Denticula, Hantzschia and Tryblionella (Bacillariaceae, Bacillariophyta) from northwestern Ceará freshwaters

Abstract Diatoms constitute a representative portion of the algal community of freshwater aquatic environments, but their studies are centered mostly in the south and southern regions of Brazil, making the microalgae biodiversity of water bodies in the semiarid practically unknown. In the state of Ceará, the few records of the Bacillariaceae are centered in the southern region of the state and are generally not identified at specific level. Denticula, Hantzschia, and Tryblionella are worldwide distributed diatom genera that belong to the family Bacillariaceae. The group is characterized by raphe located on a channel, internally supported by silica extensions, the fibulae. This is a taxonomic study on the genera Denticula, Hantzschia, and Tryblionella found in water bodies in northwestern Ceará state. Planktonic and periphytic samples were collected in four hydrographic basins, which are influenced by the semiarid climate, located in northwestern Ceará, northeastern Brazil. Fourteen infrageneric taxa were identified, described, and illustrated, all being pioneer citations for Ceará. The present study highlighted the underestimated diversity of diatoms in the semiarid region of Ceará and the need to cover undersampled or never explored regions to significantly contribute to the knowledge of the diatom flora and its distribution in Brazilian aquatic environments.

Denticula Kutzing was described in 1844.Representatives from this genus have small, solitary, or short-chained frustules with linear or lanceolate valves, obtuse or slightly rostrated ends.The main characteristic is the presence of transapical ribs crossing the valve surface, visible under light microscopy, which are extensions of the fibulae (Ludwig & Tremarin 2006;Taylor & Cocquyt 2016).
Hantzschia was proposed by Grunow, in 1877, to group forms that differed from Nitzschia Hassall by their shape, positioning of the raphe system, and arrangement of fibulae (Round et al. 1990).Hantzschia has a dorsiventral valve, concave ventral margin, and convex dorsal margin, with capitate to rostrated ends, presenting a marginal raphe system and fibulae on the ventral margin of both frustule valves.
Tryblionella was originally proposed by Smith (1853).Round et al. (1990) placed Tryblionella and Nitzschia in the same family, but in different genera.This proposal was not adopted by Krammer & Lange-Bertalot (1997), for example, who adopted the subgeneric status for Tryblionella.Molecular studies confirm the paraphyletic character of the Nitzschia lato sensu (Lundholm et al. 2002a, b) and support Tryblionella as a distinct genus, in a phylogenetic clade close to Nitzschia strictu sensu (Rimet et al. 2011).Tryblionella has elliptical, linear or panduriform valves, wedge-shaped, rounded or sub-rostrated apices, a longitudinally undulating valve face, channeled and eccentric raphe, and transapical ribs parallel to the striae (Taylor & Cocquyt 2016).
The Brazilian catalogs (Tremarin et al. 2009;Torgan et al. 1999;Silva et al. 2011) register 13 species of Hantzschia, six species of Denticula and ten Tryblionella.Recently, Bertolli et al. (2020) updated to 23 species of Tryblionella registered in Brazil.The majority of this richness was found in the south and southeast Brazil, demonstrating the lack of studies in other regions of the country.
Studies on diatoms from Ceará began with Patrick (1940a, b), recording 52 species.Amorim et al. (2013) and Vieira et al. (2013) centered their studies in the southern region of the state and recorded about 65 taxa of diatoms, generally not identified at a specific level.None of these works, however, register species of Denticula, Hantzschia or Tryblionella.
To contribute to the expansion of the diatomological composition of the state of Ceará, the registration and description of morphological and metric variations of the species of Denticula, Hantzschia, and Tryblionella found in water bodies inserted in the northwest region of the state were carried out.

Material and Methods
The state of Ceará, located in the Northeast region of Brazil, covering an área of 148,886.3 km², and is subdivided into 12 hydrographic basins which encompass rivers, streams, lakes, and dams (IPECE 2016).Regarding to the rainfall in the region, there is a predominance of the rainy season, which occurs for around three to five months, alternating with the dry period, which lasts from seven to nine months (Bastos & Cordeiro 2012).
Approximately 90% of the total area of the state of Ceará has a semi-arid climate.Most water resources are limited and unstable, showing compromising points of pollution caused by industrial and agricultural activity, and from large urban centers (IPECE 2016).
The collections were carried out between January 2016 and May 2019 in 20 locations, including dams, streams, rivers, waterfalls, and spouts inserted in hydrographic basins in northwestern Ceará (Fig. 1; Tab.S1, available on supplementary material <https://doi.org/10.6084/m9.figshare.23650905.v1>).Periphytic samples (scraping rocks, subaerial humid bryophytes, and roots of underwater plants) and planktonic (net with 20μm mesh) were collected and preserved with a 4% v/v formalin solution.Temperature, pH and conductivity values were sampled in the field (from site water, collected with a container), using GHka portable pH meter (Model PG 1400) and conductivity meter (Model CG 1400).
In the laboratory, samples were oxidized using the Simonsen method (1974) changed by Moreira-Filho & Valente-Moreira (1981).Permanent slides were prepared with the oxidized samples and mounted with Naphrax ® resin (i.r.= 1.74).Images were obtained by a DP 071 capture camera coupled to a light microscope (LM), Olympus BX 40.Subsamples of the oxidized material were deposited on aluminum supports, sputtered with gold-palladium in Balser Sputtering/SDC300, and observed in a TESCAN VEGA 3 LMU scanning electron microscope, operated at 15 kV and 8 mm away, at the Electron Microscopy Center (CME/UFPR).

