Periphytic diatoms ( Bacillariophyta ) in streams from three Conservation Units of central Brazil :

1. Parte da Dissertação de Mestrado do primeiro Autor 2. Universidade Federal de Goiás, Programa de Pós-Graduação em Biodiversidade Vegetal, 74690-900 Goiânia, GO, Brasil 3. Universidade Federal do Pará, Programa de Pós-Graduação em Ecologia, Belém, Pará, 66075-110 Belém, PA, Brasil 4. Universidade Estadual de Maringá, Programa de Pós-Graduação em Ecologia de Ambientes Aquáticos Continentais, 87020-900 Maringá, PR, Brasil 5. Universidade Católica de Brasília, Laboratório de Biodiversidade Aquática, 71966-700 Brasília, DF, Brasil 6. Corresponding author: dunck.barbara@gmail.com ABSTRACT (Periphytic diatoms (Bacillariophyta) in streams from three Conservation Units of central Brazil: Pinnularia Ehrenberg). This study aimed to survey the genus Pinnularia Ehrenberg (Bacillariophyta) in five preserved streams located in three conservation units (Brasília National Park, Chapada dos Veadeiros National Park and Terra Ronca State Park). Periphyton was collected in different substrata during the dry and rainy seasons (years 2012 to 2013), totalizing 25 sample units. Altogether, 23 taxa were identified, being P. subanglica, P. angustivalva and P. butantanum the most frequent. Pebble and sand were the richest substrates, with nine taxa each. Among the 23 taxa reported, 13 are cited for the first time for the central Brazil: P. angusta var. angusta, P. angustivalva, P. butantanum, P. divergens var. biconstricta, P. divergens var. mesoleptiformis, P. gibba var. subundulata, P. paulensis, P. persudetica var. persudetica, P. subgibba var. angustarea, P. subgibba var. capitada, P. superpaulensis, P. viridiformis var. minor and P. undula var. undula.


Introduction
Among the various groups of organisms comprising the periphyton, diatoms stand out by its abundance among the autotrophs, due to morphological adaptations that facilitate their fixation on substrates (Stevenson 1997).Several species are able to adhere to surfaces by mucilage secreted through structures present in the valve, as the raphe, pores field apical and rimoportula (Round et al. 1990), providing them adaptive advantages (Passy 2007;Law et al. 2014).The substrate variability in lotic environments is particularly important because it affects the structure of periphyton (Cattaneo et al. 1997).
The family Pinnulariaceae D.G. Man is composed of unicellular individuals that can form short chains and are characterized by having linear-lanceolate valves, rarely elliptical, with slightly rounded apical end, which can be capitate to subcapitate to lightly cuneate (Round et al. 1990, Rocha & Bicudo 2008).The striae are alveolate chamber-shaped that extend from the sternum towards the ends of the valve.These alveolate structures are open to the outside through pores with occlusion of hymen type (Round et al. 1990).This family comprises genera such as Caloneis Cleve, Diatomella Greville, Dimidiata Hajós, Ostrupia Heiden ex Hustedt and Pinnularia Ehrenberg (Tremarin et al. 2010).
Pinnularia Ehrenberg is characterized by solitary individuals, rarely colonial.The valves can be linear, lanceolate or elliptical, with straight or wavy edges and rounded apical end, rostrate or capitates (Round et al. 1990).According to Guiry & Guiry (2016), 728 taxa have been flagged as taxonomically accepted within this genus.It occurs worldwide in freshwater environments of different pH and trophic states, with preference to slightly acidic oligotrophic environments, and in a smaller proportion, in moist soils and marine coastal environments (Metzeltin & Lange-Bertalot 1998;Krammer 2000).
Taxonomic studies on periphytic diatom algal flora in central Brazil are in early stage.The works of Souza & Moreira-Filho (1999), Delgado & Souza (2007) e Silva et al. (2016) are so far the only ones with descriptions of Pinnularia.Moreover, there is no available information about diatom communities in aquatic systems of the conservation units considered in this study.Present work aimed at surveying the genus Pinnularia in the periphyton of preserved streams from central Brazil located in three conservation units.

