Diversity and ecological aspects of aquatic insect communities from montane streams in southern Brazil

Aims: In this study, the diversity of Ephemeroptera, Plecoptera, Trichoptera and Coleoptera communities was surveyed in the Toropi River basin, a watershed localized in a slope region, in southernmost Brazil. The influence of some local abiotic factors on the most common genera was also analyzed. Methods: Samplings were conducted at 40 sites in 1st-4th order streams, along a short elevation gradient (70-500 m), with a Surber sampler. Water physico-chemical factors, as well as substrate type, were obtained at each site. Results: At all, 5,320 specimens were collected, belonging to 18 families and 52 genera. The caddisflies Austrotinodes and Celaenotrichia, and an undescribed Elmidae, Genus M, are new records for the region. The caddisfly Smicridea was the most frequent genus in the study area. The mayflies Camelobaetidius, Paracloeodes and Americabaetis were influenced by stream order. Smicridea was related to air temperature, while the mayfly Thraulodes was influenced by high levels of electrical conductivity. Conclusions: The high diversity found in the study area, compared to other Brazilian regions, reflects the environmental heterogeneity in the region. These data show that hydrographic basins in slope areas from extreme Southern Brazil sustain high levels of diversity of aquatic insect communities.


Introduction
Rivers and streams are one of the most threatened ecosystems worldwide, due to human pressure on water sources (Allan and Castillo, 2007;Maloney et al., 2011).In southernmost Brazil (Rio Grande do Sul State, RS), rivers are commonly used in agriculture, especially for irrigated rice fields (Primel et al., 2005).Additionally, man-made ponds are frequent in montane areas, mainly in small properties for agricultural and domestic purposes (Beskow, 1984;Pires et al., 2013).In other words, the rich hydrographic network of the region is being converted to a mosaic of lentic and semi-lotic environments, affecting the lotic-dependent aquatic fauna.Besides, a recent state law (nº 94, December 16 th 2008) allowed stream damming for reservoir construction without demanding of environmental assessment.Thus, it is very important to survey aquatic insect community diversity in rivers and streams in Rio Grande do Sul, before many of them are extirpated, hindering obtain data related to the protection of lotic ecosystems environmental integrity.
Aquatic insects such as Ephemeroptera, Plecoptera and Trichoptera (EPT), as well as some coleopteran (hereafter C) families such as Elmidae and Psephenidae, are important components in lotic ecosystems, and generally predominate at these environments, for they are diverse and broad distributed insects.Many of these insects are sensitive to environmental perturbations and are considered indicators of water quality.The occurrence of diverse EPT larval communities, and also of elmids and psephenids, is associated with low-polluted and well-oxygenated streams (Rosenberg and Resh, 1993).They can also be associated to well preserved riparian vegetation (Bispo et al., 2006).Additionally, abiotic factors related to streams environmental condition, such as water temperature, electrical conductivity, pH, and current velocity are important drivers of their communities (Fernandez and Fonseca, 2001;Buss et al., 2004;Bispo et al., 2006;Merritt et al., 2008).Thus, the presence of EPTC may reflect the integrity of the entire aquatic insect community (Crisci-Bispo et al., 2007).
In Brazil, studies on diversity of EPTC were conducted mainly in tropical and subtropical streams, where they are found inhabiting streams in both plateau and slope areas (e.g.Bispo and Oliveira, 2007;Gonçalves and Menezes, 2011;Paciencia et al., 2011;Segura et al., 2012).The most important factors affecting this fauna are stream order, rainfall, altitude and water current (Oliveira et al., 1997;Bispo et al., 2006;Bispo and Oliveira, 2007).Additionally, substrate plays important role in structuring their communities.EPT, as well as Elmidae and Psephenidae, are commonly found in stony-bottom streams (Bispo and Oliveira, 2007;Domínguez and Fernández, 2009).However, in streams located in colder temperate regions, and with regular rainfall, like in southernmost Brazil (Nimer, 1990;Maluf, 2000), little is known about their diversity, and, especially, about the influence of environmental factors on genera occurrence and composition.Many studies have been conducted with macroinvertebrate identification at family level (e.g., Pereira and De Luca, 2003;Buckup et al., 2007;Milesi et al., 2009) , while others deals with EPT or were restricted to certain groups (e.g., Hepp et al. (2010Hepp et al. ( , 2013)); Salvarrey et al. (2014) for EPT; Spies et al. (2006) for Trichoptera; Siegloch et al. (2008) for Ephemeroptera; Salvarrey et al. (2014) for Elmidae).
This study presents the results of an inventory of EPTC genera in montane streams from southern Brazil.Also the overall EPTC diversity is compared with those of other Brazilian regions.Considering that montane areas can reach up to 500 m in the study region (Robaina et al., 2010), and that many abiotic factors are modulated by riverine longitudinal gradient (Vannote et al., 1980), the influence of abiotic factors on the most frequent EPTC genera was also analyzed.The relationship of the abiotic factors with the environmental integrity of the streams studied was also analyzed.Thus, this study also provides important information for stream conservation and environmental assessment programs.

