Taxonomic study and local environmental conditions of occurrence of Chlorophyceae (Chlorophyta) from subtropical lotic environments, Paraná, Brazil

Abstract Lotic environments are subjected to the impacts of human activities in an intense way in urban regions and one of the ways to assist in the environmental diagnosis is through the knowledge of the composition of bioindicator organisms, including microalgae. The objective of this work was to qualitatively characterize the Chlorophyceae Class Wille, providing descriptions and meristic data of the specimens as well as the environmental conditions in which the taxa were recorded. Water and phytoplankton samplings were carried out quarterly in 2020, in nine supply rivers, distributed in three river basins in the western region of Paraná (Paraná III basin, Piquiri basin and Baixo Iguaçu basin). The studied rivers were classified as oligotrophic or mesotrophic and the taxa were mostly rare. Thirty-six taxa belonging to the Chlorophyceae class were recorded, distributed in five families: Hydrodictyaceae Dumortier, Neochloridaceae Ettl & Komárek, Radiococcaceae Fott ex P.C.Silva, Scenedesmaceae Oltmanns, Selenastraceae Blackman & Tansley. These taxa have mainly cenobial representatives, with about 70% of the individuals in this thallus configuration, followed by 22% colonies and 8% unicellular thallus. Among the identified species, five occurred only in mesotrophic sites, warning for environments with tendencies to elevate their trophic, since they are genera previously associated with these conditions. Ten new citations were recorded for the State of Paraná, namely: Pseudopediastrum boryanum var. longicorne (Reinsch) P.M.Tsarenko, Radiococcus skujae I.Kostikov, T.Darienko, A.Lukesová & L.Hoffmann, Desmodesmus perforatus (Lemmermann) E.Hegewald, Desmodesmus subspicatus (Chodat) E.Hegewald & A.W.F.Schmidt, Scenedesmus indicus Philipose ex Hegewald, Engelberg & Paschma, Ankistrodesmus bernardii Komárek, Monoraphidium capricornutum (Printz) Nygaard, Monoraphidium caribeum Hindák, Raphidocelis danubiana var. elegans (Playfair) Taşkin & Alp, Selenastrum rinoi Komárek & Comas. Taxonomic studies, such as this one, are an important tool for understanding the flora, and in addition to contributing to the registration of species in aquatic ecosystems, they serve as a basis for ecological studies and other approaches used to preserve biodiversity in these places.


Introduction
The Chlorophyceae class is part of the "UTC clade" (Ulvophyceae, Trebouxiophyceae and Chlorophyceae) within the Chlorophyta division, and stands out for having its undeniable monophyly, supported by molecular and ultrastructural data (Fučíková et al. 2019).Owing to the high number of species, it is considered one of the most abundant and diverse group in Brazilian continental waters (Rodrigues et al. 2010), grouping around 563 genera and 3.797species (Guiry & Guiry 2023).The morphology of these organisms ranges from flagellated unicellular to unicellular devoid of locomotion organelles, motile or non-motile colonies, filaments and pseudoparenchyma structures (Wehr et al. 2015).The species of the Class Chlorophyceae present a wide morphometric and ecophysiological variability, being able to develop in different habitats, being influenced especially by the light exposure, availability of reactive soluble phosphorus and mixing of the water column (Happey-Wood 1988).
Increasingly, lotic environments are degraded due to urbanization and intense anthropic activities (Li et al. 2022).In this context the water for public supply is deteriorated, besides all aquatic biota that suffer the consequences of inadequate management of these environments (Peres et al. 2022).In this sense the taxonomic composition of the Class Chlorophyceae is an important tool to assist in environmental diagnosis, since the species are often associated with environments with organic pollution and potential eutrophication (Wijeyaratne & Nanayakkara 2020).The literature for the State of Paraná, mainly studies in lotic environments, is scarce when compared to lentic environments, in addition to all the richness of species that have not yet been described.Among the main works referring to the Chlorophyceae Class in rivers, we can mention: Oliveira et al. (1994) with 46 taxa distributed in 25 genera in the Paraná River; Bittencourt- Oliveira (1997) with 24 taxa distributed in the Chlorococcales, Oedogoniales and Volvocales orders in the Tibagi River; Medri et al. (2002) with the flora of the Tibagi River; Biolo et al. (2009) with the identification of 21 taxa distributed in six families in the São Francisco Falso River.
Among the most recent works we can still cite: Bortolini et al. (2010) with 28 taxa distributed in the families Hydrodictyaceae, Oocystaceae and Scenedesmaceae in the São João River; Aquino et al. (2014) with thirty taxa, distributed in six families and 16 genera in the Cascavel River; Medeiros et al. (2021) recorded 26 taxa in a subtropical river in the State of Paraná and Aquino et al. (2022) with a book chapter that synthesizes the taxa of green microalgae described in the works for western Paraná.
Thus, our study aimed to carry out a taxonomic survey of phytoplanktonic chlorophyceans in lotic environments with different physical and chemical conditions of the water of the western region of Paraná; Provide descriptions, illustrations, morphometric and meristic data of the species found along the environments; Contribute to the registration of species found on the UNOPA (herbarium of UNIOESTE -Universidade Estadual do Oeste do Paraná) species Link platform.

