Phytoplankton of two rivers in the eastern Amazon : characterization of biodiversity and new occurrences 1 Elane

During the various hydrological periods in 2011, we studied the phytoplankton along an 87-km stretch of the Araguari and Falsino rivers, which are located in the eastern Brazilian Amazon, in the state of Amapá, Brazil, the most ecologically preserved state in the country. In the study area, the aquatic ecosystem is under pressure from human activities such as, mining, hydroelectric power generation and urbanization, which contrast with the surrounding areas that are designated for biodiversity conservation. The aim of this study was to characterize the composition, frequency and richness of algae species and also to identify spatial-temporal patterns of taxa distribution. During the study period, we identified 185 taxa (136 species in 49 genera). The division Chlorophyta (class: Zygnematophyceae) presented the greatest number of taxa, whereas the division Rhodophyta accounted for only 1% of the taxa recorded. Most of the species identified (69%) were classified as sporadic in occurrence. The few taxa that were classified as common belonged mainly to the group Bacillariophyta. Species richness was greatest richness in the lentic stretches (in a reservoir) and in November (during the dry season). Of the 185 taxa identified, 174 are new records for the state of Amapá.


Introduction
Phytoplankton is composed of microscopic organisms, also known as algae, which remain at the surface of water bodies.These organisms provide essential environmental services such as oxygen production for the aquatic environment (Wehr & Descy 1998) and for the atmosphere, from which they also sequester carbon dioxide (Ohse et al. 2009).In addition, these organisms form the base of the food chain of aquatic ecosystems and supply much of the carbon dioxide that is necessary to fishes, which are a primary source of protein for human populations (Araújo-Lima et al. 1986).
The Brazilian continental waters are rich in phytoplankton and present considerable phytoplankton biodiversity, with approximately 25% of the species in the world (Agostinho et al. 2005).Knowledge of the biodiversity of these algae is, however, quite heterogeneous regarding geographic region, taxonomic group (Bicudo & Menezes 2010) and type of aquatic ecosystem (Soares et al. 2007).
In Brazil, data related to algae is generated primarily in the southern and southeastern regions of the country, followed by the northeastern region, where substantial knowledge of these organisms has been gathered, despite the scarcity of hydrological resources in the region.Very little is known regarding the biodiversity of algae in the central-west and northern regions of Brazil, where the largest watershed in the world is located (Bicudo & Menezes 2010).Specifically in the state of Amapá, knowledge of phytoplankton is even scarcer, only two studies having been conducted to date (Förster 1964;Souza & Melo 2011).
Most studies of algae in Brazil have involved small aquatic ecosystems, such as lakes, lagoons and reservoirs (Silva et al. 2001;Bozelli & Huszar 2003).The phytoplankton biodiversity in the rivers of Brazil is still poorly studied, despite the extensive river system in the country (Soares et al. 2007).This is attributable, in part, to the high cost and complex logistics of conducting research on rivers in comparison with those of studies carried out in discrete environments (small lakes and reservoirs), features that Bozelli & Huszar (2003) referred to as "methodological difficulties".
In this context, it is necessary to increase research efforts regarding phytoplankton biodiversity, especially in the Amazon, where there are plans for the construction of hydroelectric plants, which significantly alter the flow of rivers and, consequently, the dynamics of aquatic organisms (Zeng et al. 2006).In addition, this is one the least served regions of Brazil regarding sewage treatment, as noted by Cunha et al. (2004;2005) and Agostinho et al. (2005).According to those authors, the construction of dams in the region will compound the problems caused by inadequate sewage treatment.Taken separately or together, these two factors present a major threat to aquatic biodiversity, potentially resulting in local extinctions and alterations within communities.
In the region of our study site, the aquatic ecosystem is facing pressures caused by human activities such as mining, construction of medium-sized hydroelectric plants, and urbanization, activities that put the site in contrast with the surrounding areas, which are dedicated to biodiversity conservation.In addition to a recently constructed hydroelectric plant, another two hydroelectric plants are currently under construction on the same stretch of river (Cunha et al. 2011;2013).In this scenario, the objective of the present study was to record the biodiversity within this ecosystem prior to its further modification.By characterizing the composition, frequency and richness of the phytoplankton communities in two Amazonian rivers (one in a conservation area and one already under the influence of hydroelectric plants), identifying spatial and temporal patterns in the distribution of taxa, we hope to improve the knowledge of phytoplankton biodiversity in such rivers.

