Gomphonema Ehrenberg ( Bacillariophyceae , Gomphonemataceae ) of the São Francisco Falso River , Paraná , Brazil

Gomphonema Ehrenberg is a genus well represented in periphytic algal flora of epicontinental environments. The substrate colonization is facilitated by the secretion of mucilage by the pore field, allowing cell adhesion. Samples were scraped off and analyzed from vegetative portions of the macrophyte Eichhornia azurea Kunth, collected in São Francisco Falso River, located in Santa Helena City, Paraná State. The sampling occurred in four different sites, in four different periods, totaling 16 samples. The quantitative material analysis resulted in 28 identified taxa, in which Gomphonema brasiliense ssp. pacificum, Gomphonema neoapiculatum Lange-Bertalot, Reichardt & Metzeltin Gomphonema pantropicum Reichardt and Gomphonema perapicatum Metzeltin & LangeBertalot represented pioneer citations for Paraná diatomflorula. The ultrastructure observations of some species were crucial to distinguish a few taxa, as Gomphonema brasiliense and Gomphonema brasiliensoide D. Metzeltin, Lange-Bertalot & F. García-Rodríguez. We suggest future studies in this environment, once it has propitious characteristics to the development of epilithic diatoms. Moreover, phylogenetic studies are also recommended for a better distinction of taxa with a wide morphologic variation.


Introduction
In 1832, Ehrenberg proposed the genus Gomphonema, which comprises biraphide heteropolar species, with valves linear to linearlanceolate and apical ends wider than basal ends (Round et al. 1990).The raphe is central, straight or slightly sinuous, with straight or slightly curved and expanded proximal endings, and slightly curved distal endings (Ludwig & Tremarin 2006).The striae can be uniseriate or partly to entirely biseriate, and one or rarely two isolated pore occur at the central region of the valve (Round et al. 1990;Levkov et al. 2016).
Gomphonema individuals are common in haptobenthic communities of epicontinental waters, and can present epipelic, epilithic or epiphytic habit.The epiphytic habit is facilitated due to the mucilaginous stalks or pads secreted by the pore fields located in the valve basal ends (Round et al. 1990, Wojtal 2003, Cox 2015).
In Paraná State, the study about Gomphonema species carried out by Tremarin et al. (2009a) in samples collected in Maurício River -Curitiba metropolitan region -stands out, resulting in 19 identified taxa.In São Francisco Falso River, Silva et al. (2007) registered 26 Gomphonema species, presenting the occurrence in samples but not providing a taxonomic characterization of the populations or morphometric and meristic data.
We conducted a taxonomic study of Gomphonema species found adhered on the macrophyte Eichhornia azurea Kunth, using samples collected in São Francisco Falso River, in Santa Helena City, Paraná, Brazil.

Material and Methods
São Francisco Falso River (38.8 km 2 ) is the main former of the flooded area in Santa Helena City, west of Paraná.According to the park management plan, the geographic region is characterized by a transition between tropical climate, with two well defined rainy seasons, and temperate climate, in which the rain is distributed equally over the year, with average temperatures between 15 ºC and 25 ºC.The plan also characterizes the climate as humid subtropical, or mesothermal with pronounced summers (Cfa), with possibilities of a small dry during winter that would characterize the climate as Cw (Plano de Manejo, 1999).
The samples were collected in January, February, May and June 2004, in four sampling sites along São Francisco Falso River (Figure 1), with 4 collections and 16 samples.Petioles in adult stage (on average 50 cm long) were collected from the macrophyte Eirchhornia azurea Kunth., stored in 300 mL polyethylene flasks, and forwarded to the laboratory for epiphyte extraction.
The samples with epiphytic diatom were scraped off the petioles with a toothbrush and preserved in Transeau solution, in 1:1 proportion (Ludwig & Tremarin 2006).The biological material was housed at the herbarium of the Universidade Estadual do Oeste do Paraná (UNOP-Algae), Cascavel campus, and registered as shown on Table 1.
In the taxonomic analysis, subsamples were cleaned with KMnO4 and HCl (Simonsen 1974) modified by Moreira-Filho & Valente-Moreira (1981), and permanent slides were mounted using Naphrax ® .Whenever was possible, at least 10 individuals were analyzed for taxon identification.
Samples were cleaned, placed in aluminium stubs and subjected to metallization with gold, in Balzers Union SCD 030.The slides were observed in Olympus BX60 light microscope (LM) equipped with an Olympus DP71 digital camera.The scanning electron microscopy (SEM) images were performed in a JEOL JSM 6360LV microscope (Eletronic Microscopy Center -Federal University of Paraná).

