Eunotia (Bacillariophyceae) from a subtropical stream adjacent to Iguaçu National Park, Brazil, with the proposition of a new species

Abstract: Eunotia species from a stream located in the surrounding area of the Iguaçu National Park conservation unit were analyzed. Samples were monthly collected from September 2012 to August 2013. The periphytic material, manually obtained by squeezing aquatic macrophytes, was oxidized and analyzed under light and scanning electron microscopy. Twenty-one infrageneric taxa of Eunotia were identified, measured, and illustrated. Eunotia caniculoides sp. nov. is proposed as a new species, and eight taxa are new records for Paraná state.


Introduction
The genus Eunotia Ehrenberg is characterized by the valve isopolarity, apical axis asymmetry, uniseriate striae, short raphe system, usually restricted to the valve apices and the presence of rimoportulae (Kociolek & Spaulding 2003, Furey 2011. The extant species are found in freshwater environments (Round et al. 1990) but some fossil Eunotia have already been described from marine sediments in New Zealand (Novitski & Kociolek 2005). The genus includes species with ecological optimum in the epiphyton and metaphyton of dystrophic to oligotrophic waters, being valuable indicators of acidic and lowconductivity conditions (Round et al. 1990, Van Dam et al. 1994, Cantonati & Lange-Bertalot, 2011, Cox 2015. Eunotia is one of the most common and species-rich diatom genera in the Neotropics (Patrick 1940a, 1940b, Hustedt 1952a, 1952b, 1965, Sala et al. 2002a, 2002b, Faustino et al. 2016, Costa et al. 2017, with remarkable diversity in Brazilian acidic environments (Ferrari et al. 2007, Bicca et al. 2011, Canani & Torgan 2013, Dunck et al. 2013a, 2013b, Cavalcante et al. 2014). In the state of Paraná, the genus were reported by Ludwig & Valente-Moreira (1989), Fürstenberger & Valente-Moreira (2000), Tremarin et al. (2008), Faria et al. (2010), Bertolli et al. (2010), Santos et al. (2011), Bartozek et al. (2013), Marra et al. (2016), Silva-Lehmkuhl et al. (2019), and Bartozek et al. (2020) recently proposed a new species. Based on a bibliographic check-list of freshwater diatom The purpose of the present publication was to investigate the species diversity of Eunotia from a stream located in the surrounding area of the Iguaçu National Park conservation unit. A new species, Eunotia caniculoides, was formally described. The distinctive characters between similar taxa were discussed and taxonomical notes were provided. Distribution in the state of Paraná and ecological informations were also added.

Material and Methods
The present study was carried out on the Tenente João Gualberto stream, located in a conservation unit area adjacent to the Iguaçu National Park, in the municipality of São Miguel of Iguaçu (25º28'36.3"S; 54º19'40.9"W), the western region of the state of Paraná. According to Horton-Strahler criteria (Horton 1945, Strahler 1952, the stream is a major tributary of the Baixo Iguaçu Basin, having its source and part of its course among agricultural fields ( Figure 1). The regional climate is Cfa type, subtropical humid mesotherm, with well-defined summer and winter periods, and rains distributed during the year (Alvares et al. 2013).
Periphytic samples were removed by manually squeezing the aquatic macrophyte Eleocharis minima Kunth (Cyperaceae) and were preserved in Transeau solution, in 1:1 proportion (Bicudo & Menezes 2017). The organic matter was eliminated by oxidation with KMnO 4 and HCl according to Simonsen (1974) technique modified by Moreira-Filho & Valente-Moreira (1981). Cleaned material was dried onto glass coverslips and mounted on slides with Naphrax® (IR= 1.74). The taxonomic analysis was performed using an Olympus BX60 light microscope equipped with a DP71 image capture camera. Subsamples from cleaned samples were mounted on aluminum stubs and coated with gold. Micrographs were taken with JEOL JSM 6360 scanning electron microscope (SEM), operated at15 kV and 8mm working distance, housed in the Electron Microscopy Center from the Federal University of Paraná. Slides and samples were deposited in the UNOPA Herbarium, UNIOESTE, campus Cascavel and registered as shown in Table 1.
Every Eunotia species was measured at the valve apical length, transapical width and striae density in the central part of the valve. The ecological information cited for identified taxa corresponds to the abiotic parameters measured in the samples collected during this study.
Eunotia bilunaris (  Valves arched tapering toward apices, 16.2-98.7 µm long and 3.1-4.6 µm wide. Dorsal margin convex, ventral margin concave sometimes with median swelling (Figure 2e-2j). Apices rounded to acutely rounded. Terminal nodules near the apices. Distal raphe end strongly curved onto the valve face. One rimoportula per valve, near the apex. Striae parallel, denser near the apices, 12-15 in 10 µm in the middle region of the valve. Areolae rounded, not visible in LM. Cingulum composed of four open bands perforated by a linear row of pores.
Taxonomic remarks: Eunotia bilunaris is a very common periphytic species, broadly distributed in the state of Paraná, and frequently registered in Europe, Africa, Asia, North and South America (Costa et al. 2017). According to Lange-Bertalot et al. (2011), the species have a heterogeneous concept and a confusing taxonomy due to their wide morphological variability, mostly related to shape and dimensions. Specimens with uncommon morphology are common and have already been formally described as infraspecific taxa. The material analysed showed a wide morphological and metric variation of the valves, corroborating with the observations of Krammer & Lange-Bertalot (1991) and Tavares & Valente-Moreira (2000). Frustule rhomboid in lateral view. Valves dorsiventral, sometimes heteropolar, 11.1-22.5 µm long and 2.5-3.7 µm wide. Dorsal margin convex, ventral margin straight to slightly concave. Apices rounded to obtusely rounded . Terminal nodules near the apices. Distal raphe end curved onto the valve face. One small rimoportula per valve, near the apex. Striae parallel, 14-19 in 10 µm in the middle region of the valve. Areolae rounded, not visible in LM. Cingulum composed of four open bands perforated by one row of pores.

