Diversity Assessment of Benthic Macroinvertebrates, Yeasts, and Microbiological Indicators along a Longitudinal Gradient in Serra Do Cipó, Brazil

The main goals of this study were: 1) to evaluate the structure, diversity, and functional trophic group composition of benthic macroinvertebrate communities; 2) to characterize water quality in the head-waters of the Doce river watershed, based on physical, chemical, and biological parameters (benthic macroinvertebrates, fecal coliforms, heterotrophic bacteria, and yeasts); and 3) to contribute to the knowledge of the structure and function of longitudinal gradients in lotic ecosystems in Brazil. A total of 60 benthic macroinvertebrate taxa were identified, the dominant group being the aquatic insects, with 50 families distributed in 8 orders. The dry period presented higher values of taxonomic richness and total density of benthic macroinvertebrates. A decreasing gradient was observed in these variable values from the 3 rd order stretch down to the 6 th order stretch. The highest Shannon-Wiener diversity values were found in the rainy period in the 3 rd order stretches, which presented well-developed riparian forest. Besides the 3 rd order stretches, the Pielou evenness index values were also high in the 6 th order stretch. The collectors, together with the scrapers, predominated in the benthic macroinvertebrate communities in all river stretches, except in the 2 nd , 4 th , and 5 th order stretches in the rainy period, where communities were dominated by filterers. The shredders and predators presented low densities for all river stretches. All microbiological variables presented low levels. Due to the high counts of heterotrophic bacteria and coliforms, the studied river stretches presented inadequate potability but adequate balneability levels. The results suggest that the structure, diversity, and composition of the benthic macroinvertebrate communities are influenced by the trophic resource availability, seasonality, and sediment heterogeneity. The microbiological results of this study allow inferring that the waters from Serra do Cipó have excellent potential for recreational use and as future sources of water for human consumption. RESUMO Avaliação da diversidade de macroinvertebrados bentônicos, leveduras e indicadores microbiológicos ao longo de um gradiente longitudinal na Serra do Cipó, Brasil Os principais objetivos deste estudo foram: 1) avaliar a estrutura, a diversidade e a composição de grupos tróficos funcionais das comunidades de macroinvertebrados bentônicos; 2) caracterizar a qualidade das observado gradiente decrescente nos valores dessas variáveis do trecho de 3 a ordem ao trecho de 6 a ordem. Os maiores valores do índice de diversidade de Shannon-Wiener foram encontrados no período de chuvas nos trechos de 3 a ordem, que apresentaram mata ciliar …


INTRODUCTION
Within recent decades aquatic ecosystems have been altered at different scales, and registered as negative consequences of anthropogenic activities (e.g., mining, dam construction, artificial eutrophication, river canalization, and recreation).Detection of resulting impacts on streams depends on the use of biomonitors combined with physical (e.g., temperature, suspended solids) and chemical (e.g., nutrient levels, concentrations of potential toxins) data (Dudgeon, 1994).
Biological indicator use in monitoring programs provides a more exact measure of anthropogenic effects on aquatic ecosystems (Callisto & Esteves, 1995;Callisto et al., 2001a).Biological indicators have the advantage of monitoring water quality over a long period of time, thus providing a more adequate picture of level of pollutant effects on the ecosystem than is the case for chemical methods, which provide only momentary evidence of water quality (Tundisi & Barbosa, 1995).
Within the organisms commonly used as biological indicators, benthic macroinvertebrates stand out as ideal due to: relatively low mobility and long life cycles, reflecting temporal patterns and local conditions; high diversity, abundance and, consequently, in providing a wide range of responses to different environmental pollution agents; large size and easy identification at high taxonomic (such as family) resolution by non-specialists; well standardized and low-cost methodologies; and temporal and spatial stability, reflecting changes in ecosystem processes (Rosenberg & Resh, 1993).
Microbiological monitoring of organisms important in determining water contamination levels is usually done by fecal pollution indicators, represented by bacteria counts of coliform groups (Ceballos et al., 1995;Lutterback et al., 2001).This parameter is used by government agencies to classify water bodies as to use and sanitary levels (balneability and potability).Aquatic bacteria and fungi feed on dissolved organic matter, multiplying rapidly under favorable conditions.Some authors suggest that the number and composition of yeast species present in rivers and lakes can be used as organic enrichment indicators in water bodies (Rosa et al., 1995;Morais et al., 1996).Species within the genera Cryptococus, Debaryomyces, and Rhodotora are characteristically found in nonpolluted waters, while Candida and Saccharomyces species can be frequently found in eutrophic waters (Hagler et al., 1986;Rosa et al., 1995).
The main goals of this study were: 1) to evaluate the structure, diversity, and functional trophic group composition of benthic macroinvertebrate communities; 2) to characterize the water quality in the headwaters of the Doce river watershed, based on physical, chemical, and biological parameters (benthic macroinvertebrates, fecal coliforms, heterotrophic bacteria and yeasts); and 3) to contribute to knowledge of longitudinal gradient structure and function in lotic ecosystems in Brazil.

