Surface sediment and phytoplankton diatoms across a trophic gradient in tropical reservoirs : new records for Brazil and São Paulo State

(Surface sediment and phytoplankton diatoms across a trophic gradient in tropical reservoirs: new records for Brazil and São Paulo State). This,study aimed to inventory the diatom fl ora of tropical reservoirs from southeastern region of Brazil under oligoto hypereutrophic conditions. We collected diatom samples from the surface sediment and phytoplankton (summer and winter) in 41 sites (ten reservoirs). Seventy-eight taxa, distributed in 28 genera, were identifi ed in the surface sediment and phytoplankton samples. Nizschia was the most representative genus, with nine species. Six taxa represent new records for Brazil and six for São Paulo State; ten taxa were identifi ed at the genus level and probably represent new species. Additionally, this study contributed with data on the ecology of the species and evidenced the necessity of fl oristic surveys to improve the knowledge about tropical diatom biodiversity.


Introduction
Evidences show that recent species extinctions rates are exceptionally high, suggesting a probably new event of mass extinction due the environmental crisis caused by mankind (Ceballos et al. 2015).Despite the efforts on taxonomic and ecological studies, many species are extinct even before they can be described.The biota of freshwater ecosystems has been highly neglected in studies about biodiversity, especially invertebrates and microorganisms at the tropical region (Dudgeon et al. 2006).Freshwater ecosystems support a high diversity despite their reduced superfi cial area, therefore being considered hot spots for biodiversity (Strayer & Dudgeon 2010).
Diatoms are among the most species-rich group of algae and the number of species is around 100,000, including fossil species (Mann & Vanormelingen 2013).They are an abundant component of primary producers in plankton and benthos, both in marine and freshwaters (Round et al. 1990).Due to their high richness and abundance in aquatic ecosystems, diatoms represent an important account to local and regional biodiversity, which can be accessed by fl oristic surveys.Furthermore, ecological preferences of many species are relatively well know, making these group widely used to evaluate environmental conditions (e.g., Passy 2007, Bennion et al. 2014, Blanco et al. 2014).However, just about 12% of the estimated diatom fl ora is currently described (Julius & Theriot 2010).
In recent years, the number of studies about the diatom fl ora in Brazil has been increasing (e.g., Souza & Senna 2009, Bertolli et al. 2010, Silva et al. 2010, Bartozek et al. 2013).However the inclusion of surface sediment diatoms is still scarce (Fontana & Bicudo 2009, 2012, Almeida & Bicudo 2014, Faustino et al. 2016), and all of them were carried out in the state of São Paulo.Such studies have added several new records to the Brazilian diatom fl ora and new species to Science (Almeida et al. 2015, Almeida et al. 2016, Marquardt et al. 2016), demonstrating the importance of fl oristic survey of sediments.Other studies about the diatom fl ora encompassing lotic and lentic environments have been carried out in the state of São Paulo (Carneiro & Bicudo 2007, Bere & Tundisi 2010, Marquardt & Bicudo 2014, Ferreira & Bicudo 2017).The present study was carried out in ten reservoirs from three watersheds of São Paulo State (Brazil).To our knowledge, this is a pioneer diatom fl oristic survey to the study area, except for the unpublished study carried out by Silva (2017).We aimed at improving the knowledge of the diatom fl ora from surface sediments and phytoplankton, highlighting the new records for Brazil and São Paulo State.Furthermore, this study contributes to the increase of knowledge about biodiversity and ecology of tropical diatoms.

Material and methods
This study was carried out in the southeastern region of Brazil and comprises three drainage basins in the State of São Paulo (fi gure 1, table 1).We selected 10 reservoirs located in protected and highly urbanized areas, ranging from oligo-to hypereutrophic conditions and with different uses (recreational, power generation, navigation and public water supply).The reservoirs range from shallow to deep (maximum depth from 2 to 33 m) and from small to large (surface area from 0.2 to 241.3 km²).We selected three to six sampling sites (table 1) per reservoir depending on size, the main water inputs (main streams) and deepest region of the reservoirs.
