Composition, frequency and seasonal distribution of periphytic microalgae in stretches of the Curiaú River, Amapá, Eastern Amazon

Periphyton communities are composed of microorganisms loosely attached to the substrate, including fungi, bacteria, microinvertebrates, and mostly microalgae. Due to their photosynthesizing component, they are important primary producers that subsidize the aquatic trophic chain. This study aimed to verify the composition, frequency, and seasonal distribution of periphytic microalgae in stretches of the Curiaú River, located in the Environmental Protection Area (APA) of the Curiaú River, in the State of Amapá. For this, quarterly collections were carried out comprising the months of June 2016 to October 2017, during the dry and rainy seasons. The periphyton algal communities were obtained by squeezing parts of two macrophytes species, Eichhornia crassipes Mart. (Solms) and Pistia stratiotes L. The data were statistically treated using absolute values and the Kruskal-Wallis test, the latter considering the distribution of richness between sites and sampling periods (significance level of α=0.05). A total of 64 taxa were inventoried, aggregated into three taxonomic divisions: Chlorophyta (n=48; 75%), Cyanophyta (n=12; 18.75%) and Heterokontophyta (n=4; 6.25%). In class terms, Zygnematophyceae had the highest number of taxa (n=42; 65.63%), followed by Cyanophyceae (n=12; 18.75%), Chlorophyceae (n=5; 7.81%), Bacillariophyceae (n=4; 6.25%) and, finally, Ulvophyceae (n=1; 1.56%). Of the total number of inventoried taxa, 64.06% (n=41) presented sporadic frequency. Species richness was influenced by seasonality, as higher values were associated with the rainy season (p<0.05). This research is a contribution to the phycology of the Amazon region, especially in its pioneering for the state of Amapá.


INTRODUCTION
Microalgae are unicellular or multicellular microorganisms, constituents of the communities of primary production of the aquatic ecosystem, that convert sunlight into chemical energy through photosynthesis [1,2]. Among the microalgae groups commonly found in freshwater environments, we report Bacillariophyta, Chlorophyta, Chryptophyta, Chrysophyta, Cyanophyta, Dinophyta, and Euglenopyta. These organisms can be found dispersed in the water column and in the form of periphyton [3,4].
Periphyton term was first defined in 1983 at the 1st International Workshop on community adhered which living in aquatic ecosystems. Its definition includes microorganisms, such as microalgae, fungi, bacteria, and animals that adhered to organic or inorganic debris [5,6].
Microalgae are the main organisms that compose the periphyton and have a great influence on the nutritional level of associated ecosystems [5,7,8]. Periphyton studies are less developed compared to phytoplankton and only from the 1980s began to be performed more comprehensively [9].
In the periphytic community, we can find a great diversity of life forms and groups of algae. For example, talophytes can range from single-celled, colonial, pseudofilamentous, unbranched simple filamentous organisms, branched filamentous, filamentous with false branching, syphonaceous or cenocytic, to pseudoparenchytic forms [10]. Among the groups of algae found in the periphytic community, we have Cyanophyceae, Chlorophyceae, Oedogoniophyceae, Zygnematophyceae, and, for the most part, Bacillariophyceae [11][12][13].
The periphyton is an important group of organisms that compose the base of the food chain in the aquatic ecosystem. They are a source of proteins, vitamins, and minerals [14] for zooplankton, insects, and fishes that feed on them [15]. Besides that, they are recognized as a bioindicator of water quality and trophic state, since they have a high capacity to accumulate nutrients from pollutants (i.e., insecticides) in water bodies [7,12].
The aquatic ecosystem studied (Curiaú river) is at the Environmental Protection Area (APA) of the Curiaú River. This APA is composed of several environments rich in fauna, flora, and microbiota. Studies related to the inventory of this biodiversity are necessary to support the knowledge of existing species in the area and enable the development of environmental management policies for the monitoring of natural resources in legally protected areas.
In addition, the APA of the Curiaú River is located near the urban expansion area of the municipality of Macapá, the capital of the Amapá state, suffering pressure on its population and ecosystems. This pressure often is due to the disordered use of natural resources, resulting in short, medium, and long-term impacts [16].
Thus, this research aims to perform a taxonomic survey of the periphytic community in stretches of the Curiaú River (Macapá-AP), evaluating its frequency and seasonal distribution in an annual cycle.