Results and Discussion
The material analysis resulted in the determination of twenty-eight (28) Gomphonema taxa in specific level.
In SEM, stigma externally elliptic and striae uniseriate with areola in "C" shape (Figure 79).Raphe with proximal ends dilated into pores (Figure 79), curved to the opposite side of the stigma (Figure 77), and distal ends deflected and extending toward the valve mantle (Figue 78).Pore field formed by rounded poroids disposed in both sides of the terminal raphe fissure (Figure 80).
Comment: due to the wide metric amplitude already registered in many manuscripts for Gomphonema affine var.affine, we opted to follow the taxonomic review written by Reichardt (1999), since his work presents more limited meristic data (length: 36-88 µm; width: 9-13.6 Consulted literature: Reichardt (1999).
Comment: the species has valve structure similar to Gomphonema mexicanum Grunow, however some characteristics as areolae density and end morphology allow the distinction between these species, once G. affinopsis has 15-18 areolae in 10 µm and apice attenuate-rounded, while G. mexicanum has 21-25 areolae in 10 µm and apice subrostrate to rounded (Metzeltin & Lange-Bertalot 1998, Metzeltin et al. 2005).In the individuals sampled, by their characteristic attenuate-rounded apical end and by their wide variation in number of areolae 16-22 in 10 µm, the opted taxonomic identification is G. affinopsis.More specific studies are suggested with taxonomic clarification of the group.
In SEM, the raphe-sternum is ornamented by slight irregular depressions (Figure 85).Striae are uniseriate .The areolae are elongated longitudinally, except near the axial area, where they are "C" shaped .Pore field formed by rounded poroids are disposed in both sides of the terminal raphe fissure (Figure 88).
In SEM, the external view shows the raphe-sternum ornamented with weak irregular depressions (Figure 93).Striae are bisseriate along the valve and uniseriate at basal ends (Figures 93-96).The areolae are rounded to elongated longitudinally in both sides of the terminal raphe fissure (Figures 94-96).In internal view, the raphe presents proximal ends curved in sickle shape (Figure 98) and distal ends ending in prominent helictoglossa (Figure 99).
Consulted literatures: Comment: the analyzed specimens resemble the ones described by Reichardt (2001) as Gomphonema capitatum because the apical region is less constricted than Gomphonema anglicum, and rounded.G. anglicum presents a more accentuated constriction in the apical region and apice truncate-capitate, which are distinct characteristics of the studied population in this present work.
Comment: Gomphonema contraturris, proposed by Lange-Bertalot & Reichardt in Lange-Bertalot (1993) presents morphology similar to São Francisco Falso River individuals, except for the widely subrostrate to subrostrate apical end.However, the authors synonymize G. contraturris with G. acuminatum var.turris (Ehrenberg) Cleve sensu Fricke, which has apice acuminate.Despite the disagreement, the characteristics of Gomphonema contraturris population were taken into account to taxonomically identify the specimens here described.
In SEM, external view shows the proximal ends of the raphe straight to slightly curved and distal ends and stigma aperture rounded (Figures 101-103).Striae uniseriate (Figure 100).External areolae aperture mostly reniform (Figures 100-103).Internal valves view shows proximal raphe ends curved in sickle shape (Figure 106) and distal ends ending in prominent helictoglossa (Figures 105 and 107).
Comment: Jüttner et al. (2013) characterize the variety exilissimum carefully, differentiating it from Gomphonema parvulum according to the length x width ratio.In this case, the individuals that showed sizes between 4 and 6 µm were grouped as G. exilissimum, while the valves that presented ratio higher or lower were identified as G. parvulum.Gomphonema lagenula, also similar, can be differentiated by its assymetric valve shape, ends subcapitate to capitate and more pronounced, and by areolae shape, observed in SEM, generally more delicate and in "C" shape (Levkov et al. 2016).However, this separation does not solve all questions, since some individuals did not present this differentiation.Consulted literature: Krammer & Lange-Bertalot (1997) and Levkov et al. (2016).
