Melosira ( Diatomeae ) taxa from the Iguaçu River in southern Brazil

The study of the genus Melosira in plankton samples from the lower Iguaçu River revealed the presence of four taxa: Melosira undulata (Ehrenberg) Kützing var. undulata, M. undulata var. normanni Arnott, M. varians C. Agardh and M. muscigena Iwahashi. We present illustrations of the frustules using light microscopy (LM), descriptions, and comments about the morphology of the four taxa. The analysis of Melosira muscigena under scanning electron microscopy revealed unprecedented details of the ultrastructure, such as the shape and distribution of the rimoportulae at the valve mantle. This is the first record of Melosira undulata var. undulata and the second of M. muscigena in Brazil.


Introduction
Diatoms from the genus Melosira C. Agardh are characterized by cylindrical to subspherical frustule, usually united at the valve faces by mucilage pads and small irregular spines (Crawford 1975) in pairs or chains of three cells (Van Heurck 1896).The valve face can be flat or convex and may be bordered by a corona, with granules more or less developed, and a carina (collar-like structure) (Crawford 1975).The areolae are loculated with loculi open to the outside through small pores.The cingulum presents ligulate bands perforated by longitudinal rows of small pores (Round et al. 1990).Rimoportulae usually occur near the edge, scattered or grouped at the valve mantle (Round et al. 1990).
Previously, the genus Melosira included most of the centric diatoms that grew in chains with cells strongly connected by the valve face (Stoermer & Julius 2003), accommodating a wide range of taxa gradually transferred to different genera, such as: Aulacoseira Thwaites, Paralia Heiberg, Orthoseira Thwaites, and Ellerbeckia Crawford (Round et al. 1990, Houk & Klee 2007).
Here, we presented a taxonomic survey of Melosira taxa for the Iguaçu River, showing unprecedented ultrastructural details of Melosira muscigena and the first citation of M. undulata var.undulata to Brazil.