We sampled a total of forty-one sites (table 1) during two climatic periods (austral winter and summer) in 2013 and 2014.Water column samples were taken with a van Dorn bottle along the reservoir vertical profi le (subsurface, mean depth and 1 m above the sediments), and mean results of water column were used to characterize each sampling site.Conductivity, pH and water temperature were measured in fi eld using standard electrodes (Horiba U-50).The analytical methods for dissolved oxygen, dissolved inorganic nitrogen, total nitrogen, orthophosphate, total phosphorus and soluble reactive silica followed Standard Methods (APHA 2005).The reservoirs Trophic State Index (TSI) was calculated according to Lamparelli (2004) based on values of chlorophyll-a (Sartory & Grobbelaar 1984) and total phosphorus.Integrated water column samples were used to describe the phytoplankton diatom community.Surface sediment diatoms (top 2 cm) were sampled only in winter (41 samples) using a UWITEC gravity  corer.Each sampling site was sampled in triplicate to compound the site spatial heterogeneity.For diatom analyses, samples were digested using hydrogen peroxide (H 2 O 2 35%) and hydrochloric acid (HCl 37%) according to Battarbee et al. (2001).Permanent slides were prepared using Naphrax® as inclusion medium.Optical observations, measurements and micrographs were taken at a magnification of 1000× with a Zeiss Axioskop 2 plus microscope equipped with phase contrast and Axiocam ERc5s high-resolution digital camera.Taxonomy and nomenclature followed specific publications (e.g., Lange-Bertalot 1993, Metzeltin et al. 1998, Lange-Bertalot et al. 2011) and the on-line catalogue of valid names (site of California Academy of Sciences 2012).The classification systems followed Medlin & Kaczmarska (2004) for supra-ordinal taxa and Round et al. (1990) for subordinal taxa, except for genera published after to this work.
Diatoms were quantified at a magnification of 1000× and until reaching a minimum of 400 valves per slide (Battarbee et al. 2001), and at least 90% in counting efficiency (Pappas & Stoermer 1996).We included those taxa with relative abundance ≥ 2% in at least one sampling site and indicated the new records for Brazil and São Paulo state after consulting the published literature (books and articles).Descriptions, relevant taxonomical and ecological comments were provided for taxa identified in genus level.Morphometric information is provided for the new records for Brazil and São Paulo State and for genus level taxa (L: length; W: width; S: striae; A: areolae; F: fibulae).Samples were deposited at the "Herbário Científico do Estado Maria Eneyda P. Kauffmann Fidalgo" (SP), Brazil (SP469229 to SP467313, SP469453, SP469454 and SP456488 to SP469523).

Results and Discussion
The main environmental features of the study area are available in table 2. A total of seventy-eight taxa presented relative abundance ≥2%, including two non-typical varieties, distributed in 28 genera (table 3).Surface sediment and phytoplankton contributed, respectively, with 60 and 70 taxa.Nitzschia Hassal was the genera with higher number of taxa (nine species) followed by Achnanthidium Kützing (eight species), Aulacoseira Thwaites (eight species) and Fragilaria Lyngbye (eight species).Aulacoseira ambigua and A. granulata var.angustissima were the most spread species in the surface sediment, occurring in 100% of samples and with the highest abundance in eutrophic conditions (63.6% and 63.7%, respectively).The most frequent species in the phytoplankton were Aulacoseira ambigua and Discostella stelligera, both with an occurrence of 90.2% in samples and higher abundances in oligo-and mesotrophic and meso-eutrotrophic conditions, respectively (63.6% and 92.0%).All inventoried taxa and their relative abundances according to the trophic state are presented in table 3  Short and narrow specimens of F. grunowii may be mistaken with large specimens of Fragilaria tenuissima Lange-Bertalot & Ulrich.However, the specimens found in this