Material and methods
The state of Amapá is located in the extreme north of Brazil, in the eastern portion of the Amazon basin, and 62% of its territory is set aside through the establishment of constructs such as conservation units, environmentally protected areas, legal reserves, remaining territories of quilombos (Brazilian settlements founded by escaped or freed slaves) and lands reserved for use by indigenous tribes, making it the most ecologically preserved state in the country (Brito, 2008).
The Araguari and Falsino rivers are part of the watershed of the Araguari river, the largest watershed exclusive to the state of Amapá.These are considered "clear-water" rivers, because of the small quantity of suspended particles (Junk et al. 2012), are acidic (like the majority of Amazonian rivers) and contain 6-8 mg/L of dissolved oxygen (Bárbara et al. 2010;Cunha et al. 2011).According to the Brazilian National Environmental Council, these rivers are categorized as class II, meaning that their water is potable (after conventional treatment); their aquatic species are protected; and they are open to recreation, irrigation, aquiculture and fishing (Brasil, 2005).These rivers are turbulent, because the declivity is 0.50% at the beginning of the stretch (site F1), resulting in high flow rates, which decrease near the Coaracy Nunes Hydroelectric Plant (CNHP) reservoir, where the declivity drops to 0.35%, and are considered to have a high self-purification capacity (Bárbara et al. 2010;Cunha et al. 2011;2013).
The stretch of river selected for this study is 87 km long, beginning at the Amapá National Forest, on the Falsino river, and ending at the CNHP reservoir on the Araguari river (Fig. 1).This stretch is strategically important regarding land use, which varies from conservation units to human activities that are common in the Amazon basin, such as mining, including pebble extraction (Bárbara et al. 2010); urbanization (small-to medium-sized cities); and, mainly, the generation of hydroelectric energy (Cunha et al. 2011).
We conducted phytoplankton surveys at 14 sampling sites arranged as indicated in Tab. 1.These sites-designated F1 (within a conservation unit on the Falsino river); P1-P4 and P6-P9 (on lands used for various purposes along both rivers); and R1-R5 (on the CNHP reservoir, Araguari river)-were chosen in order to continue the historic water quality series developed by Bárbara et al. (2010) and Cunha et al. (2010) on the Araguari and Falsino rivers.Our surveys were conducted in February, May, August and November of 2011, corresponding to the following hydrological periods, respectively: dry-wet seasonal transition, wet season, wetdry seasonal transition and dry season.
We obtained phytoplankton samples using a 20 μm mesh plankton net.A total of 56 samples were collected.The samples were subsequently fixed in Transeau's solution (Bicudo & Menezes 2006).The taxa were identified under standard light microscopy with the aid of specialized bibliography, which included but was not limited to Grönblad (1945), Förster (1963;1964), Komárek & Anagnostidis (1999;2005) and Bicudo & Menezes (2006).Whenever possible, specimens were identified down to the species level.We used the system devised by Hoek et al. (1995) to arrange the taxa in divisions and classes, except for the division Heterokontophyta, which was separated into Bacillariophyta and Chrysophyta.This procedure was adopted to facilitate the comparison with similar studies, because most still make use of this nomenclature.The classification of diatoms followed Medlin & Kaczmarska (2004).
To determine whether phytoplankton composition was similar among sampling sites, we conducted a cluster analysis with Ward's method and determined Euclidean distances using the software R-Project 2.14 (R Development Core Team 2012).
To verify new records for the state of Amapá, we reviewed studies on continental microalgae carried out in the state (Förster 1964;Souza & Melo 2011).
The samples were deposited in the liquid collection of the herbarium of the Institute for Scientific and Technologi-cal Research in the State of Amapá (code, HAMAB) under the accession numbers 0000001 through 0000056.

Results
We recorded 185 taxa (Tab.2)-49 identified down to the genus level and 136 identified down to the species level-within five divisions and ten classes.The divisions that were the most well-represented were Chlorophyta (with 71.9% of the taxa), Cyanophyta (with 14.6%) and Bacillariophyta (with 10.9%), Chrysophyta and Rhodophyta accounting for only 1.6% and 1.0%, respectively (Fig. 2).
The class Zygnematophyceae had the most taxa (n = 100), followed by Chlorophyceae (n = 29).Those two classes collectively accounted for 70% of the taxa, which explains the high representation of the division Chlorophyta.The group Cladophorophyceae had the smallest number of representatives (n=2).
The majority of taxa showed a low frequency: 69.0% were categorized as sporadic and 25.0% were categorized as relatively uncommon.Only 4.0% and 2.0% of the taxa were categorized as relatively common and quite common, respectively (Fig. 3).
As previously mentioned, there have been only two studies on continental microalgae conducted in the state of Amapá (Förster 1964;Souza & Melo 2011).Our study had 11 taxa in common with those two works (Tab.3).Therefore, of the 185 taxa identified in the present study, 174 represent new records for the state (Tab.2).

