brazil. Samplings were performed in 20 sampling sites within the lake, in the limnetic and littoral regions in April 2015, using a 58 μm-meshplankton net. We identified 20 taxa of testate amoebae belonging to four families: Arcellidae (8 taxa), Centropyxidae (4 taxa), Difflugiidae (7 taxa) and Lesquereusiidae (2 taxa). The littoral region showed the highest number of taxa (n = 20), whereas in the limnetic region 11 taxa were registered. Therefore, our results evidenced the importance of testate amoebae in aquatic systems, and that further research, taxonomic and/or ecological, in those environments should include these organisms in their investigation. Moreover, we suggest that future research with greater sampling effort is necessary to expand the identification of possible cryptic species in this environment.

In aquatic food webs, testate amoebae are important because they occupy different trophic functions, which can range from decomposers to consumers at different trophic levels, and are also considered key predators in microbial food webs (Jassey, Chiapusto, Binet, Buttler, & Laggoun-Defarge, 2013).Moreover, testate amoebae are sensitive to environmental changes, and for this reason, may be considered environmental bioindicators (Schwind, Arrieira, Simões, Bonecker, & Lansac-Tôha, 2017).Therefore, the accurate taxonomic identification of those organisms may provide information on its biology, and consequently, help understand ecological processes and define their ecological functions in aquatic ecosystems (Arrieira et al., 2016a).
The Testate amoebae have a large species richness in South America.However, when considering all regions of Brazil, the amount of research is still very limited, and the few existing studies are restricted to certain regions (Lansac-Tôha, Zimmermann-Callegari, Alves, Velho, & Fulone, 2007).According to Lansac-Tôha et al. (2008), the spatial gaps of those studies hinder the understanding of the real species richness of testate amoebae in Brazilian environments.Lake Monte Alegre has been studied since 1985, and so far, the composition of planktonic and benthic communities, besides the horizontal distribution of planktonic microcrustaceans and its predators were evaluated (Arcifa, Gomes, & Meschiatti, 1992;Arcifa, Bunioto, Peticarrari, & Minto, 2013).However, no study approaching the testate amoebae community has been performed in this study area.Therefore, we aimed to describe and illustrated the testate amoebae from a lake of São Paulo State and to contribute to the knowledge about the occurrence and distribution of these protists in Brazil

Study area
Lake Monte Alegre was created in 1942 by damming the Laureano stream, which belongs to the Pardo River basin (Figure 1).The study area is located in the campus of Universidade de São Paulo (USP), Ribeirão Preto city, São Paulo state, Brazil (21 o 10' S, 47 o 51' W).This aquatic ecosystem is eutrophic and similar to an artificial lake, with and area of 7 hectares, maximum depth of 5 m and mean depth of 2.9 m (Silva, 1999).The lake shore is dominated by grass, aquatic macrophytes and sugar cane plantation due to agricultural activities.The region has a tropical climate with two well-defined seasons, cold-dry and warm-rainy, and temperature varying from 18 to 31ºC in the surface of the lake (Rangel, Silva, Arcifa, & Perticarrari, 2009).

Sampling design
Testate amoebae samplings were performed in 21 sampling sites in Lake Monte Alegre, on the subsurface of the limnetic region (8 sampling sites) and in the littoral region (13 sampling sites).Samples were taken in a single sampling period, in April 2015, by filtering 150 L of water per sample through a 58 μm-mesh plankton net, using a suction pump.Samples were fixed with 4% calcium carbonate-buffered formaldehyde solution.For each sample, identification was performed by analyzing subsamplings in Sedgewick-Rafter chamber under optic microscope.Specimens were sorted and stored in slides containing glycerol (90%) for further manipulation and visualization of taxonomic characters.
Species richness was estimated by analyzing chambers subsequently until no new individuals were found.Afterwards, three more chambers were analyzed to confirm the stabilization of species richness.The taxonomic classification was based on Adl et al. (2012) proposed guidelines.Specimens were photographed using a digital camera (Samsung Galaxy Camera) coupled to an optic microscope (Nikon Eclipse E100), in lateral and ventral views showing the morphological variation of structures relevant to the identification of the organisms (Figure 2).Measurements of shell width and height and pseudostome diameter were performed using software Axio Vision through images obtained using an optic microscope (Olympus BX41) coupled to an image capture device.