study are wider than the type population of F. tenuissima (W: 1.6-2.8µm; Lange-Bertalot & Ulrich 2014).Specimens shorter than F. grunowii type population (L: 100.0-380.0µm) were presently found, increasing the length range for this species.Silva (2017) recorded this species in surface sediment and planktonic samples in six of the reservoirs included in this study.This author estimated that this species has preference for slightly acid (pH: 6.8) and low nutrient content waters (total nitrogen: 417.7 µg L -1 and total phosphorus: 15.0 µg L -1 ).In this study, considering surface sediment samples, F. grunowii was found in one mesotrophic reservoir (Itupararanga, maximum abundance: 2.7%), and, considering planktonic samples, it was found in three reservoirs ranging from oligo-to eutrophic conditions (Santa Helena, Itupararanga and Hedberg, maximum abundance: 2.9%), but with higher frequency of occurrence in mesotrophic waters (37.1% of samples).This is the second register of the species in Brazil and the first published taxonomical register of this species.F. tenera var.nanana presents lanceolate valves, inflated and capitated ends and shorter measures (L: 50.0-70.0µm; W: 2.0 µm), while F. tenera var.tenera presents valves (sub)linear, mainly in the proximal region, slightly inflated ends, larger length (L: 60.0-120.0µm) and shorter width (W: 1.8-2.5 µm).F. tenera var.lemanensis is characterized by longer (L: 97.0-102.0µm) and narrower valves (W: 1.5-1.7 µm).This taxon was registered by Silva (2017) as Fragilaria tenera (Smith) Lange-Bertalot in the surface sediment and phytoplankton in seven of the studied reservoirs, with preferences for slightly acid to neutral waters (pH: 6.6-7.5) and low to moderate nutrients content (total nitrogen: 284.0-957.6 µg L -1 and total phosphorus: 0.1-52.7 µg L -1 ).In this study, it was observed in mesotrophic conditions in the surface sediment of one reservoir (Ipaneminha, maximum abundance: 3.9%) and in phytoplankton of three meso-and hypereutrophic reservoirs (Itupararanga, Ipaneminha and Barra Bonita, maximum abundance: 30.4%), but with higher frequency in mesotrophic conditions (25.7%), corroborating previous information.This is the second register of the species in Brazil and the first published taxonomical register of this species.
This taxon resembles F. parva (Grunow) Tuji & Williams, which however presents more pronounced swellings and capitate ends (Tuji & Williams 2008) than Fragilaria sp.This species was recorded in the surface sediment of one mesotrophic reservoir (Tatu, maximum abundance: 5.0%), and in phytoplankton of four reservoirs ranging from oligo-to mesotrophic conditions (Atibainha, Paiva Castro, Cachoeira and Tatu, maximum abundance: 18.3%).The highest frequency in samples was in oligotrophic conditions (22.0%).
This taxon was identified as belonging to the genus Pseudostaurosira due to the uniseriate striae and wide axial area.Other features, such as presence of spines and absent or reduced apical pore fields are only visible in SEM (Morales 2001).Pseustaurosira sp.resembles Fragilaria canariensis Lange-Bertalot due to their similar valve shape.Neverthless, the second species is shorter (L: 4.0-7.0µm), wider (W: 3.0-4.0µm) and exhibits smaller striae density (S: 12-15 in 10 µm) than Pseustaurosira sp (Lange-Bertalot 1993).It can be also misidentified as Staurosirella sp.(see taxon bellow), however, Pseudostaurosira sp.presents shorter striae and wider axial area.This is probably a new diatom species for science, since its general features differs from others species available in the literature.The species was documented in the surface sediment of four reservoirs with trophic state ranging from oligo-to eutrophic (Santa Helena, Itupararanga, Ipaneminha, and Hedberg, maximum abundance: 3.0%), and in the phytoplankton of four reservoirs ranging from oligo-to eutrophic conditions (Santa Helena, Itupararanga, Ipaneminha and Hedberg, maximum abundance: 3.8%).The highest frequency occurred in mesotrophic conditions (37.1%).