Study area
Located south of the Espinhaço Cordillera (19 o -20 o S and 43 o -44 o W) in the center of Minas Gerais State, Serra do Cipó is a watershed of the Doce river and São Francisco river basins.The vegetation is composed of tropical savanna (cerrado) at the lower altitudes (700-900 m a.s.l.), rupestrian fields in the highest portions (above 1,200 m a.s.l.), and riparian forest in the humid valleys along the rivers.The climate is classified as Cwb (Köppen), with rainy summers and dry winters, and presents an annual mean pluviosity of 1,500 mm/year (Galvão & Nimer, 1965).The longitudinal gradient was studied in the Indaiá stream (1 st up to 4 th orders) and Peixe river (5 th and 6 th orders), both belonging to the Doce river watershed.

Physical and chemical characterization of the waters
Chemical and physical variables measured with a Horiba multiprobe were: depth (m), temperature ( o C), pH, electrical conductivity (µS/cm), and dissolved oxygen (mg/L).Concentrations of total phosphorus, orthophosphate, ammonium, nitrate/ nitrite, total nitrogen, and reactive soluble silica were determined in the Laboratory of Limnology (Institute of Biological Science, Federal University of Minas Gerais), using water samples collected bimonthly in the water sub-surface, according to Golterman et al. (1978) and Mackereth et al. (1978).The total alkalinity was determined by the Gran method, modified by Carmouze (1994).

Ecological characteristics of watershed stretches
To evaluate ecologically the sampling stations and their surroundings we used a rapid evaluation protocol of ecological conditions and habitat diver-sity in watershed stretches, proposed by Callisto et al. (2002).This protocol evaluates not only freshwater environments but also land use and occupation of surrounding areas of drainage basins.The major variables assessed were river width and depth (m); riparian vegetation composition; dominant fraction of organic matter (coarse/fine particulate organic matter as proposed by Ward, 1992); habitat diversity (riffles/pools); sediment composition (bedrock, cobbles, pebbles, gravel, sand).The water current velocity was measured using a Global Water digital fluxmeter.

Benthic macroinvertebrate communities
Samplings were performed during the rainy (February) and dry (June) periods of 2000, along a longitudinal gradient in the Indaiá stream (1 st up to 4 th order stretches) and Peixe River (5 th and 6 th order stretches).Samples were collected using Surber samplers (0.250 mm mesh) from a 0.0625 m 2 area on the gravel/sand substrate, submerged leaves/ branches of the riparian vegetation substrate; and 0.01 m 2 from remaining substrates.The samples were then immediately fixed with 10% formaline.A total of 120 samples were collected (60 samples in each sampling period) (Table 1).
In the laboratory, the samples were washed using 0.250 mm sieves, sorted and, under stereomicroscope and with the use of appropriate literature (Dominguez et al., 2001;Pérez, 1988;Wiggins, 1977), identified.Identified organisms were preserved in 70% ethanol and deposited in the Reference Collection of Benthic Macroinvertebrates at the Institute of Biological Sciences, Federal University of Minas Gerais, according to Callisto et al. (1998).
Taxonomic richness was preferentially recorded at the family level, and the evenness and diversity indexes were calculated for each sample period, river stretch, and basin using the Shannon-Wiener diversity index and Pielou evenness index (Magurran, 1991).

Microbiological indicators
For microbiologic analyses, sub-surface water samples were immediately put into sterile glass bottles and returned to the laboratory on ice within 8 hours for processing.Total and fecal coliforms were determined using the standard most probable number method (MNP); heterotrophic bacteria counts were performed using the pour plate method on NWRI agar-HPCA (peptone 0.3%, soluble casein 0.05%, K 2 HPO 4 0.02%, MgSO 4 0.005%, FECL 3 0.0001%, agar 1.5%) after 2-5 days incubation at 22%-28 o C (Greenberg et al., 1998).For yeast counts (CFU), aliquots of 0.1 ml of the samples were plated in triplicates on YM agar with antibiotic (glucose 2.0%, peptone 1.0%, yeast extract 0.3%, malt extract 0.3%, agar 2.0%, and cloramphenicol 20 mg%).Plates were incubated at room temperature and counts were obtained after 3 to 5 days.Each morphological yeast biotype was counted and representative colonies were isolated, purified, and characterized according to standard methods (Yarrow, 1998).Yeasts were identified by keys in Kurtzman & Fell (1998).