Comment: Hustedt (1965) proposed the species basing on Brazilian material (São Paulo -Itatiaia), musciculous, at 1000 m elevation.The sizes described by the author are: length 44 µm, width 10 µm, 12-15 striae and 28 areolae in 10 µm; reasonably close measurements to the ones found in São Francisco Falso River material.The individuals found in our study presented lower density of areolae than those registered by Reichardt (2015a).However, the author indicates that the striae of G. graciloides has variable areolae number.Besides, the population from São Francisco Falso river has individuals with ends more apiculate in comparison to the ones illustrated by Reichardt (2015a), although the valve morphology and sizes are similar alike (length: 22-56 µm, width: 7.5-9.4µm, 12-16 striae in 10 µm).
In SEM, the distal raphe ends are curved to the opposite side of the stigma, while the proximal ends are dilated to pores, curved toward the stigma (Figures 112-115).The raphe is filiform and strongly sinuous (Figure 112).The striae are uniseriate over the valve and the areolae elongated longitudinally (Figures 113-115).The stigma is delicate and rounded (Figure 114).The pore field is formed by rounded poroids, disposed in both sides of the terminal raphe fissure (Figure 115).
In SEM, the raphe presents proximal ends dilated into pores (Figure 118) and distal ends curved to the opposite side of the stigma (Figures 117 and 119).Striae uniseriate .Pore field formed by rounded poroids that are disposed in both sides of the terminal raphe fissure (Figure 119).
Comment: the species presents similar morphology to Gomphonema parvulum (Kützing) Kützing.However, it differs by the rostrate apical end presented by G. parvulum and subcapitate to capitate showed by G. lagenula (Abarca et al. 2014).This species has large morphological variation in the analyzed community as well as in the literature, therefore making the taxonomic identification confuse and complex.Studies about the molecular biology of the valve morphology can assist future identification of species.
In SEM, external view shows proximal ends of the raphe straight to slightly curved and distal ends and stigma delicated and rounded (Figures 121-123).Striae uniseriate with areola in "C" shape (Figure 120).Internal valve view shows proximal raphe ends hooked and stigma elongated .
In SEM, the species presents stigma externally elliptic (Figure 129).Raphe slightly lateral and sinuous (Figure 127), with proximal ends dilated into pores (Figure 129) and distal ends extending on valve mantle, both curved to the same side of the stigma (Figures 128 and  130).Striae uniseriate, areolae "C" shaped (Figures 127-130).Pore field formed by rounded poroids, disposed in both sides of the terminal raphe fissure (Figure 130).
Comment: G. pantropicum Reichardt was considered until just recently as G. subtile Ehrenberg.Marquardt & Bicudo (2014) described this last taxon with apical end rostrate to capitate, characterized by the strong constriction of the valve close to the ends.According to the review written by Reichardt (2015b), this characteristic belongs to G. pantropicum, once G. subtile has apical end extended and widely capitate.
The identification of G. pantropicum was identified as G. subtile by Marra et al. (2016), also reflect the recent alteration of this taxon.Roy & Keshri (2015) describe a similar taxon, identifying it as Gomphonema cf.pantropicum.The individuals analyzed in this work match with the description given by Reichardt (2015b) Valves lanceolate to elliptic-lanceolate, apical ends subrostrate and basal ends attenuate-rounded.Raphe-sternum linear, narrow.Central area irregular and narrow, limited by a irregular shortening of a median stria.Raphe straight to slightly sinuous, proximal ends curved to the stigma side.Striae parallel to slightly radiate at the ends.Areolae inconspicuous.Stigma at the median stria end.Length: 18-31 µm; width: 6-7.5 µm; length/width ratio: 2.9-4.3;10-12 striae in 10 µm.
In SEM, the species presents stigma externally elliptic (Figure 137).Striae uniseriate, areolae "C" shaped .Pore field formed by rounded poroids disposed in both sides of the terminal raphe fissure (Figure 138).
In SEM, internal valve view shows proximal raphe ends curved in sickle shape and distal ends curved to opposite side of the stigma , ending in prominent helictoglossa (Figure 140) and stigma elongated (Figure 141).

