Fragilaria rinoi sp. nov. (Fragilariales, Fragilariophyceae) from periphytic river samples in Central Portugal

A new benthic freshwater diatom, Fragilaria rinoi Almeida & C.Delgado sp. nov., is described from river periphyton samples in Portugal. Fragilaria rinoi sp. nov. is illustrated and discussed based on populations collected from the Vouga, Mondego and Lis river basins in central Portugal and compared with the type material of Fragilaria vaucheriae (Kutz.) J.B.Petersen. The morphological features of the new diatom species are documented through light and scanning electron micrographs, including a comparative analysis with related species of the genus ( F. candidagilae Almeida, C.Delgado, Novais & S.Blanco, F. intermedia Grunow in Van Heurck, F. neointermedia Tuji & D.M.Williams, F. recapitellata Lange-Bert. & Metzeltin, F. perminuta (Grunow) Lange-Bert., F. vaucheriae and F. microvaucheriae C.E.Wetzel & Ector). Fragilaria rinoi sp. nov. is characterized by solitary cells without spines, lanceolate valves with slightly rostrate apices, a narrow, linear axial area, and a large, unilateral central area. Fragilaria rinoi sp. nov. may be confused with F. microvaucheriae in terms of length, striae density and outline, although a morphometric analysis revealed that F. rinoi sp. nov. is significantly wider. Fragilaria rinoi sp. nov. is present in rivers with high dissolved oxygen concentrations, medium to high conductivity, neutral to slightly alkaline pH and high mean values of nitrates and ammonium.


Introduction
Diatoms are very important and well represented in all freshwater ecosystems, being among the four biological quality elements required in the Water Framework Directive (WFD, The European Parliament and European Council 2000) for freshwater biomonitoring, and are known to be sensitive to a wide variety of pressures (Kelly & Whitton 1995;Almeida & Gil 2001;Nunes et al. 2003). In the years 2011 and 2012, as part of a monitoring program in the rivers of Central Portugal, the epilithic diatom flora was sampled in the Vouga, Mondego and Lis river basins. During this project, identification problems were encountered within Fragilaria species.
During the last decades, a number of taxonomic studies have focused on Fragilaria taxa, this genus being the object of several reviews (Poulin et al. 1986;Williams & Round 1986, 1987. As a consequence, the taxonomy and systematics of Fragilariaceae has changed greatly over the years. Different authors have been using contrasting characters in order to delimitate species within this genus (Ognjanova-Rumenova et al. 1994;Morales 2003), although the systematic position of certain related genera is still not well resolved (e.g., Round 1984;Williams 2006).
The detailed study of the type material of Fragilaria sheds light on this complex genus, with few distinct structures. In this regard, light and scanning electron microscopy studies are very useful, together with the careful examination of the environmental characteristics of the sites. Therefore, the aim of this work is to describe Fragilaria rinoi Almeida & C.Delgado sp. nov., based on its ecology and detailed morphological features using light and scanning electron microscopy.

Study area and sampling
The study area located in Central Portugal is a region with a high economic value, but where anthropogenic activities have caused strong environmental pressures (Feio et al. 2007). The 51 samples analysed in this study were collected in 26 watercourses from three adjacent river catchments (total area of 11 215 km 2 ) in Central Portugal: Mondego, Vouga, and Lis river basins (Fig. 1). Water samples were collected in spring and autumn during the years 2011 and 2012. Environmental parameters such as water temperature (°C), pH, dissolved oxygen (mg.l -1 ), oxygen saturation (% O 2 ) and electric conductivity (µS.cm -1 ) were measured in situ with a Multiparameter Probe 3430 WTW portable meter. Water samples were collected into polypropylene bottles, stored at 4°C in darkness, and transported to the laboratory for analysis of the nutrients and cations according to conventional methods (APHA 1995).
Epilithic biofilms were collected simultaneously with the water samples. The sampling design, treatment procedure and study of diatom communities were based on European standards (CEN TC230 N68 2003), and consisted in removing epilithic biofilm from stones, by scraping their upper surface with a toothbrush. An aliquot of each sample (ca 3 ml), preserved in 4% formalin, was washed with distilled water and treated with HNO 3 (65%) and potassium dichromate (K 2 Cr 2 O 7 ) at room temperature for 24 hours. At least three centrifugations (1500 rpm) took place followed by rinsing with distilled water to remove oxidation by-products, and permanent slides were mounted with Naphrax®.