Material and Methods
The Iguaçu River runs westward 1275 km from its source in the Serra do Mar to its confluence with the Paraná River (Paraná 2010).Considered a large river, part of the river is located in Iguaçu National Park, a protected area important for its great biodiversity (Paraná 2010).
Station 1 (S1) is located upstream of the falls (25º39'12,8" S and 54º25'34,0" W), where the river is wider, with variable depth from 1.4 m to 3.0 m.The station was classified as oligo-mesotrophic for the sampling period, with maximum and minimum values according the Table 1 (Nardelli et al. 2016).
Station 2 (S2) is downstream from the falls (25º38'55,4" S and 54º27'31,0" W).In this region, the river is narrower, deeper (11.7 to 27.4 m), and flows faster than at Station 1. Station 2 was classified as ultra-oligotrophic to oligotrophic for the sampling period, with maximum and minimum chemical and physical values according the Table 1 (Nardelli et al. 2016).
Monthly collections were performed between September 2010 and September 2011, from the subsurface water of the river with a phytoplankton net.Samples were cleaned according to the Simonsen (1974) method modified by Moreira-Filho & Valente-Moreira (1981).Permanent slides were mounted with Naphrax  (RI = 1.73) and analyzed with an Olympus  BX60 microscope.Images were obtained with an Olympus DP71 digital camera.The slides are deposited in the herbarium of the Universidade Estadual do Oeste do Paraná (UNOP-Algae, Western Paraná State University), Cascavel campus (Table 2).
Part of the oxidized material was placed on aluminum stubs and sputter-coated with gold in a Balzers SCD 030 at 1 kV for 5 min.Scanning electron microscopy (SEM) analysis was performed in a JEOL JSM 6360LV microscope operating at 15 kV and 8 mm working distance.The terminology used in the species descriptions follows Round et al. (1990) and Houk & Klee (2007).
LM observations.Cylindrical frustules, with thick wall of uneven structure forming two layers, the external view cylindrical and the internal view more or less elliptical.Valve mantle formed with a reticulated pattern in polygonal chambers, decreasing toward collar.Circular valve view, reticulated striation pattern, composed of polygonal areolae decreasing in diameter toward the center, changing to radial rows of small pattern, scored lengthwise, hyaline central area.Cells solitary or joined by the valve face, forming pairs or short chains of three cells.Mantle height: 14.3-15.5 µm; diameter: 15.7-27.9µm; 19-21 striae in 10 µm (Figures 1-10).
SEM observations.Flat valve face with short marginal spines.Rimoportulae scattered on the valve face and disposed in a ring around the mantle, not equidistant from each other.Rimoportula with external openings rounded or elongated, 0.5 µm in diameter, and internally sessile.Collar with short pervalvar wrinkles at the valve margin, and often oblique.Connective bands open, ornamented by delicate striae, 9-10 per 1 µm, composed by rounded pores, 10-11 per 1 µm.Prominent ligule and rounded antiligule.Fimbriate valvocopula and pleura thinner than the other bands (Figures 11-18).Comments.This is the first ultrastructure documentation of a brazilian population of Melosira muscigena.Analysis via SEM revealed unprecedented details of the ultrastructure, such as the shape and distribution of the rimoportulae at the valve mantle.The species was recorded by Koide (1987), as M. ruttneri, by Houk & Klee (2007) and Nardelli et al. (2014).Koide (1987) compared individuals identified as M. ruttneri from Japan (pl. 1, figures 1-7; diameter: 7-33 µm; mantle height: 5-25 µm) with the type material illustrations present by Iwahashi (1936) of M. muscigena (diameter: 16-33 µm; mantle height: 12-20 µm) and verified that it was the same taxon.The only difference found in the Hustedt (1937) and Iwahashi (1936) descriptions was regarding the presence or absence of thin spines on the rim of the valve face.The first describes the spines as not being well distinguished, and the latter makes no mention of them.As the M. muscigena name is older, it has priority over M. ruttneri.
We suggested the investigation of Melosira muscigena and Melosira ruttneri type materials since they were originally described based on optically limited characteristics.Also the original illustrations are uninformative.The two taxa are ambiguous, showing many similarities in their morphology.The frustule ultrastructure would properly ascertain other morphologically diagnostic features between the species.
Melosira muscigena is morphologically similar to M. anastomosans Grunow in the reticulate pattern of the valve face but differs in the mantle outline, which is rounded and more similar to M. undulata (Houk & Klee 2007).
Houk & Klee (2007) describe M. ruttneri as rare in waterfalls, springs, and streams.It is frequently found in moss, and also a good indicator of alkaline waters (Koide 1987).However, in the present study, the species was found in the Iguaçu River, a place of flow and large water falls.
LM observations.Cylindrical frustules (Figure 19), usually solitary or united in pairs or short chains, valves wider than higher, internally Comments.Krammer & Lange-Bertalot (1991) describe M. undulata var.undulata as presenting a rounded valve, with internal margin the of valve face with polygonal circumference different from the internal rounded shape of M. undulata var.normanii Arnott. Ferguson Wood et al. (1959) described individuals with polygonal morphology with six to twelve internal projections, however they did not illustrate specimens with these characteristics.Such internal projections are consistent with the sessile rimoportulae present in the valve mantle.Rimoportulae may also be found in the central region of the valve mantle (Bahls 2012).
LM observations.Cylindrical frustules, united in pairs or short chains.Rounded valve face with rounded internal wall, ornamented with areolate striae in radial rows, sometimes ramified and with a spiral arrangement in the central region.Diameter: 42.3-89.4µm; 10-11 striae in 10 µm on the valve face; 12-14 areolae in 10 µm on the valve face .
Comments.Melosira undulata var.normanii differs from the typical variety by the spiral arrangement of central striae on the valve face as also the different internal margin the valve face described above (Krammer & Lange-Bertalot 1991, Brassac et al. 1999).Our study also found a smaller number of areolae (12-14 in 10 µm) in the var.normanii compared to the typical variety (17-18 in 10 µm).
Krammer & Lange-Bertalot (1991) comment that the species is taxonomically closer to the genus Orthoseira Thwaites due the structure of valve surface, requiring a more detailed taxonomic study.However, Garcia (2009) did a taxonomic comparison of Melosira undulata var.normanii with the genera Orthoseira, Paralia Heiberg, Ellerbeckia Crawford, Podosira Ehrenberg, and Hyalodiscus Ehrenberg and concluded that the species features are in accordance with the genus Melosira.
Comments.Melosira varians occurs mainly in alkaline conditions (pH 7-8.5), in oligotrophic to heterotrophic environments, with moderate oxygen, and requires periodically high levels of nitrogen.Occurs mainly in water bodies, but it is also regularly found in humid environments (Soltanpour-Gargari et al. 2011).
Melosira varians is a very common species in freshwater, occurring in considerable abundance in streams and lakes, naturally eutrophic to polluted, throughout North America (Stoermer & Julius 2003).It is also very common in Brazilian waters, with more than 30 records from the Paraná (Tremarin et al. 2009) as opposed to M. undulata and M. muscigena Iwahashi, which have few taxonomic records around the world.

Conclusion
This is an important study about the knowledge of Melosira diversity in Brazil and taxonomy of the genus as a whole.Melosira undulata var.normanii and M. mucigena are taxa with restricted distribution, which have few taxonomic records around the world.We did not find enough individuals in the samples to analyze in electron microscopy the taxon M. undulata var.normanni as also the typical variety, for highlighting the differences between both.There is a need for new studies under scanning electron microscopy, with a larger number of individuals, in order to discuss details of the ultrastructure.