Study area
The Toropi River basin is located in extreme southern Brazil (Rio Grande do Sul State, RS) (28°30'-31°S; 53°30'-57°W), covering a 47,740 km 2 catchment area.The headwaters are located in the Southern Plateau (Planalto Meridional), reaching ca.500 m altitude, although most streams run through the slope areas.The Toropi is a 6 th order river near the mouth, and flows into the Ibicuí River, in a lowland region, the Central Depression (Depressão Central, ca.70 m altitude, Hundertmarck and Miorin, 2001).Through its course, the Toropi River runs through basaltic rocks in the Plateau, a mixture of sedimentary rocks and basalt in the slope, and alluvial-origin sands in the lowland (Robaina et al., 2010).The climate is characterized by regular rainfall through the year, with annual accumulated precipitation range of 1,250-2,000 mm (Silva et al., 2006;Buriol, 2007).The mean temperature in the warmest months is superior to 22°C, and in the coldest ones, near 13°C (Maluf, 2000).The original vegetation in the region is located in a transitional zone between Seasonal Decidual Forest (Atlantic Forest biome) in the slope, and Savannah (grassland) in the Central Depression (Quadros and Pillar, 2001;Kilca and Longhi, 2011).Nowadays, most part of the original landscape was converted to agricultural activities, mainly rice fields in the floodplains (Pedron et al., 2006).

Sampling sites and abiotic factors
Sampling was conducted in October and November 2010, when droughts and floods are scarce in the region (Maluf, 2000), a suitable period for macroinvertebrate sampling (Bispo et al., 2001).Samplings took place in 40 sites (Figure 1), in 1 st to 4 th order streams, distributed along the altitudinal gradient from the Plateau to the Depression (ca.500 m).All sites had riparian vegetation in both stream banks.The altitude was taken with a GPS (Garmin model), and stream order, through consultation to cartographic charts (scale 1:50,000).Air and water temperatures (AT and WT, respectively, alcohol 0-50°C thermometer), mean depth (m) and water current velocity (WC, floating method) were taken at each site.Stream bed sediment grain size was determined based on Wentworth scale, through calculation of % gravel directly at each site and the rest in the lab.Dissolved oxygen (DO, mg.l -1 ), pH (mol.dm - ), electrical conductivity (EC, µS.cm -1 ) and biochemical oxygen demand (DBO, mg O 2 .l - ) were taken with a multiparameter probe Concentration of calcium and iron ions (mg.l -1 ) were also measured (atomic absorption spectrophotometer).Data on accumulated precipitation (mm) and mean month temperature (°C) for the study region were taken from the Departamento de Fitotecnia of UFSM.Samplings were carried out preferably in streams margins, at less than 1 m depth sites.For the collection a Surber sampler was used (area = 0.1 m 2 ; mesh = 0.25 mm).Three subsamples were taken at each site, distant each other at least 10 m, and posteriorly pooled in one single sample.The individuals collected were fixed with 70% ethyl alcohol.

Sampling methods and identification
The specimens were identified up to genus level, through consultation to specialized bibliography (Angrisano and Korob, 2001;Lecci and Froehlich, 2007;Mariano and Froehlich, 2007;Domínguez and Fernández, 2009;Segura et al., 2011).Voucher specimens are deposited in the Coleção de Macroinvertebrados Aquáticos of the Departamento de Biologia of the UFSM.