The species resembles Rimoneis inanis Garcia in relation to the valve shape.However, R. inanis presents larger valve length (L: 17.0-25.0µm, Garcia 2010).This author points out that R. inanis presented restricted distribution to freshwater/brackish water of the Lagoa dos Patos lagoon.Rimoneis sp. is described as a new species in the revision carried out by (Silva 2017) and mainly associated to low nutrient content environments.In this study, it occurred in the surface sediment of five reservoirs ranging from oligo-to mesotrophic conditions (Santa Helena, Atibainha, Cachoeira, Paiva Castro and Itupararanga, maximum abundance: 7.0%) and in the phytoplankton of mesotrophic reservoir Itupararanga (maximum abundance: 1.4%).Nevertheless, highest frequency in samples was observed in mesotrophic conditions (20.0%).This taxon resembles Staurosira altiplanensis Lange-Bertalot & Rumrich due to the overlapping measures (L: 4.5 µm; W: 2.8-3.6 µm) and similar valve outline.However, S. altiplanensis presents valves strictly elliptical and was described for the high altitude of the Altiplano of South America (Lange-Bertalot 2000).Furthermore, Staurosirella sp. was identified as Staurosirella pinnata (Ehrenberg) Williams & Round by Ribeiro et al. (2008) and Nardelli et al. (2014).However, Morales et al. (2013) demonstrated that S. pinnata type material, originally described as Fragilaria pinnata Ehrenberg, represents a species of the genus Denticula Kützing.Therefore, for these authors, a prior careful taxonomical and ecological analysis of this species is necessary.It was presently found in the surface sediment of four reservoirs with trophic states from oligo-to eutrophic conditions (Santa Helena, Itupararanga, Ipaneminha and Hedberg, maximum abundance: 4.0%), and in the phytoplankton of three reservoirs ranging from oligoto eutrophic conditions (Santa Helena, Itupararanga and Hedberg, maximum abundance: 2.3%), and with higher frequency in mesotrophic waters (34.3%).
C. affinis var.neoprocera is the synonymous of Cymbella excisa var.procera Krammer.This taxon can be compared to Cymbella tropica Krammer, however, our species exhibits more slightly protracted ends than C. tropica (Silva et al. 2015).This taxon was recorded in the Paraná state by Ludwig et al. (2005) as Cymbella affinis Kützing.According to Krammer (2002), this taxon is usually abundant in eutrophic waters.In this study, it occurred in oligo-to eutrophic conditions in three reservoirs (Santa Helena, Ipaneminha and Hedberg) for both surface sediment and phytoplankton (maximum abundance: 7.0% and 0.5%, respectively), with higher frequency in eutrophic waters (10.5%), corroborating previous finding.Valve strongly dorsiventral, semi-lanceolate, with dorsal margin strongly convex and ventral margin straight to slightly concave and expanded in the middle portion; ends ventrally bent; axial area straight and linear; raphe straight, external ends deflected to the ventral side and proximal ends deflected to the dorsal side.L: 11.0-22.2µm; W: 4.0-5.8µm; S: 10-13 in 10 µm.
This taxon resembles E. simile Krammer in valve shape and measures (L: 16.0-25.5µm; W: 5.1-6.0 µm; S: 10-13 in 10 µm; Krammer 1997), however Encyonema sp.presents thinner and slightly more capitate ends.Further analyses (e.g., MEV) are required to confirm if this taxon is a new species.In this study, it was found in the surface sediment of three oligotrophic reservoirs (Atibainha, Cachoeira and Paiva Castro, maximum abundance: 2.2%), and in the phytoplankton of three reservoirs ranging from oligo-to mesotrophic conditions (Cachoeira, Paiva Castro and Tatu, maximum abundance: 4.8), and presented higher frequency of occurrence in oligotrophic conditions (26.0%).Krammer (1997) this species is found in springs and falls.In this study, it was recorded exclusively in the phytoplankton of two oligo-and mesotrophic reservoirs (Santa Helena and Itupararanga, maximum abundance: 3.6%), with higher frequency in mesotrophic conditions (5.7%).Gomphonema naviculoides belongs to Gomphonema gracile complex sensu Grunow et sensu auct, and according to Reichardt (2015) both species can be easily mistaken.However, this author highlights that G. naviculoides presents valves lanceolate to rhombiclanceolate and almost naviculoid-symmetrical, while G. gracile presents valves lanceolate, gomphonemoidclavate shaped with bluntly rounded poles.In this study, the species occurred in the surface sediment of two oligo-and mesotrophic reservoirs (Paiva Castro and Tatu, maximum abundance: 3.0%), and in the phytoplankton of three oligo-and mesotrophic reservoirs (Cachoeira, Paiva Castro and Tatu, maximum abundance: 2.3%), and with higher frequency in mesotrophic conditions (14.3%).L: 24.2-33.8µm; W: 9.7-11.2µm; S: 10-14 in 10 µm.This taxon resembles Placoneis constans (Husted) Cox var.symmetrica (Hustedt) Kobayasi due to their lanceolate valves and rostrate ends.However, the second species is smaller (L: 19.3-25.6 µm, W: 8.1-9.3 µm) than P. exigua and presents a central area with a longer striae surrounded by shorter striae (Marquardt & Bicudo 2014).Placoneis exigua is reported as an indicator of eutrophic conditions (Van Dam et al. 1994, Besse-Lototskaya et al. 2011).In this study it occurred in surface sediment of eu-and hypereutrophic reservoirs (Hedberg and Barra Bonita, maximum abundance: 3.7%), and in the phytoplankton of eutrophic reservoir Hedberg (maximum abundance: 0.5%), with 5.7% frequency in samples, corroborating previous finding.
This taxon was identified as A. cf.macrocephalum because our specimens showed ends slightly less rounded than those presented in Simonsen (1987).Individuals similar to ours were recorded by Taylor et al. (2007) and Marra et al. (2016) as A. macrocephalum.Our taxon can be distinguished from A. minutissimum (Kützing) Czarnecki mainly due to its capitate ends (Potapova & Hamilton 2007).According to these authors, A. macrocephalum is an alkaliphilous species, however, they recorded other more capitate specimens in slightly acid and nutrient-poor waters of North America.Marra et al. (2016) recorded this species among the ten most frequent species in a mesotrophic subtropical reservoir.In this study, it was found in phytoplankton of five reservoirs ranging from oligo-to eutrophic conditions (Atibainha,Cachoeira,Paiva Castro,Tatu and Salto Grande, Valves linear-lanceolate with rounded to subrostrate ends; raphe valves with narrow and linear axial area.L: 11.5-18.0µm; W: 2.4-2.8µm; inconspicuous striae. The principal feature to distinguish this species from A. minutissimum is its narrower width measures (Hlúbiková et al. 2011;W: 2.5-3.1 µm).Further studies, such as SEM are necessary to a detailed analysis and to investigate whether Achnanthidium sp. 1 represents a new species.In the present study, this species was recorded in surface sediment of four reservoirs ranging from oligo-to eutrophic conditions (Santa Helena, Itupararanga, Ipaneminha and Hedberg, maximum abundance: 6.3%), and in the phytoplankton of the mesotrophic reservoir Ipaneminha (maximum abundance: 0.8%), presenting higher frequency in mesotrophic waters (17.1%).Valves rhombic-lanceolate with ends strongly capitate; axial area is narrow and linear, central area is small and rhombic; raphe is filiform; striae are slightly radiated in middle portion becoming parallel toward the ends.L: 13.5-27.0µm; W: 3.2-5.0µm; S: 32-35 in 10 µm.
Brachysira sp. is similar to Brachysira microcephala (Grunow) Compère "morphotype 2" presented by Siver et al. (2005) due to the distinctly capitate ends.However, Brachysira sp.differs due to its shorter width and valves becoming narrower toward the ends.Besides, Brachysira sp.maintains the valve shape (capitate) in smaller specimens.In the present study, this taxon was recorded in surface sediment of three reservoirs (Atibainha, Paiva Castro and Tatu, maximum abundance: 5.3%), however, it exhibited higher frequency in oligotrophic samples (16.0%).