Water physical and chemical characteristics
The studied stretches presented acid to slightly neutral waters, with pH varying between 4.2 in the 2 nd order stretch (rainy period) and 7.3 in the 5 th and 6 th order stretches (dry period), and high dissolved oxygen concentrations (6.7 mg/L in the 3 rd order stretch, closed canopy section in the dry period, and up to 9.1 mg/L in the 5 th order stretch, rainy period).Low electrical conductivity values (< 15 µS/cm in the dry period and < 25 µS/cm in the rainy period) and low water column turbidity (0 NTU to 34 NTU) were found.Temperature varied along the gradient, with higher values in the rainy period in stretches presenting open riparian vegetation (composed by rupestrian fields or degraded by pastures) and lower values in the well-developed riparian forest stretches (in the 3 rd order stretches) (Table 2).

Assessment of the ecological characteristics of basin stretches
Along the longitudinal gradient in the Indaiá stream an increase in the mean river width from the 1 st up to the 6 th order was observed.On the other hand, the depth varied as a result of terrain unevenness and channel structure (Table 2).Riparian vegetation was composed of rupestrian field in the 1 st , 2 nd , and 4 th order stretches.In 3 rd order stretches, this vegetation type was replaced by riparian forest, while in 5 th and 6 th order stretches, isolated secondary forest fragments and large pastures were seen.The fine particulate organic matter (FPOM) source followed the distribution of riparian vegetation and presence of aquatic macrophytes.In 3 rd order stretches, coarse particulate organic matter was the dominant form (CPOM), while in the remaining stretches FPOM predominated.Regarding sediment composition, riffle/pool sequences, and river channel morphology, the river stretches were grouped into four types by an ecological conditions characterization protocol (Callisto et al., 2002): • 1 st order stretch: bottom formed by 100% bedrock; the stream is constituted by a small water thread during rainy period, while in the dry period, isolated pools with large FPOM deposits are formed; • 2 nd and 4 th order stretches: bottom of over 70% bedrock; riffle/pool sequences welldefined, with riffles formed by steep slopes, and deep pools having gravel and coarse sand deposits near the river margins; • 3 rd order stretches: bottom composed mainly by rocks, cobbles, and pebbles, with bedrock in the pools; riffle/pool sequences scattered, with short riffle stretches and long, shallow pools; • 5 th order stretch: bottom of rocks, cobbles, pebbles, gravel, and sand; riffle/pool sequences are substituted by erosion/deposi-tional zones, with erosion zones located mainly in the river channel, and deposition zones along the margins; • 6 th order stretch: bottom of bedrocks covered with gravel, sand, and silt deposits throughout the channel extension; the riffle/ pool sequences are substituted by erosion/ depositional zones, with erosion and depositional zones located at the river curves.

Benthic macroinvertebrate communities
Sixty macroinvertebrate benthic taxa were identified, the dominant group being the aquatic insects, with 50 families distributed in 8 orders.Taxonomic richness varied largely along the gradient, with lower values in the rainy period and a decreasing gradient from the 3 rd order (open canopy) down to the 6 th order stretch (Fig. 1).The highest taxonomic richness values were found in the dry period in the 2 nd (42 taxa) and 3 rd order (open canopy, 43 taxa) stretches, while the 6 th order stretch presented the lowest taxonomic richness values (19 taxa) (Table 3).The Shannon-Wiener diversity index values presented little variation for the dry period, with only a small decrease in the 1 st down to the 2 nd order stretches and from the 5 th down to the 6 th order stretches.The rainy period also presented little variation between river stretches, except for the 3 rd (open canopy) and 5 th order stretches where an increase and a large decrease, respectively, were     observed in the diversity values (Fig. 1).The highest values were found in the rainy period in the 3 rd order stretches (open canopy, H´ = 2.246 and closed canopy, H´ = 1.972), with the lowest value found in the 5 th order stretch (H´ = 0.621) in the same period (Table 3).