Material and methods
This study was carried out in five low order streams located in three conservation units from central Brazil: Brasilia National Park (hereafter, BNP), Chapada dos Veadeiros National Park (hereafter, CVNP) and Terra Ronca State Park (hereafter, TRSP) (figure 1).The region is occupied by the Cerrado domain (Brazilian savanna), where climate is rainy tropical, with strong seasonality.There is a rainy season from October to April concentrating up to 80% of annual precipitation (mean temperature around 29 o C), and a dry season from May to September when precipitation ranges from zero to below 50 mm (mean temperature around 18 o C) (Silva et al. 2008).
Between streams, BNP is located in the Federal District of Brazil, near the Brazilian capital, Brasília.CVNP and TRSP are located in Goiás State (table 1).TRSP comprises the largest cave complex in South America, and the streams sampled in this area crosses caves with their respective names (Lapa and São Mateus).The total number of sampling units was 25, considering different substrates and two seasons, as will be described below.
Periphyton was collected during the dry (August) and rainy (March) seasons, between the years 2012 and 2013, according to the protocols established by the ComCerrado Network (unpublished data).In each stream it was delimited a 50 m longitudinal transect, where the following substrates, wherever possible, were considered: leaf litter (L), macrophytes (M), roots (R), trunk (T), pebble (P) and sand (S) (table 1).It was used a syringe to collect sand; macrophytes were squeezed and the other substrates were scraped with a toothbrush, using distilled water gentle jets.Samples were preserved in 3-5% formalin solution and deposited in the Herbarium of the University of Goiás (UFG).Along with periphyton, some abiotic variables (water temperature, dissolved oxygen, pH and electrical conductivity) were measured in the field using standard electrodes (Yellow Spring Instruments) (table 2).
Periphyton samples were cleaned using the oxidation method of Simonsen (1974) modified by  Moreira -Filho & Valente-Moreira (1981).The samples were fixed on permanent mounts with Naphrax® resin.Taxonomical analyzes were performed using a light microscope Leica DM500 equipped with digital camera ICC50Hd.The identification was based on valve morphological and morphometric features, using both classic (e.g., Hustedt 1965, Krammer 2000, Metzeltin & Lange-Bertalot 1998, 2007) and regional literature (e.g., Rocha & Bicudo 2008, Tremarin et al. 2010, Pereira et al. 2012).We observed at least five individuals of each species to the identification.The classification system used was Round (1991).

Results and Discussion
Altogether 23 taxa were recorded for genus Pinnularia (table 3).This genus is the second with higher species richness in Brazil (125 species) according to Menezes et al. (2015), followed only by Cosmarium ex Ralfs (Conjugatophyceae) with 156 species.Our study contributed with the record of 13 taxa not yet reported for the central Brazil, and this number is very representative to Pinnularia species compared to 53 species cited by Silva et al. (2011) for the Central-West region.
The most frequent species were P. subanglica, P. angustivalva and P. butantanum, present in more than 20% of the samples.Among the streams analyzed, Córrego 1 and Estiva were the richest ones, with 11 and nine taxa, respectively (table 3).These streams are located at CVNP, and presented the highest variety of substrates.Pebble and sand were the richest substrates, with nine taxa of Pinnularia, followed by trunk (seven), macrophytes (seven), leaf litter (six) and roots (four).
An identification key is presented with the identified taxa, followed by description, comments, distribution in the central Brazil and illustration of the examined material.Pinnularia angusta var.angusta (Cleve) Krammer, In Krammer, Biblioth.Diatomol. 26:122. 1992.Figure 2 The valves are linear with triundulate margins and a slight inflation in the middle region; apices are capitate to subcapitate; raphe filiform; central area asymmetric; Striae are often slightly bent, radiate near the valve center and become convergent in the end.Apical axis: 48.88 µm; transapical axis: 7.74 µm; rate apical axis/transapical: 6.31 µm; 13 striae in 10 µm.

Identification key of species of
Pinnularia angusta var.angusta has morphological characteristics such as frustule shape and similar striae  as Pinnularia grunowii Krammer and Pinnularia pluvianiformis Krammer.These two species have differences mainly in the the rate apical axis/transapical.Krammer (2000) describes the rates between 3.7-5.4for Pinnularia grunowii and 4.5 for Pinnularia pluvianiformis, and P. angusta is between 5.5-8.5.In northern Europe, the distribution related to this species is mainly in oligotrophic environments with low electrolytes (Krammer 2000).
Midwest region records: pioneer reference.The valves are lanceolate with a slightly convex side; apices are subcapitate to capitate; raphe is straight with a narrow sternum; axial area is lanceolate widening into the middle region; central area is semirhombic formed by shortening of the middle striae and a siliceous space reaching the valve margin; striae are radiate and convergent near the apices.Apical axis: 53.18-63.32µm; transapical axis: 9.18-10.86µm; rate apical axis/transapical: 5.18-6.34µm; 10-12 striae in 10 µm.
Pinnularia subcapitada Gregory is close to P. brauniana because their valves are lanceolate with subcapitate to capitate apices, but they differ on a narrow axial area and a small transapical axis (Krammer 1992).
The specimes of this study are smaller than that illustrated by Tremarin et al. (2010) for P. divergens var.biconstricta.They are similar in the valves characteristics, striation and rhombic central area.According to Krammer (2000), the valves are linear and the valves margins commonly parallel, similar to that found in our study.The specimes differ from P. divergens var.malayensis mainly by the undulation on the valves margin, as illustrated by Tremarin et al. (2010) and Rocha et al. (2008) for P. divergens var.malayensis.Pinnularia divergens var.biconstricta have planktonic habitat and is found in standing water, but can occur running water (Moro & Fürstenberger 1997).
Midwest region records: pioneer reference.
This variety differs from the variety type, by presenting a smaller apical and transapical axis (Krammer 2000).Pinnularia divergens var.media prefers oligotrophic environments with low mineral contents (Krammer 2000).The observed specimens had shown a bigger transapical axis and less striae compared with specimens found in Guaraguaçu's river (apical axis 33.1-49.4µm; 12-14 striae in 10 µm) by Tremarin et al. (2010), but the dimensions fit with the description of the species and the original illustrations.
The original description of P. divergens var.mesoleptiformis put this species similar to P. divergens var.laticeps Franquelli, showing only difference on the transapical axis' measures, which in Pinnularia divergens var.laticepes always is smaller, 13,4 µm against 20 µm.Although, illustrated in Metzeltin & Lange-Bertalot (1998) and Frenguelli (1933) we realize that besides the length range of the valves, both varieties differs on the apices' shape and type of the raphe.At Pinnularia divergens var.laticeps the apices are capitate with a small flatness and the raphe is filiform, yet Pinnularia divergens var.mesoleptiformis has apices capitate and stretched and the raphe is complex.
Midwest region records: pioneer reference.
Midwest region records: pioneer reference.