Data analysis
In order to facilitate the comparison of EPTC diversity patterns with subtropical and tropical areas of the country, a brief inventory on genera richness per group studied (Ephemeroptera, Plecoptera, Trichoptera, Elmidae and Psephenidae) is provided, based on previous community studies selected from the literature, conducted with similar sampling effort in other regions of the Brazilian territory.
Community diversity was analyzed in relation to richness (S), number of individuals (N), relative frequency (%) and dominance.The accumulated richness in the study area was estimated through the collector's curve, obtained after the generation of 500 curves by random addition of samples.The program EstimateS 8.0 (Colwell, 2006) was used for this analysis.
The influence of abiotic factors on the communities was assessed by a Redundancy Analysis (RDA; Legendre and Legendre, 1998).For the analysis, the following variables were used: stream order, altitude, AT, WT, WC, pH, DO, EC, DBO, calcium and iron concentration, and granulometric composition (Sand, Gravel and Mud).The forward stepwise procedure was chosen to select the most important variables for the model (p<0.05,after Monte Carlo randomizations).Variables with inflation factor >20 were excluded from the analysis (ter Braak and Šmilauer, 2002).The variables retained in the model were EC, AT and stream order.Rare taxa (<10 individuals) were excluded from the biotic matrix, which was Hellinger-transformed (Legendre and Gallagher, 2001).Monte Carlo randomization test (999 permutations) was used to assess the statistical significance of the canonical axes generated (ter Braak and Šmilauer, 2002).

Results
Overall, 5,320 individuals were collected, belonging to 18 families and 52 genera (Table 1).The collector's curve did not reach the asymptote but showed a trend to stabilization (Figure 2), suggesting that a small increase in richness may be achieved with additional samplings.Ephemeroptera was represented by four families, Plecoptera, by two, and Trichoptera, by ten.The most diverse families were Elmidae (eight genera), followed by Leptohyphidae (six genera).The most abundant genera were Smicridea McLachlan, 1871, Americabaetis Kluge, 1992, Thraulodes Ulmer, 1920 and Baetodes Needham and Murphy, 1924, which represented 59% of the total of individuals (Table 1).The most well distributed genera were Americabetis and Neoelmis Musgrave, 1935, which occurred in more than 90% of the sites.Seventeen genera were rare, occurring at two sites at most (Table 1).Table 2 compares genera richness per group studied (Ephemeroptera, Plecoptera, Trichoptera, Elmidae and Psephenidae) with inventories conducted in other regions of Brazil.

Influence of abiotic factors on community composition
The model generated by the RDA was significantly different from chance (F=2.62;p=0.005).The two first axes summarized 16% of the variance in genera abundance data, and explained 89% of their relation with abiotic factors measured (Table 3).The first axis was negatively correlated with AT and EC, while the second one, positively correlated with stream order and EC (Figure 3a; Table 4).In general, the first axis of the ordination segregated sites in relation to their altitudinal variation (from 0-300 m to 300-500 m).The second axis summarized part of the environmental variation of samples, segregating samples in relation to EC and stream order (Figure 3a).
Few relationships of some taxa with abiotic factors could be derived in the ordination diagram (Figure 3b).Smicridea was positively related to AT. Thraulodes was positively to EC, while Camelobaetidius Demoulin, 1966 and Paracleodes, negatively.Americabaetis and Paracloeodes were negatively related to stream order.