Selection, location and characterization of study sites
We selected nine rivers used to capture water for public supply in the western region of Paraná, along the Lower Iguaçu River, Paraná III and Piquiri river basins (Table 1), which were distributed in nine municipalities: Guaraniaçu, Catanduvas, Três Barras do Paraná, Boa Vista Aparecida, Foz do Iguaçu, Medianeira, Santa Tereza do Oeste, Cascavel and Toledo (Figure 1).
The water sampling for physicochemical and biological analyses was performed in two sites in each river.These sites are similar in terms of flow, riparian vegetation and human influence, however, they were descrições e dados merísticos dos espécimes bem como as condições ambientais em que os táxons foram registrados.Foram realizadas amostragens de água e de fitoplâncton trimestralmente no ano de 2020, em nove rios de abastecimento, distribuídos em três bacias hidrográficas da região oeste do Paraná (bacia do Paraná III, bacia do Piquiri e bacia do Baixo Iguaçu).Os rios estudados foram enquadrados como oligotróficos ou mesotróficos e os táxons apresentaram em sua maioria ocorrência rara.selected taking into consideration the characteristic of lotic systems, where the water flow carries dissolved materials including pollutants, making a comparison between the two possible (Vannote et al. 1980).Sampling was carried out during the year 2020 in all four seasons.All samples were deposited in the herbarium of UNIOESTE -Universidade Estadual do Oeste do Paraná -UNOPA, Campus Cascavel, connected to the Brazilian Network of Herbaria and the data were computerized and made available on speciesLink (www.splink.cria.org.br).

Sampling and analysis of environmental variables in rivers
Data were obtained on water temperature (Temp -°C), dissolved oxygen (DO -mg L -¹), pH, electrical conductivity (Condut -mS/cm -¹) and turbidity (Turb -NTU), measured at the moment of the samples through the multiparameter probe Horiba U-5000.The data referring to the flow (m³ s) and maximum depth were collected with the aid of a ruler, measuring tape and a floating object, considering the multiplication between the average speed resulting from the displacement of the object and the cross-sectional area at the site to calculate the flow, measured in situ.
For chemical analysis, water samples were collected by subsurface immersion of polyethylene bottles, being kept properly refrigerated and in the dark until their destination.Estimates of concentrations of nitrate (NO 3 -mg L -¹), ammonia nitrogen (N-NH3 -mg L -¹), total phosphorus (TP-mg L -¹), chlorophyll a (CLa -mg L -1 ), were performed following the standardized methods in Standard Methods (APHA 2017).

Phytoplankton community
For the qualitative analysis, phytoplankton samples were obtained using a 25 µm mesh size plankton, were preserved in Transeau solution (Bicudo & Menezes 2017) in order to concentrate the phytoplanktonic material.The qualitative study of phytoplankton was carried out using an Olympus CX41 photomicroscope, coupled with an Olympus SC30 camera, and the morphometry of the taxa was performed at 40× and 100× magnification.The taxa were identified based on specialized literature, classification used follows Krienitz & Bock (2012).In order to verify the occurrence of taxa in the Paraná State, we considered only taxonomic studies with description, measures and/or illustrations, in lotic and lentic environments.The measurements (in μm) are represented by Llength; W -width; T -thorn; D -diameter; De -dent; Co -coenobium; P -process.Constancy is a measure of species occurrence (C) and was expressed as follows: constant (C ≥ 70%), common (30% ≥ C ≤ 70%), sporadic (10% ≥ C ≤ 30%) and rare (C ≤ 10%) (Dajoz 2005).