Gomphonema pseudoaugur
In SEM, external view shows proximal ends of the raphe straight to slightly curved and distal ends and stigma aperture rounded .Striae uniseriate (Figure 142).External areolae aperture mostly reniform .Pore field formed by rounded poroids disposed in both sides of the terminal raphe fissure.(Figure 145).
Occurrence radiate, tending to parallel close to the ends.Areolae inconspicuous.
Comment: Gomphonema subclavatum has wide morphological variation, but the only specimen found possesses similarity with other registered individuals in Krammer & Lange-Bertalot (1991) and Levkov et al. (2016).
Occurrence in samples: UNOP-Algae 2264.Comment: the species differs from its typical variety by the valve outline characteristically claviform, wider valves, shorter apices and elliptic central area.Tremarin et al. (2009) presented similar specimens to the ones in the population, describring them as Gomphonema turris var.coarctata morphotype 1 and 2, the first with valves more apiculate, apice less highlighted and central area elliptic, and the second with valves clavate, apice highlighted and central area linear.The population from São Francisco Falso River showed both morphological variations.It is recommended further and more specified studies in order to distinguish the morphotypes.

Final considerations
Overall, we observed large morphological variations in the Gomphonema populations.Gomphonema parvulum, Gomphonema exilissimum and Gomphonema lagenula were often difficult to distinguish from other similar species.The description and comparison of specimens with specialized literature (Table 2) were decisive for taxa description.As a criterion for Gomphonema exilissimum definition we used length/width ratio and for Gomphonema parvulum the rostrate apical and basal ends.To differentiate species from Gomphonema lagenula, which is also similar, we defined as criteria the assymetric valve contour, ends subcaptate to capitate more pronounced, and areolae shapes, observed in SEM.
In some specific cases, the ultrastructure analysis was necessary to define the individual taxon, as Gomphonema brasiliense and Gomphonema brasiliensoide that were separated mostly by their uniseriate and biseriate striae, respectively, and Gomphonema brasiliense ssp.pacificum, which possesses more delicate and elongated areolae than the type species.
Among the diatom floristic survey studies in Paraná, only Silva et al. (2007) discussed exclusively about the genus Gomphonema.Tremarin et al. (2009a) performed the taxonomic survey of Gomphonema and Gomphosphenia.Concerning of the West side of the State, these studies represented the largest genus diversity of the last couple years (26 and 19 taxa, respectively).Recent papers in Paraná comprising the epiphytic diatom taxonomy as Bertolli et al. (2010), Silva et al. (2010), Santos et al. (2011), Moresco & Rodrigues (2013), and Moresco & Rodrigues (2016) did not exceed 11 Gomphonema registered species, highlighting that the 28 taxa registered in this paper respond to the analyzed environment specificity.
In qualitative analysis of two collections from São Francisco Falso River (January and June), also comprised in this current study, Silva et al. (2007) listed the Gomphonema taxa and their respective temporal and spatial distributions, but without taxonomic comments and illustrating only a few of them.Moreover, four species (Gomphonema subtile, Gomphonema mexicanum, Gomphonema affine and Gomphonema gracile) were differently identified in this paper (as Gomphonema pantropicum, Gomphonema affinopsis, Gomphonema graciloides e Gomphonema guaraniarum, respectively), mostly because of the criteria used, as the apical ending shapes and valve length.
This study broadens the knowledge about ultrastructure of several Gomphonema species, due to the lack of more specific taxonomic descriptions and illustrations involving SEM.
Future similar studies in this environment need to be conducted in order to continue to explore the diatom Brazilian flora, since it has propitious characteristics to the development of epilithic diatoms.
We also emphasize that studies using molecular tools are fundamental to better circumscribe Gomphonema species, due to the wide morphological variation of valve shape.
Table 2. Valve morphology and morphometric and meristic limits of Gomphonema species found in São Francisco Falso River compared with data from the literature used for taxonomic classification.Similar taxa are grouped for better comparison.

Table 1 .
Reference of the São Francisco Falso river examined material housed at the herbarium of Universidade Estadual do Oeste do Paraná (UNOP-Algae), Cascavel campus, and its respective register number.