Pinnularia meridiana var. meridiana
P. meridiana var.meridiana distinguishes from P. var.parallela, by showing parallel to slightly convex margins and apices are slightly rounded, while P. var.parallela has parallel margins and apices strongly rounded (Metzeltin & Lange-Bertalot 1998).The specimen of P. meridian var.meridian found on this study has similarity with the illustrated in Metzeltin & Krammer (1998), differing on the transapical axis, which according to the author are 12 to 13 µm.
The specimens are similar to that illustrated by Krammer & Metzeltin (1998, table 172 pg 578, figs. 6-8) manly in the valve format, striae and the straight proximal raphe ends.This is cosmopolite species, have been found in oligotrophic waters with low electrolytes (Krammer 2000).
Midwest region records: pioneer reference.
Midwest region records: pioneer reference.
The illustrated individuals in this study, shows morphological characteristics as valves shape, apices, raphe, central area and striae similar to the description in Krammer (2000), but differs on the transapical axis measure (11.6-13.7The valves are elliptic-lanceolate with lightly convex margins; apices are acuminate-rounded; raphe is lateral and slightly undulate with distal ends deflected to the same direction in bayonet shape; central area is rounded; striae are radiate and slightly convergent on the apices.Apical axis: 78.61 µm; transapical axis: 14.88 µm; rate apical axis/transapical: 5.28 µm; 10 striae in 10 µm. P. viridiformis var.minor differs from Pinnularia viridiformis var.viridiformis Krammer because the valve size is different and the raphe is complex.The illustrated individuals in this study showed less striae, when compared with the original description by Krammer (2000) which has between 7-9 striae in The valves are lanceolate; apices are rounded and slightly cuneate; raphe is filiform slightly undulate with distal ends in hook shape; central area is slightly rounded in one side of the valve and a small siliceous space in the center; striae are shorter in an irregular form, slightly radiate to convergent on the apices.Apical axis: 48.67-54.33µm; transapical axis: 9.7-9.92µm; rate apical axis/transapical: 4.90-5.60µm; 11 striae in 10 µm.

Pinnularia sp. 4 Figura 44
The valves are linear; apices are rostrate to cuneate; raphe is straight with distal ends in bayonet shape and deflected to the same side; central area is asymmetric formed by a slightly shortening of the striae in the center; striae are radiate in the center and slightly convergent on the apices.Apical axis: 47.39 µm; transapical axis: 9.39 µm; rate apical axis/ transapical: 5.04 µm; 11 striae in 10 µm.

Pinnularia sp. 5 Figures 14-15
The valves are linear to elliptic-lanceolate and weakly convex; the apices are conic and does not differentiated; raphe is filiform; central area is rounded developed in a fascia that can appear in one side or both sides; striae are weakly radiate in the middle region turning into weakly convergent towards to the apices.Apical axis: 28.82-34.92µm; transapical axis: 5.51-5.95µm; rate apical axis/transapical: 4.96-6.06µm; 12-13 striae in 10 µm.
The illustrated individuals on this study shown similarity with Pinnularia castraregina on the

Table 3 .
Occurrence of Pinnularia Ehrenberg in Cerrado streams during dry and rainy seasons, in different substrates and species richness by stream (L: leaf litter, M: macrophytes, P: pebble, R: roots, S: sand, T: trunk).
Krammer (2000) subgibba var.capitada differs from the complex species type P. subgibba, by presenting an undulation to triundulation on the margins with a slightly inflation in the middle region, apices are slightly rounded.Midwest region records: pioneer reference.Occurence in the samples: BRAZIL.Goiás.Parque Nacional da Chapada dos Veadeiros, Córrego 1, A.A.The valves are linear with an expansion in the center of the valve; apices are slightly cuneate-rounded; raphe is straight and filiform with distal end in a hook shape; central area is slightly rounded in one side of the valve; striae are shorter throughout all the complex of the valve, slightly radiate and convergent.Apical axis: 128.67-129.38 µm; transapical axis: 14.49-14.63µm;rateapicalaxis/transapical: 8.84-8.87µm;14striae in 10 µm.According toKrammer (2000)Pinnularia paulensis Grunow differs from Pinnularia superpaulensis because the raphe is simple and the striae are shorter.