Results
Were  2: A Flat coenobium, with circular to oval shape; formed by 16 cells, clathrated; marginal cells in an asymmetrical "H" shape, concave base, two slender, long processes of equal length, ending in a slightly rectused papilla, deeply excavated "U" incision; inner cells similar to outer ones; chloroplast with the shape of the cell, one central pyrenoid.Morphometric data: Co = 32.5-62.5 μm; L = 12.5-15.0μm; W = 5.0-10.0μm.Paraná State citation: Aquino et al. (2014)    Taxonomic remarks: the lineage of Pseudopediastrum boryanum (Turpin) E.Hegewald is divided into varieties through a set of morphological characters, such as the number of coenobium cells, the size and shape of the marginal cells, the absence of perforations between the cells and the density of the granules from the surface (Lenarczyk & Saluga 2018).The specimens of this work were identified as  Taxonomic remarks: in the species proposition, some specimens from the sampling had been identified as Pediastrum tetras, however, Buchheim et al. (2005) proposed the transference of Pediastrum tetras to the genus Stauridium Corda from molecular analyses.Despite being a well-defined species, it can present considerable morphological variation (Ramos et al. 2016), and therefore the dimensions of the specimens also vary, as verified in Aquino et al. (2022).Paraná State citation: Picelli-Vicentim (1985, 1987), Rodrigues & Train (1993), Oliveira et al. (1994) -Vicentim (1987), Rodrigues & Train (1993), Oliveira et al. (1994), Bittencourt-Oliveira (1997), Train et al. (2001), Borges et al. (2003), Train et al. (2003)  Selenastrum rinoi Komárek & Comas, 272, 276, figure 10, 1982.The trophic state index indicated that seven of the nine sampled municipalities fall into oligotrophic conditions, while the remaining two are considered mesotrophic.Regarding the limnological conditions we observed a variation in the flow of the sampled rivers, where the lowest flow recorded is in Rio Tamanduá (0.09 m³ s) and the highest is in Rio Toledo (1.39 m³ s).Water temperature varied from 15°C to 20°C in Baú and Jacutinga Rivers respectively.The pH value in all sampled rivers was considered neutral (6).The electrical conductivity varied from (0.02 mS cm -1 ) in the Toledo River to (0.08 mS cm -1 ) in the Baú River.As for turbidity, the lowest value recorded was in Rio Baú (6.41 NTU) and the highest in Rio Jacutinga (23.35 NTU).Dissolved oxygen varied from (6.39 mg L -1 ) in Gonçalves Dias River to (14.95 mg L -1 ) in Baú River.The lowest value of ammonia nitrogen (0.01 mg L -1 ) was constant in four rivers, namely Baú, Itaguaçu, Gonçalves Dias, and Jacutinga, while the highest value (0.14 mg L -1 ) was registered in the Cascavel River.The values of total phosphorous were similar in all sampled rivers.Nitrate varied from (0.53 mg L -1 ) at Gonçalves Dias to (1.83 mg L -1 ) at Toledo River (Table 2).All sampled rivers had at least two species recorded, however, approximately 70% of the occurrences were in oligotrophic environments.The rivers with higher occurrences recorded were Cascavel (31%), Passo Liso (23%) and Toledo (17%), the first two being oligotrophic and the third mesotrophic.