Discussion
The collector's curve did not reach the asymptote, but tended to stabilization.Although collections were restricted to one period only, the sampling season (spring months) favored the occurrence of many insects which are recruited in the larval stage in warmer months (Bemvenuti, 1998).Yet, many taxa might have not been sampled due to stochastic events that affect macroinvertebrate communities, and could not be detected by sampling.Precipitation, for example, tends to interact with local factors, and destabilize the lotic ecosystem through alterations in stream flow and velocity, causing individuals drift (Bispo et al., 2001;Yokoyama et al., 2012).
The genera richness found in the study area (18 Ephemeroptera, five Plecoptera, 18 Trichoptera, eight Elmidae and one Psephenidae) might be considered high when compared to studies dealing with these taxa, conducted with similar sampling effort in subtropical regions of the country (Table 2).This result might have been determined by the location of the Toropi River basin in a slope region, a condition that enables the basin to host considerable environmental heterogeneity.In such regions, streams tend to show great variability in abiotic factors through their courses, especially in river bed sediment grain size (Alan and Castillo, 2007), providing a high number of suitable habitats for the establishment of more diverse macroinvertebrate communities (Principe and Corigliano, 2006).In lowland courses, the occurrence of macrophytes can also be expressive (Sá et al., 2013).Aquatic vegetation represents one of the main factors providing habitat and refugia for macroinvertebrates, favoring an increment in richness (Taniguchi and Tokeshi, 2004).Besides, other factors vary along the altitudinal gradient, like stream width and depth, water turbidity and coverage of riparian vegetation (Vannote et al., 1980).However, overall richness of some groups studied here is at times lower when compared to tropical climate regions (e.g.Savannah and Amazon regions; Table 2).Concerning genera richness, some aquatic insects are more diversified in tropical or subtropical climate regions, such as Ephemeroptera and Elmidae (Barber-James et al., 2008;Jäch and Balke, 2008).The relationship between warmer temperatures and higher richness are well known from the literature for many animal communities  1; subtitles for taxa: numeration follows the exact same order of taxa in Table 1.(e.g., Allen et al., 2002;Wiensa and Donoghueb, 2004;Mccain, 2005).On the other hand, Trichoptera, which is evolutionarily original from cold and temperate regions (Ross, 1967), tends to be more diverse in regions with similar conditions, as in southern Brazil, due to adaptive and historic factors.Thus, the results of this study corroborates trends suggested in previous studies that lotic ecosystems from temperate regions sustain richer communities of certain groups of macroinvertebrates than warmer regions (e.g., McKie et al., 2005;Floss et al., 2012).
The diversity patterns concerning the families representing EPT in the Neotropics were maintained in relation to studies conducted in other regions of the country (Bispo and Oliveira, 2007;Righi-Cavallaro et al., 2010).The Plecoptera is represented by six families in the Neotropics (Stark et al., 2009), but only two (Gripopterygidae and Perlidae) occur in Brazil (Froehlich, 2011); both recorded in this study.Fourteen families of Ephemeroptera are recorded in the Neotropics, ten of them in Brazil (Salles et al., 2004).In the study area only four families were recorded, and all of them were already assigned to the state (Pereira and De Luca, 2003;König et al., 2008;Siegloch et al., 2008;Milesi et al., 2009;Hepp et al., 2013).The families lacking are Polymitarcyidae, Euthyplociidae, Ephemeridae, Oligoneuriidae, Coryphoridae and Melanemerellidae).From the six families not found in the area, four (Ephemeridae, Oligoneuriidae, Coryphoridae and Melanemerellidae) are few diversified and/or more common in subtropical areas (Salles et al., 2004).The Trichoptera has 24 families recorded for the Neotropics (Flint et al., 1999), 16 in Brazil (Paprocki et al., 2004).In this study, ten families occurred.From the six families not found in the area, four (Anomalopsychidae, Atriplectidade, Limnephilidae, and Xiphocentronidae) are typical of warmer, tropical regions (states of Minas Gerais and Rio de Janeiro; Paprocki et al. (2004)).The other two (Odontoceridae and Sericostomatidae) had been registered during the 1980's in states of Santa Catarina and Paraná, which are similar in climate with Rio Grande do Sul, indicating that the occurrence of such families in the state is predictable.
The most diverse families in relation to number of genera in the Toropi River were Elmidae (eight genera), Baetidae, Leptohyphidae and Hydroptilidae (six each), which were also the most diverse in other regions of Brazil (Bispo and Oliveira, 2007;Righi-Cavallaro et al., 2010;Segura et al., 2012).Elmidae are abundant and diverse in many Brazilian streams (Segura et al., 2012), for their larvae are well-adapted to riffle areas and high levels of dissolved oxygen (Brown, 1987).Baetidae are found in many freshwater ecosystems (from riffles to temporary ponds), and even in highly impacted limnetic systems (Callisto et al., 2001).Hydroptilidae are generally found in stony substrate streams, and some genera of this family are scrapers and collector-gatherers, adhering to the substrate to feed (Stehr, 1987;Merritt and Cummins, 1996), making them typical of lotic areas.Similarly, Leptohyphidae nymphs are poor swimmers and therefore must gather to rocks or to the vegetation to feed (Stehr, 1987).
The most abundant genera in this study (Smicridea, Americabetis and Thraulodes) are also dominant in various Brazilian regions (Crisci-Bispo et al., 2007;Righi-Cavallaro et al., 2010).In southern Brazil they are also frequent (Spies et al., 2006;Siegloch et al., 2008).The caddisfly Smicridea is generally abundant in streams, occurring from headwaters to large rivers (Flint et al., 1999), and their individuals are typical of stony substrate streams (Spies and Froehlich, 2009), like the streams of this study.Thraulodes is a mayfly typical of low-order and well-preserved streams, living on rocks (Cardoso et al., 1997).Thus, the occurrence of this genus in the study area reflects not only its preference for the order of the streams sampled, but also the environmental condition of some of them.Many streams and tributaries in the Toropi River basin still present well-preserved banks.The other mayfly (Americabaetis) is also widely distributed and found in many habitats, from sites with riparian and aquatic vegetation to highly impacted ones (Siegloch et al., 2008;Domínguez et al., 2006).Despite Ephemeroptera be considered characteristic of 'clean' waters, Baetidae (the family which Americabaetis belongs to) is classified as 'sensitive' in biotic indexes (Hilsenhoff, 1988).This moderate sensitivity and tolerance possibly explains the elevated abundance of this genus in the study region.
The caddisfly Austrotinodes Schmid, 1955 and Celaenotrichia Mosely, 1934 are recorded for the first time in Rio Grande do Sul.To date, Austrotinodes had only been recorded in tropical and subtropical areas of the country, and Celaenotrichia had not been recorded for the southern region of Brazil (Paprocki et al., 2004).The elmid Genus M, which had only been recorded for the São Paulo state (Segura et al., 2011), is also a first record for Rio Grande do Sul.