*Nitzschia semirobusta
This species can be confused with Nitzschia amphibia Grunow due to the same valve shape.However, N. semirobusta can be distinguished by its more extended fibulae (Lange-Bertalot 1993).The taxon also resembles Denticula kuetzingii Grunow, nevertheless, the fibulae of the second species is extended from margin to margin with a similar thickness, while the fibulae of N. semirobusta decrease in thickness as they extend across the valve from one margin to another (Underwood 2017).In the present study, it was observed in surface sediment of five reservoirs ranging from oligo-to eutrophic conditions (Atibainha, Cachoeira, Paiva Castro, Tatu and Salto Grande, maximum abundance: 4.3%), and in phytoplankton of four reservoirs ranging from oligoto mesotrophic conditions (Atibainha, Cachoeira, Paiva Castro and Tatu, maximum abundance: 5.0%).Higher frequency occurred in oligotrophic conditions (74.0%).Nitzschia sp. 2 resembles N. bryophila (Hustedt) Hustedt because of their similar valve shape.Nevertheless, N. bryophila is wider (W: 5.0 µm), presents higher striae density (S: 31-33 in 10 µm) and more prominent ends (Simonsen 1987).In the present study, it was found in surface sediment of mesotrophic reservoir Tatu (maximum abundance: 20.2%), and in the phytoplankton of the same reservoir (maximum abundance: 3.3%), with a frequency of 11.4% in the mesotrophic samples.
Regarding the twelve species registered as new records for Brazil and São Paulo State, fi ve of them presented wide distribution in relation to the trophic conditions, occurring from oligo-to eutrophic conditions (Achnanthidium jackii, A. cf.macrocephalum, Cymbella affi nis var.neoprocera, Fragilaria grunowii and Sellaphora rostrata).Other four taxa occurred in oligo-mesotrophic conditions (Encyonopsis thienemannii, Gomphonema naviculoides, Nitzschia perminuta and N. semirobusta), while Achnanthidium lineare occurred in mesoeutrophic and Placoneis exigua was only recorded in eutrophic conditions.Two of the new records were exclusively found in the surface sediment (Achnanthidium lineare and Nitzschia perminuta) and other two only in the phytoplankton (Achnanthidium cf.macrocephalum and Encyonopsis thienenmannii).Finally, this study increased the information on the ecology and distribution of these species, particularly in tropical reservoirs, and highlights the need of floristic surveys to improve the knowledge of biodiversity of freshwater tropical diatoms.
FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, AcquaSed Project, nº 2009/53898-9).ECRB and SZA thanks their doctoral fellowship (FAPESP process 2013/14337-7 and2013/23703-7, respectively).Funding was also provided by CNPq, Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grant 310404/2016-9 to DCB).We appreciate the valuable assistance of personnel from the agency in charge of the public water supply in São Paulo -SABESP (Companhia de Saneamento do Estado de São Paulo), Barco Escola da Natureza Association, Companhia Energética Salto do Lobo, Floresta Nacional do Ipanema and Votorantin Energia for their valuable logistical support during the fieldwork.We deeply thank all the students from the Department of Ecology involved in field and laboratory work and those who collaborated in the construction of the AcquaSed database.We are also grateful to Prof. William de Queiróz (Universidade de Guarulhos, Laboratório de Geoprocessamento) for the study area map.

Figure 1 .
Figure 1.Location of the ten studied reservoirs in southeastern region of Brazil.Black lines represent the boundaries of the basins.Modifi ed from Bicudo et al. (2016).

Table 1 .
Watersheds, reservoirs, number and codes of sampling sites and number of samples collected from surface sediments (SS) and phytoplankton (P) in each reservoir.

Table 4 .
Minimum (min) and maximum (max) relative abundances of diatoms from surface sediment and phytoplankton in summer and winter.-: taxon did not occur in sample; taxa in bold are detailed in the present study; *: first record for Brazil; **: first record for São Paulo State.Ulrich (2014) provided new illustrations of F. tenera from the lectotype slide and presented three varieties: F. tenera (Smith) Lange-Bertalot var.tenera, F. tenera var.nanana (Lange-Bertalot) Lange-Bertalot & Ulrich and F. tenera var.lemanensis Druart, Lavigne & Robert.