Discussion
In our study we recorded 36 taxa of chlorophyceans belonging to the order Sphaeropleales.The Scenedesmaceae family was the most representative with 20 taxa (55%), followed by Selenastraceae with 10 taxa (27.7%) and Hydrodictyaceae with 4 taxa (11%).We also highlight the record of 10 new citations for the State of Paraná, thus expanding the knowledge of the flora of Chlorophyceae for lotic environments: Ankistrodesmus bernardii, Desmodesmus perforatus, Desmodesmus subspicatus, Monoraphidium capricornutum, Monoraphidium caribeum, Pseudopediastrum boryanum var.longicorne, Raphidocelis danubiana var.elegans, Radiococcus skujae, Scenedesmus indicus and Selenastrum rinoi.The class Chorophyceae develops in wide environmental variation, but they are important in oligotrophic aquatic environments and described as cosmopolitan, being bioindicators of water quality (D'Alessandro & Nogueira 2017).
The genus with the highest number of taxa was Desmodesmus with 9 species (25%), followed by Coelastrum with 5 species (13%) and Ankistrodesmus with 4 species (11%).The higher occurrence of Desmodesmus compared to the others may be associated with the fact that this genus is common in most aquatic environments, from eutrophic to oligotrophic (Borges et al. 2008, Hentschke & Torgan 2010, Domingues & Torgan 2012, Rosini et al. 2012, Aquino et al. 2014), which can be observed in the Cascavel (Municipality of Cascavel) and Arroio Passo Liso (Municipality of Catanduvas) rivers, both in the watershed of the lower Iguaçu River, considered oligotrophic where high contributions of the genera Coelastrum sp. and Desmodesmus sp. were observed.
Among the 36 taxa identified in this study, five species occurred only under mesotrophic conditions, namely: Desmodesmus brasiliensis, Desmodesmus subspicatus, Scenedesmus obtusus, Monoraphidium caribeum, and Raphidocelis denubiana var.elegans.The occurrence of Desmodesmus in nutrient-rich environments, as mentioned earlier, is due to it being a genus adaptable to diverse environmental conditions, being common at all trophic levels (Phinyo et al. 2017).The genus Scenedesmus has already been associated with sites with mild to moderate organic pollution, warning for environments with tendencies to elevate their trophy (Sabkie et al. 2020).We can also mention Monoraphidium caribeum, which, although cosmopolitan, is found mostly in eutrophic aquatic environments.The other occurrences were in oligo to mesotrophic conditions, which is in accordance with what is expected for the group (Comas 1996).
The Cascavel River showed the highest species richness (21 taxa).This environment was related to one of the lowest values of turbidity (8.29 NTU) in the area sampled.The Jacutinga River had the highest value for this variable (23.35 NTU), represented by only two species.The correlation of turbidity with Chlorophyceae representatives has been reported previously, where this variable negatively influenced the diversity of the group (Gogoi et al. 2019;Kumar et al. 2020).This relationship is due to the limitation of light penetration into the water, which reduces phytoplankton photosynthesis, in addition to altering the other limnological conditions (Nunes et al. 2022).
In our study, 94% of the taxa found had a rare frequency of occurrence, and only 6% were classified as sporadic.The rarity of the species in lotic environments may be related to the local hydrodynamics, where the greater flow of water prevents the establishment and development of potamoplankton, promoting a constant transport of taxa downstream (Medeiros et al. 2020).Phytoplankton is considered a group of key organisms to indicate changes in aquatic environments due to changes in the structure of biota, in response to physical and chemical factors in water bodies (Wojciechowski et al. 2017).Since 2007, 91 taxa have been recorded for the Chlorophyceae class in the State of Paraná, and according to the flora of Brazil (2020), 362 species were recorded throughout the country, with publications in São Paulo and Rio de Janeiro predominating, thus showing the need for further studies in Paraná.In conclusion, we can emphasize the importance of taxonomic studies, which serve as a basis for knowledge of biodiversity, providing essential information for ecological, bioindicator and conservation approaches.

Table 1 .
Herbarium sample number (UNOPA), weather station, geographic coordinates, river and watershed of the water samples for microalgae analysis.
Kostikov et al. (2002)the specialized literature, some authors identified R. skujae as Thorakochloris nygaardii Komárek, however this taxon was transferred to the genus Hindakochloris nygaardii (Komárek) Comas.Kostikov et al. (2002)considered this genus as a synonym of R. skujae, due to the cell shape and reproductive behavior, with Radiococcus being the most used nowadays.
Tucci et al. (2019)wever, currently the current name is Desmodesmus subspicatus as seen inTucci et al. (2019).Scenedesmus indicus is easily differentiated from the other species due to the morphology of the coenobium, in which the cells are arranged in an alternating manner.

Paraná State citation: first record. Taxonomic remarks: recently
Ramos et al. (2012))imiiTeiling, Svensk Bot.Tidskr., 10 (1):59-66, 1916.Figure4: K Coenobium formed by up to four cells; two are in the same plane and two are arranged vertically, joined by the poles; lunate or approximately reniform cells with rounded apices; parietal chloroplast with one pyrenoid.Ankistrodesmus bernardii Komárek can be confused with Ankistrodesmus densus Korshikov, as both form colonies with many cells that are variable in length but differ in that A. densus has curved or sigmoid cells throughout.Taxonomic remarks: the specimens in this work have larger dimensions than those recorded inAquino et al. (2022), however they are in agreement with the specimens observed inRamos et al. (2012).M. caribeum can be confused with Ankistrodesmus arcuatus Korshikov, however the species differ by the size of the cells, where A. arcuatus has larger dimensions.the genus Kirchneriella contorta var.elegans (Playfair) Komárek changed to the taxon Raphidocelis danubiana var.elegans, which can be found in the literature in the first way.