Influence of abiotic factors on community composition
The influence of climatic, spatial, and anthropogenic drivers on EPTC communities was observed, although it had been stronger on few genera.The positive relation of Smicridea with AT is better understood by analyzing its distribution in Brazil.Smicridea can be found in warmer regions of the country (Oliveira and Bispo, 2001;Pes et al., 2005;Souza et al., 2013), and some of its species (e.g., Smicridea truncata Flint, 1974) occur in the tropical Amazon region (Pes et al., 2008).Three genera were inversely related to stream order.Species of Camelobaetidius are sensitive to pollution and commonly found at well-preserved and coarser substrate sites (Buss and Salles, 2007), usual features of low-order streams.Paracloeodes can also be frequent in low-order sites (Salvarrey et al., 2014).Americabaetis, although a normally widely distributed genus, is also associated to the presence of riparian vegetation (Domínguez et al., 2006), which is usually better preserved in low-order stretches (Vannote et al., 1980).In fact, those conditions (coarser substrate, presence of riparian vegetation and lower pollution) are typical from higher elevations (Vannote et al., 1980).
Thraulodes was positively influenced by EC, despite being also found at well-preserved sites (Cardoso et al., 1997).High EC values are normally related to anthropic activities, like agriculture (Stewart et al., 2000), which is more common in low elevation areas (Lenat and Crawford, 1994), indicating that this genus might be tolerant to human activities.Therefore, the spatial distribution of both the fauna and abiotic drivers in this study is linked to the elevation gradient of the region.Modifications in abiotic community drivers in lotic ecosystems according to altitude are well known in literature, due to combining roles of watershed geology, climatic constraints and distribution of human activities (Miserendino, 2001;Jacobsen, 2004Jacobsen, , 2008)).

Final Remarks
The Toropi River basin hosts high diversity of genera of Ephemeroptera, Plecoptera, Trichoptera and Elmidae coleopterans in relation to other basins from Rio Grande do Sul and other states of Brazil.Although collections were temporally restricted, 52 genera were found, and three of them are new records for the state.The elevated number of genera found in the study area is due to the environmental heterogeneity of the streams located in slope areas, as observed in other studies (Floss et al., 2012;Sá et al., 2013).Additionally, it is verified that the elevation gradient of the study area affected climatic, anthropic and spatial drivers, such as air temperature, stream order and electrical conductivity, which, in turn, influenced the distribution of some genera (e.g., Camelobaetidius, Paracloeodes, Americabaetis, Smicridea and Thraulodes).Thus, this study evidences that streams from slope areas in the Southern Plateau (Planalto Meridional) deserve special attention from future aquatic conservation and assessment programs, for they host high levels of biodiversity.

Figure 1 .
Figure 1.Location of the study area and the sampling sites.

Figure 3 .
Figure 3. Ordination diagrams of the Redundancy Analysis of Ephemeroptera, Plecoptera, Trichoptera and Coleoptera (Elmidae and Psephenidae).a) biplot of abiotic factors and sampling sites; b) genera distribution in the canonical space.Subtitles for sites: see Table1; subtitles for taxa: numeration follows the exact same order of taxa in Table1.

Table 1 .
Composition, number of individuals (N), frequency (%) and number of occurrences in relation to the total number of sampling sites (NO) of Plecoptera, Ephemeroptera, Trichoptera and Coleoptera (Elmidae and Psephenidae) genera in the Toropi River basin, southern Brazil.(Ab = genera name abreviation).

Table 3 .
Eigenvalues, taxon-environment correlation coefficients and explained cumulative percentage of variance of the first two axes of RDA of EPT and Coleoptera (Elmidae and Psephenidae) communities in the Toropi River basin, southern Brazil.

Table 4 .
Inter-set correlations between the two first axes of the RDA and the abiotic factors measured at Toropi River basin, southern Brazil.