Study area
The Curiaú River APA is situated 8 km 2 north of the city of Macapá, in the State of Amapá, Brazil, with a total of 216.76 km 2 , with demarcated boundaries between the Campina Grande community (north), BR-156 Highway (west), Macapá city (south) and the Amazon River (east) [16,17]. The local scenery presents numerous landscapes, with a variety of fauna, flora, and associated microbiota [18] The Curiaú river basin ( Figure 1) is interconnected to permanent and temporary lakes influenced by tidal regimes, characteristic of the Amazon region. It presents two distinct seasonal periods: a) dry, comprising the months of July to December; b) rainy, comprising the months of January to June [19]. It is mainly used for recreation, plantation irrigation, artisanal fishing, and river routes [18]. Studies indicate that water from the Curiaú River presents acid pH, low electrical conductivity, and dissolved oxygen concentration between 4 and 7 mg/L, with transparency of ± 100 cm [20,21]

Collection and sampling of periphyton
The collection of microalgae from the periphyton was carried out in quarterly campaigns, between the months of June 2016 to October 2017, totaling six collections (six replicates per sampling site) and considering the following seasonal periods: D (dry); D-R (dry-rainy); R (rainy); R-D (rainy-dry). The sampling sites (n=3), located in the Curiaú River basin between the geographic coordinates 0°08'13.69 "N; 51°02'53.35 "W and 0°08'55.86 "N; 51°02'23.55 "W (see Figure 1; Table 1), were defined due to their access logistics and presence of aquatic macrophyte banks.  The periphyton algal communities were obtained by squeezing parts of two macrophytes species, Eichhornia crassipes Mart. (Solms) and Pistia stratiotes L. The collected material was fixed and preserved with Transeau's solution, according to the usual methodology [22].

Taxonomic identification and frequency
The taxonomic identification of periphyton was carried out by optical microscopy at the Environmental Sanitation laboratory of the Federal University of Amapá, with the aid of specialized bibliography and identification keys [22][23][24][25][26].
The frequency (%) was determined according to Matteucci and Colma (1981) [27]. In this study, the result was obtained using the following formula: (n*100)/N, where n= number of samples in which the taxa occurred and N= the total number of samples (space-time) analyzed in the study period. In the end, the taxa were classified as quite common (≥70%), relatively common (<70% and ≥40%), relatively uncommon (<40% and ≥10%), and sporadic (<10%).

Statistical analysis
Data were tabulated in an Excel spreadsheet for the generation of statistical analyses through the R-statistics Software [28]. Data (raw and transformed) was submitted to Shapiro-Wilk and Levene-Bartlett normality tests to assess homogeneity and homoscedasticity, respectively. As the data were not normal, we used the non-parametric Kruskal-Wallis test (α=0.05) to assess the spatial and seasonal variation in the species richness.

Figure 2: Distribution of taxa by division and taxonomic class, showing frequencies (light bars) and absolute values (grey bars).
Most of the taxa presented a sporadic frequency of occurrence (S) (n=41; 64.06%), characterizing an environment with a local variety of rare species. In turn, 34.48% (n=22) were relatively uncommon (RU) and only one taxon (Spyrogira sp.) was considered relatively common (RC) in the sampling period (n=1; 1.56%) (Figure 3). Regarding the space-seasonal fluctuation, there was no significant variation between the sites of sampling for the richness of periphyton (Figure 4). However, a significant variation was observed between sampling periods (p<0.05), with the highest richness values associated with the rainy season (Figure 4).