Discussion
The division Chlorophyta had the highest number of taxa, which occurred mostly in the CNHP reservoir.This environment provided conditions that were more favorable for the development of the phytoplankton than were those elsewhere on the river, which is quite turbulent, a condition to which few species can adapt.Other rivers with dams display this same pattern: the Contas river, in the state of Bahia (Fuentes et al. 2010); the Corumbá river, in the state of Goiás (Silva et al. 2001); and the Paranapanema river, in the southeast of Brazil (Nogueira et al. 2010).
The class Zygnematophyceae had the largest number of taxa.That is probably attributable the characteristics of the two studied rivers.According to Coesel (1982), oligotrophic waters, with the presence of macrophytes and low electric conductivity, have a high richness of desmids.Bárbara et al. (2010) and Cunha et al. (2011) both classified the Araguari and Falsino rivers as oligotrophic and found that the CNHP reservoir, where desmids were more numerous, had aquatic macrophytes.In the Amazon basin, there is considerable desmid diversity, making it one of the best known groups in the region (Grönblad 1945;Förster 1963Förster , 1964Förster , 1969Förster , 1974;;Scott et al. 1965;Thomasson 1971;    Sophia & Huszar 1996;Lopes & Bicudo 2003;Melo & Souza 2009;Souza & Melo 2011).
The majority of the most common taxa belonged to the division Bacillariophyta.This result was expected, because diatoms are typical of predominantly lotic environments (Soares et al. 2007).These organisms did not exhibit the highest richness, but they occurred in almost all samples.In a study conducted on the Arataï river in French Guiana, which shares a border with the state of Amapá, Tudesque et al. (2012) reported that Surirella sp., the most common genus in our study, is adapted to the turbulence of rivers and possesses high mobility.Furthermore, these authors observed the diatoms Eunotia sp., Fragilaria sp. and Surirella sp. in high frequency, similar to our findings on the Araguari and Falsino rivers.Diatoms have also been reported to be the most common group on other Amazonian rivers such as the Guamá and Caeté, in the state of Pará (Monteiro et al. 2009;Pereira et al. 2010).
The high frequency of diatoms in the present study might be explained by their ability to adapt to conditions of high turbulence (Reynolds et al.1994), a typical characteristic of rivers (Press et al. 2003).For the stretch of river studied Cunha et al. (2010;2011) reported the mean water velocity to be 0.57-0.92m/s in the lotic stretch (upstream) and 0.04 m/s in the lentic stretch (CNHP reservoir).Branco et al. (1986) categorized the water velocity as: quite rapid (> 0.5 m/s), or moderate-to-slow (≤ 0.5 m/s).Therefore, the studied stretch of river can be considered mostly turbulent because of its high water velocity, which tends to naturally decrease toward the dam.In the CNHP reservoir, however, the speed is not uniform as in totally lentic environments, because its water retention time is only two to five days, and its characteristics therefore resemble those of a river more than those of a lake (Bárbara et al. 2010;Cunha et al. 2013).
In the present study, most of the taxa were classified as sporadic, occurring in less than 10% of the samples.According to Townsed et al.(2008), this pattern is normal, because these species in general are naturally rare.In studies conducted on the Guamá and Paranaíba rivers (Monteiro et al. 2009;Oliveira et al. 2011), the majority of taxa were also classified as sporadic.
The spatial distribution of taxa was aggregate in the CNHP reservoir.The majority of these taxa are desmids, which present slow growth and require lentic environments for their development (Coesel, 1982).The dendrogram of the cluster analysis (Fig. 5) indicated one cluster associated with the reservoir sampling sites and two additional large clusters, one associated with the sites near the beginning of the stretch of river studied and another associated with sites intermediate between the two ends of the stretch.Considering the species composition, the locations of these clusters might be related to the declivity of the land at the sampling sites and, consequently, to the water velocity, the declivity being similar among sites within the same group.The sites at the beginning of the stretch (F1 and P1-P4) had high declivity, the remaining sites along the stretch (P6-P9 and R1) had intermediate declivity, and the sites at the reservoir had low declivity, resulting in low water velocity (Cunha et al. 2011), the last creating the most favorable conditions for the development and reproduction of algae (Coesel, 1982).
Because of the presence of a reservoir in the stretch of river studied, we expected more taxa of the division Cyanophyta, or that their distribution would be more restricted to the reservoir, because these organisms typically occur in lentic environments, with a long water retention time (Chorus & Bartram, 1999).However, the CNHP reservoir has a water retention time of two to five days; that is, all the water in the reservoir is renewed in a relatively short period of time, even when considering the complete hydrological cycle (Cunha et al. 2011).Much longer water retention times have been reported for the reservoirs of other hydroelectric plants in the Amazon basin (Tucci, 1998): 50, 107 and 578 days, respectively, at the Tucuruí, Samuel and Balbina reservoirs.This fast turnover prevents the occurrence of algal blooms, which, according to Chorus & Bartram (1999) do not usually occur in environments where the water retention time is shorter than five days.However, these characteristics might be altered with the construction of new reservoirs, and two additional hydroelectric plants are currently under construction on the stretch of river studied (Cunha et al. 2010;2011;2013).In fact, the water retention time might increase considerably.On rivers with dams, such as the Paraibuna river (in the state of Minas Gerais) and the Paranapaíba river (in the states of Goiás and Minas Gerais), Cyanophyta is the most well-represented division, in terms of the number of species (Soares et al. 2007;Oliveira et al. 2011).The increase in species richness seen during the dry season in the present study might be related to the physical characteristics of that season, such as greater solar radiation, higher temperatures and less precipitation, all of which favor the growth of algae (Kivrak 2006;Marques et al. 2012).In addition, water velocity tends to decrease during the dry season (Nogueira 2000;Zeng et al. 2006;Cunha et al. 2013).The occurrence of a larger number of taxa during the dry season has also been reported for other Brazilian rivers, such as the Urubuí (Aprile & Mera 2007), Paraibuna and Pomba (Soares et al. 2007).
Reviewing the literature, we found only two studies of phytoplankton in the state of Amapá: Förster (1964), who studied desmids on the Oiapoque river and recorded five taxa; and Souza & Melo (2011), who conducted a survey of desmids in Amapá lake and recorded 35 taxa.Therefore, in the present study, we have identified the largest number of taxa recorded to date for the state of Amapá (185), of which 172 (93%) represent new records for the state.