Paraná State citations: cited as E. intermedia (Krasske ex Hustedt)
Taxonomic remarks: Eunotia botulitropica was recently described based on samples collected in reservoirs from São Paulo state. According to Costa et al. (2017), E. botulitropica differs from E. botuliformis Wild, Nörpel & Lange-Bertalot by its "narrower valve, lower striae density, rounded and more tapered ends" (Lange-Bertalot et al. 2011). However, the measurements and number of striae overlap, and E. botuliformis can be separated by the parallel valvar margins and the regular distribution of the striae along the valve. Eunotia botulitropica shows rhomboid girdle view, resembling E. rhomboidea Hustedt, but the latter is clearly a heteropolar species and presents larger valves (Costa et al. 2017

Occurrence in samples
Eunotia ambivalens  Figures 2k-2o, 5a-5d. Valves dorsiventral, 30.0-58.5 µm long and 3.5-4.9 µm wide. Dorsal margin slightly convex, to almost straight in the larger valves; ventral margin straight to subtly concave. Apices acutely rounded, nose-like, quite protracted from the main body, deflected to ventral side. Pseudosepta in the valve ends. Terminal nodules ventral, somewhat apart from the apices. Distal raphe ends simple, not reaching the valve center. Raphe fissure obliquely curved toward ventral mantle, with proximal ends dilated in pore. One small rimoportula per valve, at the center of the apex. Striae parallel to radiate, denser near the apices, 10-14 in 10 µm in the middle region of the valve. Areolae round, 38-40 in 10 µm, not visible in LM. Cingulum composed of four open bands perforated by a single linear row of pores. On the mantle, at the middle of raphe branch, 3 areolae between valve face margin and raphe fissure; 2 to 3 areolae between the raphe branch and lower mantle margin.
Frequency of occurrence: constant (Table 3). (length 17-48 µm, width 4.0-5.0 µm) and striae density (11-13 in 10 µm) (Table 2) but the apices are more rounded, less protracted, and not flexed to ventral side as in E. caniculoides (Lange-Bertalot & Metzeltin 2009). E. sioliopsis shows similar dimensions and apices shape, but striae and areolae densities are lower and dorsal margins are more convex (see Table 2  E. sioliopsis, the areola number in the middle of the raphe branch in the mantle, between the raphe and the mantle margin is higher (4 to 5) (Moser et al. 1998), and the stability of this character in the epitheca had been reported to the genus by Mayama (2001).
Valves dorsiventral, 23.5-59.0 µm long and 4.0-5.2 µm wide. Dorsal margin straight, slightly undulate; ventral margin straight to subtly concave. Apices nose-like. Terminal nodules near the apices. Striae parallel to radiate, denser near the apices, 12-17 in 10 µm in the middle region of the valve. Rimoportula and areolae not visible in LM.
Frequency of occurrence: common (Table 3) Frequency of occurrence: common (Table 3) Taxonomic remarks: E. maior (W. Smith) Rabenhorst is easily distinguished from E. monodon by the clearly set off capitate ends and by the more convex dorsal margin, but parallel to the ventral margin. Eunotia monodon usually has dorsal margin more strongly convex and ventral concave, but not parallel, rounded apices, slightly differentiated from the main body (Patrick & Reimer 1966 nose-like, differentiated from the main body. Terminal nodules near the apices. Distal raphe end short, obliquely curved onto the valve face. One rimoportula per valve, near the apex. Striae parallel to radiate, denser near the apices, 11-15 in 10 µm in the middle region of the valve. Areolae rounded, ca. 38 in 10 µm. Frequency of occurrence: common (Table 3). Paraná State citations: Bartozek et al. (2013), Tremarin et al. (2009), Marra et al. (2016, Silva-Lehmkuhl et al. (2019).
Taxonomic remarks: The population of Eunotia pseudosudetica analyzed here is similar to type material from Uruguay (Metzeltin et al. 2005) and to southern Brazilian specimens described and illustrated by Cavalcante et al. (2014).
Figures 3ac-3ad, 6f. Valves slightly arched, 22.9-32.7 µm long and 6.5-8.5 µm wide. Dorsal margin convex with three undulations being the median undulation more pronounced than the others; ventral margin concave. Apices broadly rounded. Terminal nodules in the apices. Striae parallel to radiate, denser near the apices, 12-14 in 10 µm in the middle region of the valve. Rimoportula and areolae not visible in LM. In SEM, 40 areolae in 10 µm.
The more common taxa in the studied river, occurring in at least 50% of the samples, were: E. bilunaris, E. ambivalens, E. longicamelus, E. karenae, E. minor, E. pseudosudetica var. rotundata, E. monodon, E. juettnerae, E. pileus, E. rabenhorstii var. monodon, E. rabenhorstii var. triodon, E. botulitropica and E. sedina. 66.6% of identified species were widely distributed in the study area and considered of common occurrence, 19.04% were constant, 9.52% were sporadic and 4.76% were rare. In general, the high frequencies of occurrence of the periphytic Eunotia community were in the spring period (76%) and in October (71%).