Type material
For comparative purposes, the type material of similar Fragilaria taxa was analysed. The type material of F. vaucheriae, deposited in the Natural History Museum (London) (Fig. 2) was observed and photographed under a Zeiss Ultra Plus VP FEG SEM operated at 7 kV and 20 mm distance. The dried sample was coated with a gold-palladium mixture before SEM observations.

Portuguese samples
Diatoms were observed under the light microscope Leitz Biomed 20 EB equipped with an immersion objective of 100× and NA 1.32. Light micrographs were taken using an Olympus DP70 camera attached to a Zeiss Axioplan 2 imaging microscope with differential interference contrast (DIC) and a 100× immersion objective (NA 1.40). Measurements of valve length, width and number of striae/10 µm were taken of at least 30 specimens per species, under the light microscope. A Hitachi SU-70 electron microscope operated at 7 kV and 10 mm distance was used for image acquisition of organic free samples, which were air-dried on a metal stub coated with a thin pellicle of graphite (EMITECH K 950X). The dried sample was finally coated with a gold-palladium mixture (Polaron equipment limited SEM coating unit E5000) before SEM observations. Ultrastructural analysis of areolae types, number and placement of rimoportulae, girdle bands, and apical pore fields were checked in SEM micrographs. Plates containing LM and SEM pictures were mounted using CorelDraw X5®.

Valve shape analysis
Geometric morphometry was used to assess differences between taxa and/or populations based on valve shape. To perform this analysis, the valve outline was captured as a geometric configuration of 400 non-homologous pseudolandmarks per individual using CLIC software (Dujardin et al. 2010), directly digitized from the LM micrographs. Illustrations of the type material of comparable taxa available in the literature were also digitized and used in this study: 1) Photographs and measurements of In total, 246 images were processed. The Cartesian coordinates of the pseudolandmarks were aligned (translated, rotated and scaled) by the Procrustes generalized orthogonal least-squared superimposition procedure (Rohlf & Slice 1990). Size, rotation and scale were additionally standardized by Bookstein's transformation, forcing the two first pseudolandmarks onto the coordinates (0.0) and (1.0). A non-metric multidimensional scaling (NMDS) was carried out on the resulting normalized coordinates by means of the software PAST version 2.17 (Hammer et al. 2001), using Spearman's rank correlation coefficient (ρ) as the similarity measure. To visualize the size and shape of the scatter plot for each predefined group, the resulting groups were fitted to 95% confidence ellipses. Finally, a one-way non-parametric multivariate analysis of variance test (NPMANOVA, Anderson 2001) was performed between the transformed coordinates in order to test for significant differences between the a priori defined groups.
Main morphometric parameters (valve length and width, striae and areolae density) were measured directly on LM/SEM micrographs. The type material of comparable taxa illustrated in the literature were also measured and compared to the parameters obtained in the populations here described by means of a NPMANOVA (ρ distance metric), in order to test the null hypothesis of no differences between the morphometry of the populations under study and that of the types of similar species.

Etymology
The new species is dedicated to Prof. Jorge Rino (Aveiro, Portugal) who carried out valuable research on the ecology and biology of freshwater algae during the second half of the twentieth century. His passionate and unique way of teaching encouraged students to pursue this study area.

Type material
Holotype Slide BM 101 794, prepared with material from the sample collected in Mogofores, housed in the Natural History Museum, London (UK), illustrated in Figs 3-62. The valve representing the holotype is illustrated in Fig. 9.