Conclusion
We found that the species richness of the phytoplankton community of the Araguari and Falsino rivers was high in comparison with that reported in similar studies for the Amazon and for other regions of Brazil.This suggests that, in addition to the natural tendency toward an increase in species richness toward the tropics, the sampling effort was satisfactory, because we identified spatio-temporal variations within the microalgae community.We observed a large number of taxa belonging to the division Chlorophyta, class Zygnematophyceae, and most of the taxa identified were relatively rare.However, most of the taxa classified as common were diatoms.When we examined the spatiotemporal dynamics, we found that species richness was lowest at the sampling sites close to conservation units (sites with high declivity and high water velocity) and increased progressively at each site nearer to the CNHP reservoir (sites at which the declivity and water velocity were low).In addition, the number of taxa was highest in dry season.The 174 new records generated for the state of Amapá constitute a significant contribution to expanding the knowledge of aquatic biodiversity in the Brazilian Amazon.

Figure 1 .
Figure 1.Map of the study site.Sampling sites on the Araguari and Falsino rivers.

Figure 2 .
Figure 2. Distribution of taxa by division (left side) and class (right side), showing frequencies (light bars) and absolute values (grey bars).

Figure 4 .
Figure 4. Distribution of taxa among the sampling sites.

Figure 6 .
Figure 6.Distribution of taxa by hydrological period.DW -dry-wet seasonal transition; W -wet season; WD -wet-dry seasonal transition; and D -dry season.

Figure 5 .
Figure 5. Cluster analysis: groupings according to the similarity of species composition among the sampling sites.

Table 1 .
Description of the sampling sites on the Araguari and Falsino rivers.

Table 2 .
Complete list of taxa with their respective frequency classes and site of occurrence (n of samples = 56).

Table 3 .
Previous studies of continental microalgae in the state of Amapá: taxa in common with the present study.