Discussion
The number of taxa found in the present study (21) can be considered high when compared to Eunotia studies carried out in other Brazilian regions that used a similar methodology, obtaining samples by squeezing an aquatic macrophyte. We highlight Ludwig & Valente-Moreira (1989) that identified 24 Eunotia and Santos et al. (2011) with 18 species. Other studies about diatom flora based on samples collected in the state of Paraná included 44 taxa (Tremarin et al. 2008), 16 taxa (Marra et al. 2016), 13 taxa (Bichoff et al. 2016), 10 taxa (Fürstenberger & Valente-Moreira 2000), and 8 taxa (Bertolli et al. 2010). Bicudo et al. (1995) identified 14 taxa on samples from Mato Grosso wetland, Cavalcante et al. (2014) found 12 taxa in the state of Santa Catarina, and Ferrari et al. (2007) registered 23 species in amazonian water bodies.
Despite numerous investigations of diatom diversity in the state of Paraná, the taxonomic study of the Eunotia based on 12 samples from the Tenente João Gualberto stream resulted in eight new citations for the State, and Eunotia caniculoides was proposed as new. The more common taxa in the studied river, occurring in at least 50% of the samples, were: E. bilunaris, E. ambivalens, E. longicamelus, E. karenae, E. pseudosudetica var. rotundata, E. monodon, E. juettnerae, E. pileus, E. rabenhorstii var. monodon, E. rabenhorstii var. triodon, E. botulitropica and E. sedina. 66.6% of identified species were widely distributed in the study area and considered of common occurrence, 19.04% were constant, 9.52% were sporadic and 4.76% were rare. In general, the high frequencies of occurrence of the periphytic Eunotia community were in the spring period (76%) and in October (71%).
Species of Eunotiaceae are influenced by pH and prefer acidic water (Patrick & Reimer 1966, Cantonati & Lange-Bertalot 2011. They are typical components of the diatom flora from tropical and subtropical areas, frequently associated with acid waters and oligotrophic or dystrophic habitats , Kociolek & Spaulding 2003, Burliga et al. 2013. During this study period, the annual pH mean value was predominantly acidic (6.3), an environmental condition that may have favored the species richness of Eunotia. The months of September and October 2012, April and June 2013, with greater richness of Eunotia taxa, showed higher concentrations of dissolved oxygen (11.05 -22.89 mg.L -1 ). Of the months cited, those of 2012 had higher concentrations of TP and NO 3 -(0.0198-0.0523 mg.L -1 and 0.365-0.3775 mg.L -1 respectively), and lower depth and flow values (0.16-0.21 m and 0.16-0.20 m 3 .s -1 respectively).
In conclusion, Eunotia is of great taxonomic complexity and studies are still needed, leading to more complete knowledge of species diversity and distribution in Brazil. In addition, taxonomic knowledge of Eunotia species is essential due to their already known ecological preferences, being useful information for ecological researches aimed at the conservation and management of aquatic environments.