Isotype
Slide ZU10/14, prepared from the sample Mogofores, housed at the Friedrich Hustedt Diatom Collection, Alfred-Wegener-Institut für Polar-und Meeresforschung, Bremerhaven, Germany (BRM).  (Figs 3-77). Fragilaria rinoi sp. nov. is characterized by the presence of solitary cells with lanceolate valves and slightly rostrate apices in larger specimens to rhombic lanceolate in smaller specimens. Frustules rectangular in girdle view with interruption of striation in the middle portion. Axial

Associated diatoms
The assemblage of the type locality ( The type material of Fragilaria vaucheriae (Kützing 1833b) was published in Kützing's exiccata set Algarum Aquae Dulcis Germanicarum (Decas III, No. 24) without either description or figure. This material was observed by Lange-Bertalot (1980, pl. 4, figs 82-94, 97-102) with a broader concept that was later narrowed in Krammer & Lange-Bertalot (2004: pl. 108, figs 10-15). In the present study, the type material from the Natural History Museum of London was photographed with a scanning electron microscope (Figs 83-90). Micrographs from the recently published paper by Wetzel & Ector (2015), which were acquired from Van Heurck's collection housed at the Botanic Garden Meise, Belgium (BR) were also used for the morphometrical measurements of F. vaucheriae.

Description
Fragilaria vaucheriae is characterized by the presence solitary cells with valves linear and narrow and rostrate to subcapitate ends (Fig. 83). Frustules rectangular in girdle view with interruption of striation in the middle portion (Figs 84-85). Axial area narrow, linear, central area larger than F. rinoi sp. nov., unilateral in all specimens. Striae coarse, uniseriate, parallel to the transapical axis and slightly radiate at the poles (Figs 83, 88). External valve face presents small spines in some specimens (Figs 83, 88). A single rimoportula is present at one pole, aligned with the first stria at the valve face apex (Figs 83,  88). Girdle bands are open (Figs 84, 85, 87), with small, unoccluded perforations. Striae composed of round areolae (12-13 areolae in 1 μm) on both valves (Figs 83, 89). Each valve has two apical pore fields (APF) composed of simple fine porelli arranged in regular rows parallel to the apical axis (Fig.  83) and made up of 6 rows, each composed of 10 to 11 poroids (Figs 85-88). Outer areolar are closed with siliceous depositions (Figs 83-90 Table 2). The shape and morphometry of Fragilaria rinoi sp. nov. is statistically different from the one of F. recapitellata and F. neointermedia. Compared to F. vaucheriae, the species here described is statistically different in all the parameters except in outline (Table 2). Fragilaria rinoi sp. nov. has similarities in length with F. candidagilae and F. perminuta, but is statistically different in width, striae density and outline (p = 0.0015, Table 2). Fragilaria rinoi sp. nov. is only similar to F. capucina in striae density and to F. intermedia in width.
The morphology of F. rinoi sp. nov. overlaps with the one of F. candidagilae in terms of length, but the new species is slightly wider (4.2-5.6 µm vs 4.5-5.0 µm), has denser striation than F. candidagilae (14-16 vs 12-14 striae in 10 µm) and slightly rostrate apices, instead of the typical strongly capitate apices present in F. candidagilae.
The ecology of F. rinoi sp. nov. is different from that of F. microvaucheriae because F. microvaucheriae is present in rivers with low conductivity, never over 230 µS cm -1 and low nutrient concentration (Wetzel & Ector 2015); while F. rinoi sp. nov. is present in waters with medium to high conductivity and high nutrient concentration.

Conclusions
Fragilaria rinoi sp. nov. is a new diatom species that was differentiated from similar species using valve morphology and geometric morphometric comparisons using light and scanning electron microscopy. This study shows that F. rinoi sp. nov. is not abundant in central Portuguese rivers, but can appear in several rivers from adjacent basins. An additional difficulty was the low number of specimens and images used in the literature for the description of new taxa or even for the redefinition of others. Fragilaria rinoi sp. nov. is present in rivers with high dissolved oxygen concentrations, medium to high conductivity, neutral to slightly alkaline pH and high nutrients concentrations. This study contributes to improving the knowledge of the benthic diatom flora in Portugal.