Results and Discussion
Based on the analysis of 20 samples from the northwest region of Ceará, mostly of periphytic origin, fourteen infrageneric taxa were observed.Tryblionella was the most representative genus, with eight taxa, followed by Hantzschia (five taxa) and Denticula (one taxa).
Denticula ranierensis is described with acutely to broadly rounded ends and slightly radiating fibulae (costae) at the apices, in contrast to D. subtilis with very sharp apices and parallel fibulae throughout the valve (Lange-Bertalot & Krammer 1993;Johansen et al. 1994).Furthermore, the density of striae (26-30 in 10 µm) assigned to D. subtilis is much lower than the range reported to D. ranierensis (45-52 in 10 µm) (Lange-Bertalot & Krammer 1993;Johansen et al. 1994).The shape of the apices, the fibulae pattern and the striae density of the population studied here are closer to the circumscription of D. ranierensis.However, as we observed by SEM (Fig. 2i-j), the striae of Ceará population are organized in double rows throughout the valve.In D. ranierensis, as presented in Johansen et al. (1994), the striae in the type material are composed of irregular rows of areolae, showing a distinct striation pattern.Lange-Bertalot & Krammer (1993, figs. 39-43) presented a population from Ulla River (Spain), very similar to the individuals found in the present study, with striae in double rows, identified as D. sundaysensis Archibald.However, D. sundaysensis is originally described by Archibald (1982), as having striae in double rows composed of much larger areolae, consequently, lower striation density (ca.20 in 10 µm).More detailed studies comparing type materials of mentioned species complex are necessary to resolve the precise identification of the taxon described and illustrated here (Fig. 2a-j).Hantzschia abundans is similar to H. amphioxys (Ehrenberg) Grunow due to its valve shape, differing basically in terms of dimensions, striae density, and raphe structure (Zidarova et al. 2010).H. abundans is 40-80 µm long and 7-10 µm wide, with 5-8 fibulae and 15-20 striae and 40 areolae in 10 µm.The dimensions of H. amphioxys are 15-50 µm in length and 5-7 µm in width, in addition to 20-29 striae and 40-50 areolae in 10 µm (Lange-Bertalot 1993).Observations of the frustule in scanning electron microscopy help to delimit the species Hantzschia amphioxys and Hantzschia abundans.The proximal raphe endings in H. amphioxys have an "L" shape directed to opposite sides and in H. abundans the termination is rounded directed to the same side, almost straight (Zidarova et al. 2010;Bulínová et al. 2018).It was not possible to get images of the raphe endings.Although different shapes are still found in literature, the length, valve width and density of striae were decisive to differentiate them.In a recent study (Maltsev et al. 2021), H. abundans was described according to all the main morphological characteristics already documented by Lange-Bertalot (1993), except for some smaller strains (37.5-39 µm and 6-7 µm).Also, the molecular data of H. abundans studied strains shared 95.61-96.67%similarities with those of H. amphioxys, and, according to Maltsev et al. (2021), H. abundans needs to have a taxonomic revision.
Hantzschia calcifuga differ from H. amphioxys and H. abundans by the most elongated shape, the more concave median region of the ventral margin, and by the apices facing the dorsal side (Lange-Bertalot et al. 2017).
All taxa are first citations to the state of Ceará and Tryblionella was the most representative genus with eight species documented.Analyzing the diatomological studies in the Northeast of Brazil (Gomes et al. 2003;Santiago-Hussein & Oliveira, 2006;Sá et al. 2018;Aquino et al. 2015;Ferrari et al. 2014;Cavalcante et al. 2014;Dantas 2010;Dantas et al. 2008;Souza et al. 2007;Azevedo 1999), we noticed the lack of registry entries of the documented taxa for the region.Only three had been previously cataloged: Tryblionella hungarica and T. granulata, registered in the archipelago of Fernando de Noronha, state of Pernambuco (Costa et al. 2009), and T. persuadens, registered in the state of Bahia (Cavalcante et al. 2013).Hantzschia calcifuga and Tryblionella balatonis were registered for the first time in Brazil.Denticula aff ranierensis needs more detailed studies comparing type The caatinga is one of the most extensive semi-arid regions in the world.Unfortunately, in Brazil, this important environment is suffering anthropic impacts with widespread devastation and compromise of water resources.We highlight the importance of carrying out inventories of species, detailed taxonomic studies, fundamental information in environmental assessments and in the knowledge of biodiversity in regions that are still little or not explored, spread across this huge country.

Figure 2
Figure 2 -a-j.Denticula ranierensis -a-h.valves in LM; i-j.valves in SEM -i.external view of the whole valve, transapical fibulae with thickenings near the valve margins and raphe canal open to the valve interior by elliptical portulae; j. internal view of the whole valve showing striation pattern and raphe endings.Bars: a-h = 10 µm; i-j = 2 µm.