River stretches
The Pielou evenness values presented a pattern similar to that of the Shannon-Wiener diversity index, with higher values in the dry periods and an abrupt decrease in the 5 th order stretch (Fig. 1).The highest values were found in the 6 th order (J´ = 0.652), and in the 3 rd order (open canopy, J´ = 0.642), both in the rainy period.The lowest value was found in the rainy period in the 5 th order stretch (Table 3).
The highest density values were found in the dry period, except for the 2 nd order stretch, where an increase was shown in the rainy period (Fig. 1).The 4 th (64,775 ind/m 2 ) and 5 th order (90,255 ind/m 2 ) stretches presented highest values in the dry period (Table 3).The lowest value was found in the rainy period in the 3 rd order (closed canopy; 6,488 ind/m 2 ).
The collectors, together with the scrapers, predominated in the benthic macroinvertebrate communities in all the river stretches, except for the 2 nd , 4 th , and 5 th order stretches in the rainy period, where communities were predominantly filterers (Fig. 2).In these river stretches, the dominant organisms were Simuliidae (Diptera) larvae, and also filterer nymphs of Leptophlebiidae, found in lower densities (e.g., Hermanella).In the remaining stretches, the main dominant groups in both periods were the Chironomidae (Diptera) larvae and Baetidae (Ephemeroptera) nymphs (Table 3).In contrast, the shredders presented the lowest densities within the trophic groups, with abundances of lower than 15%.The predators also presented low densities, except in the dry period in the first order stretch (Fig. 2) where the benthic macroinvertebrate communities were dominated by this trophic group, represented mainly by Corixidae, Veliidae, and predator larvae of Tanypodinae (Chironomidae) (Table 3).The predator density was related positively to total prey density (p < 0.05; r = 0.82).

Microbiological indicators
The Indaiá stream presented low fecal and total coliform counts along the longitudinal gradient in both sampling periods, except for the 5 th and 6 th order stretches, which showed high counts in the rainy period (Table 4).Regarding the heterotrophic bac-terial densities, the highest values were found in the rainy period, except for the 3 rd (open canopy) and 4 th order stretches, which showed higher values in the dry period (Table 4).The assessment of yeast diversity revealed low microrganism densities along the longitudinal gradient, with eight yeast species being identified (Table 4).

DISCUSSION
In studying the structure, diversity, and functional trophic group composition of the benthic macroinvertebrate communities along the longitudinal gradient, the results indicated that, as verified in other studies, the major influences were seasonality (Oliveira et al., 1997;Bispo & Oliveira, 1998), habitat and microhabitat diversity (Galdean et al., 2000(Galdean et al., , 2001;;Callisto et al., 2001b), and the canopy cover of the riparian vegetation along the river bed (Aguiar et al., 2002).
The rainy period in the Serra do Cipó region is characterized by fierce storms that influence the whole aquatic biota, with rapid increase of water current velocity and flow, and water level rising up to 5 meters above normal values.This significant flow and water velocity increase may lead to a dislodgment of resident benthic fauna through physical disturbance of the substrate (Poff & Ward, 1991).A decrease in taxonomic richness and total density of benthic macroinvertebrate communities was observed in the rainy period in almost all river stretches.In studying the spatial-temporal distribution of benthic macroinvertebrate communities in streams in Goiânia Ecological Park (Goiás State, Brazil), Bispo & Oliveira (1998) found similar results: density and taxonomic richness reductions during the rainy periods.
However, the diversity and evenness indexes increased during the rainy period, except for the 1 st and 5 th order stretches.This may have happened because of food resource increase and density reductions in the benthic macroinvertebrates, which resulted in a well-structured community.The first order stretch is characterized by 100% bedrock sediment and large FPOM deposits.During the rainy period part of this material is carried off, reducing food availability and, consequently, taxonomic richness, diversity, and evenness of the benthic macroinvertebrate communities.On the other hand, in the 5 th order stretch, which presented   lower values, reduction in the diversity and evenness values is due to the increase in densities of Simuliidae (Diptera) larvae.These organisms feed on suspended FPOM, the most abundant food resource in this river stretch during the rainy period, as pointed out by Galdean et al. (2001).
Collectors predominated in all river stretches and both sampling periods.But in the dry period the benthic macroinvertebrates were found to be composed mainly of collectors and scrapers while during the rainy period filterers increased considerably in numbers in all stretches, dominating the communities in the 2 nd , 4 th , and 5 th order stretches.
Structural and compositional modifications of the benthic macroinvertebrate communities probably occur due to a change in trophic resource availability (Callisto et al., 2001b).The physical disturbance of the substrate observed in the rainy period caused the sediment to revolve, thus resuspending the FPOM deposited on the bottom.Besides that, in aquatic ecosystems pluvial increase leads to a larger input of allochthonous organic matter including both coarse and fine material (Allan, 1995).Therefore, during the rainy period an increase was found in suspended FPOM, which modified the functional trophic group composition of the benthic communities.
The shredders and predators presented low densities in both sampling periods.According to temperate region models such as the river continuum concept (Vannote et al., 1980), low-order streams located in forested areas present restricted primary production due to riparian shading.Consequently, much of the energy demand by consumers is met by allochthonous sources (Dudgeon & Bretschko, 1995) and these stretches present high densities of organisms such as the shredders (e.g., Calamoceratidae: Trichoptera), capable of processing these coarse particulate organic matter resources.
Unlike aquatic ecosystems in temperate regions, most of the streams (specially in the upper stretches,  (Mathuriau & Chauvet, 2002;Ramirez & Pringle, 1998).Regarding predator densities, it is known that these organisms are naturally less abundant in the environment, and their distribution and numbers are usually related to prey abundance (Begon et al., 1996).