Discussion
Families Arcellidae and Difflugiidae showed the greatest number of taxa.The same pattern was also observed in several studies performed in the floodplain lakes of Pantanal, Araguaia, Amazon and Paraná, and also in São Paulo state (Lansac-Tôha et al., 2008;2009;Lima, Loverde-Oliveira, Silva, & Oliveira, 2012;Arrieira et al., 2016b).The results observed for species richness were superior to that of other microbial taxa traditionally studied in this lake, such as the rotifer community (14 taxa) (Soares, Tundisi, & Matsumura-Tundisi, 2011), cladocerans and copepods, showing low representativeness with only three and one taxa, respectively (Meschiatti & Arcifa, 2002).Similar results were observed in a study performed in Bonita Lake (SP), in which testate amoebae species richness was higher than that observed for other planktonic groups (Alarcão et al., 2014).
In a survey of testate amoebae species in São Paulo state, a total of 67 taxa were registered (Regali-Seleghim, Godinho, & Tundisi, 2010).According to this study, our results showed 33% of the total taxa registered in that state (20 taxa), in addition to a new record, Arcella vulgaris var.penardi.A. vulgaris is a commonly found taxonin samples from lentic environments (Lansac-Tôha et al., 2007;Maia-Barbosa et al., 2014;Silva et al., 2016).Regali-Seleghim et al. (2010) registered the occurrence of this taxon in 15 lakes located in São Paulo state.A. vulgaris var.penardi shell is angled in lateral view (Figure 3, 6a-6b; Figure 4, 6a-6b), a remarkable characteristic for its identification (Velho et al., 1996).Therefore, we verified that the observation of morphological characteristics is fundamental for the correct identification of testate amoebae species, thus demonstrating the importance of taxonomic studies to obtain data regarding the biogeography and biological diversity of those organisms.
The species richness found in this study can be considered relevant if compared to other studies performed in aquatic environments of São Paulo region, in which the sampling effort was greater than that of the present study, such as the study performed by Fulone, Vieira, Velho, and Lima (2008), in which 21 taxa of testate amoebae were registered in two streams, sampled monthly for a year.Moreover, Lahr and Lopes (2009) found only 13 taxa in samplings performed at the Tiete River Ecological Park.
Regarding the lake compartments, in the littoral region we found all taxa registered in the study, whereas in the limnetic region 11 taxa were registered.The limnetic region show limitations to the permanence of certain testate amoebae taxa, for example the higher flow velocity, in which only adapted taxa can remain at the plankton for a long period of time, such as those presenting gas vacuoles (Velho, Lansac-Tôha, & Bini, 2003;Arrieira et al., 2016b).The littoral region is usually structured by grass and aquatic macrophytes, providing a higher complexity and environmental heterogeneity to this biotope, increasing the number of ecological niches available (Landa & Mourgués-Schurter, 2000), and consequently favouring a higher species richness of testate amoebae.Moreover, this region may provide a higher abundance of food resources for testate amoebae, due to higher nutrient concentrations of allochthonous origin and elevated primary productivity (Arrieira et al., 2015).

Conclusion
Our results evidenced the importance of taxonomic studies of testate amoebae in aquatic ecosystems.This study presents the first taxonomic survey for Lake Monte Alegre and showed an elevated species richness of those organisms compared to taxa of other groups traditionally studied in this environment.Therefore, we emphasize that testate amoebae may have an important role in aquatic ecosystem functioning and that further studies should consider these organisms in their investigation.
We believe that testate amoebae diversity in this environment may be even higher than that registered at the moment, considering that this study was performed in a single sampling period.Thus, we suggest that additional studies should be performed with greater sampling effort, considering the temporal and spatial aspects, to expand the identification of possible cryptic species in the environment.

Figure 1 .
Figure 1.Map of the study area: Lake Monte Alegre, Brazil.Source: Author.
, Figure a-b.Diagnosis: Shell subspherical with very short neck and a furrow marking the shell, composed of siliceous rods.Dimensions (n = 7): shell, mean width = 99.48μm; mean height = 125.12μm.

Table 1 .
List of testate amoebae taxa registered in Lake Monte Alegre.