DISCUSSION
The composition of the periphyton was distributed in three taxonomic divisions, mostly Chlorophyta. This division, despite occurring in marine environments, prevails in continental aquatic ecosystems. They can still inhabit tree trunks, soils and live-in symbiosis with fungi, protozoa, and other animals [29]. This division presents a cosmopolitan distribution but shows a greater dispersion in species richness moving towards the equator, which results in a remarkably diverse tropical environment. In addition, the thermal amplitude (25-30ºC) found in the tropics is a promising condition for the development of this group [30].
There are many species belonging to Chlorophyta that form the periphyton, being associated with aquatic macrophytes, in which they spend much of their life cycle [8]. Emphasis is given to Zygnematophyceae group, which presents morphological adaptations such as thorns, processes, warts, mucilage, among others, which help in their permanence at the substrate, such as desmids species [7].
The Zygnematophyceae class, which presented the greatest richness for the sampled stretches of the Curiaú River, represents a remarkably diverse group, with many species and genera, especially in the periphytic community [8,31,32]. This is due to the relatively large size that this group presents, which hinders herbivory, in addition to the high capacity of nutrient cycling [31,33]. For this reason, it is widely used as an environmental bioindicator for the management and conservation processes of aquatic ecosystems.
The Zygnematophyceae class also represents a group overly sensitive to environmental changes, being a common class in oligotrophic environments and mesotrophic, with acidic water and low electrical conductivity [32]. This explains the expressive richness of this group in the Curiaú River, which presents such limnological characteristics, as well as other Amazonian rivers [34,35].
The second most representative taxonomic class was Cyanophyceae. This class is formed by microorganisms, also called Cyanobacteria, and usually configures one of the most found groups in phytoplankton and periphyton [36,37]. Cyanobacteria species, especially filamentous, are common in tropical aquatic ecosystems, growing in masses on the sediment or aquatic macrophytes, mainly in unpolluted environments [7], which may be the case in the sections studied in the Curiaú River.
However, it is known that cyanobacterial blooms occur more often in freshwater environments, due to eutrophication and global warming. This can reduce water quality with the release of toxins (e.g., microcystin), causing adverse impacts on aquatic ecosystems and human health with economic and ecological consequences [37]. Therefore, greater attention should be paid to the constant monitoring of water from the Curiaú River, which is used for balneability and irrigation in agriculture [18].
Most of the taxa presented a sporadic frequency. A similar result was observed by Bastos and Zorro (2011) [38] in the middle sector of the Xingu River, where 73 species were classified as sporadic (67.59%), 32 species as relatively uncommon (29.63%), and only 3 taxa were classified as relatively common (2.78%). The presence of rare species of microalgae in natural ecosystems often occurs. Studies show that the occurrence of sporadic species is more related to their dispersal capacity in the environment than to their affinity to the conditions found in aquatic ecosystems [39].
Regarding seasonal variation, in Amazônia the pattern of variation in rainfall indexes is remarkable, denoting two distinct periods: rainy and dry. In addition, rainfall is one of the most important variables in climate studies in the region, due to its wide variability in the temporal and spatial scales, which has been defining climatological patterns of precipitation in the rainy (January to May) and dry (July to November) regimes. This promotes pronounced variability in climatic characteristics (intensity and geographical arrangement of the maximum and minimum rainfall) in the Amazônia [40].
Due to this, species richness showed a significant variation between rainy and dry periods, with higher values associated with the rainy season. The periphytic community, in general, presents a clear spatial and temporal heterogeneity, which influences the variation of the composition, density, productivity, and biomass [41].
Factors such as water flows, seasonality, temperature, nutrient concentration, and herbivory also promote this variation [38]. Pieroni et al. (2015) [42] state that periods with higher rainfall rates tend to promote a higher occurrence of species, due to the increase in leached nutritional burden for these ecosystems, denoting a more expressive taxonomic richness, as observed in the present study.

CONCLUSION
The composition of the periphyton was represented by 64 taxa, with a higher presence of species of the Zygnematophyceae class, sporadic frequency, and the tendency to increase the richness associated with the rainy season. We also verified that local studies involving the periphytic community, mainly in natural substrates, are still quite scarce, showing the relevance of research development such as these for the region. This study is an important contribution to the ficology of the Brazilian Amazon, especially for its pioneering for the state of Amapá.