Water quality in the headwaters of Doce river
The standards established by regulatory agencies that supervise recreational water use are based upon counts of the coliform group, which includes pathogenic bacteria such as Salmonella spp., present in domestic sewage.The Indaiá stream presented low densities of coliforms along the gradient, however it should be emphasized that in the 5 th and 6 th order stretches in the Peixe river a considerable increase occurred in the numbers of this microorganism.This may indicate contamination of these waters by warmblooded animal feces, an expected finding since this river is surrounded by cattle farms and small towns.
The heterotrophic bacteria counts may indicate the water-quality degradation degree, since these organisms are frequently associated with organic matter concentrations in water (Greenberg et al., 1998).The low heterotrophic bacteria counts along the longitudinal gradient reflect the low waterdegradation degree.
We also observed that in the 1 st to the 4 th order stretches the counts were close to the ones found in the 5 th and 6 th order stretches.This similarity is probably related to organic matter input from surrounding areas, while in the studied stretches in the Peixe river, the larger organic matter input came from non-treated effluents of farms and small towns, in addition to livestock feces.
Besides heterotrophic bacterial counts, counting and identify yeasts can indicate the eutrophication level of aquatic environments, due to the strong association of yeast with organic matter concentrations in the water (Rosa et al., 1990;Peçanha et al., 1996;Morais et al., 1996).Along the longitudinal gradient in the Indaiá stream and Peixe River low counts of these microorganisms were found, confirming the conservation level of these ecosystems.
According to a Brazilian law (number 1469, of December 29, 2000) concerning potability levels of water, all of the studied stretches in the Indaiá stream and Peixe river are inadequate for human consumption without previous treatment, due to the presence of heterotrophic bacteria.Regarding balneability, all river stretches were considered appropriate for bathing.The microbiological results of this study allow us to infer that the waters from Serra do Cipó have excellent potential for recreational use and as future sources of drinking water.

Final comments
In altitudinal lotic ecosystems the energy conversion and organic matter degradation processes are sustained mainly by algae biofilm and aquatic microbiota, are intimately related to maintenance of the physical environment, and have fundamental consequences for aquatic community structure and diversity.Studies regarding lotic ecosystem structure and functioning should include, in addition to physical characterization of the environment (canopy covering, sediment composition, forms and distribution of available organic matter, etc.) and characterization of physical (e.g., temperature, water flow) and chemical (dissolved oxygen, pH) water variables, also the identification and structural characterization of the biological communities, especially the benthic macroinvertebrate and microorganism communities.
Biodiversity assessment in protected areas such as the Federal Conservation Units, which is the case of Serra do Cipó National Park, involves the study of structural characteristics and diversity of biological communities in their natural environments.The results of such studies can be used in determining restoration measures relating to these communities in anthropogenically disturbed sites.Aquatic biological communities can be preserved by creating and maintaining conservation units, implementing conservation measures outside these areas, and restorating degraded habitats (Primack & Rodrigues, 2001).
Understanding aquatic community structural factors depends on knowing the autoecology of these organisms, as well as their use of food resources, substrate associations, and longitudinal distribution.The study of benthic macroinvertebrate community structure and composition along longitudinal gradients constitutes an important tool for aquatic ecosystem conservation in the Serra do Cipó.The importance of lateral and longitudinal dimensions of lotic ecosystems makes immediate large-scale approaches (in terms both of time and space) urgent if the elements and ecological processes supporting the biota are to be conservated and managed effectively.Besides that, use of the drainage basin as a study unit and middleand long-term monitoring program implementation are extremely necessary.

Fig. 1 -
Fig. 1 -Taxonomic richness (A), total density (B), Pielou evenness index (C), and Shannon-Wiener diversity index (D) of the benthic macroinvertebrate communities during the rainy and dry periods of 2000, along the longitudinal gradient in the Serra do Cipó.

Fig. 2 -
Fig. 2 -Functional trophic groups composition of the benthic macroinvertebrate communities along a longitudinal gradient in the Indaiá stream and Peixe river, in the rainy and dry periods of 2000.