Habitat heterogeneity on feeding habit of two sympatric and congeneric characidae fi shes in two tropical reservoirs

Food fl exibility and omnivory are important features pronounced in Neotropical freshwater fi sh species, particularly for Astyanax species. Traditionally most fi sh diet resources are known to be originated in the aquatic environment, however recent studies have pointed to the importance of allochthonous sources. Besides, the colonization of macrophytes, common at several tropical reservoirs, may enhance insectivory in fi sh diet expanding or concentrating the area of available resources for feeding. Here we employed stomach contents analysis of two sympatric Astyanax species to access the importance of habitat diff erentiations as spatial complexity in two tropical reservoirs with diff erent environmental features. The NMDS analysis indicated separation in the diet of these species between reservoirs (Stress= 8.28%). Additionally, analysis of variance revealed a signifi cative diff erence in the volume of food itens ingested between the reservoirs (Anova one-way F(1, 132)=4.4446; p= 0.037). This points out the importance of environmental conditions on the composition of the diet of fi shes. This study highlighted the insectivorous feeding habit of Astyanax species and revealed diff erent feeding strategies between sympatric fi shes despite high niche overlap in both environments. Habitat heterogeneity increasing food resources availability plays an important role in the diet strategy of these Astyanax species and on their constant maintenance in the two diff erent reservoirs.

ABSTRACT.Food fl exibility and omnivory are important features pronounced in Neotropical freshwater fi sh species, particularly for Astyanax species.Traditionally most fi sh diet resources are known to be originated in the aquatic environment, however recent studies have pointed to the importance of allochthonous sources.Besides, the colonization of macrophytes, common at several tropical reservoirs, may enhance insectivory in fi sh diet expanding or concentrating the area of available resources for feeding.Here we employed stomach contents analysis of two sympatric Astyanax species to access the importance of habitat diff erentiations as spatial complexity in two tropical reservoirs with diff erent environmental features.The NMDS analysis indicated separation in the diet of these species between reservoirs (Stress= 8.28%).Additionally, analysis of variance revealed a signifi cative diff erence in the volume of food itens ingested between the reservoirs (Anova one-way F(1, 132)=4.4446;p= 0.037).This points out the importance of environmental conditions on the composition of the diet of fi shes.This study highlighted the insectivorous feeding habit of Astyanax species and revealed diff erent feeding strategies between sympatric fi shes despite high niche overlap in both environments.Habitat heterogeneity increasing food resources availability plays an important role in the diet strategy of these Astyanax species and on their constant maintenance in the two diff erent reservoirs.
Trophic interactions and quantifying spatial and temporal variability in fi sh diet remains a challenge for ecologists (Scheffer & Carpenter, 2003;McCann et al., 2005).Food fl exibility and omnivory are important features particularly pronounced in Neotropical freshwater fi sh species (Lowe-McConnell, 1999;González-Bergonzoni et al., 2012).The food variety in the tropics can be associated with environmental conditions of the aquatic ecosystem.May be infl uenced directly by characteristics of land, vegetation, human activities in the drainage systems and the driving forces of dry and rainy period (Manna et al., 2012;Xu et al., 2012).Furthermore, the fi sh capacity to explore this broad food supply is directly connected with the biology of species (Abelha et al., 2001).
Many resources sustain fish fauna in tropical reservoirs and most of them originate in the aquatic environment (Araújo-Lima et al., 1995).However, morphometric features of the reservoir, such as perimeter and depth, retention time, characteristics of the area flooded and elapsed time from filling can influence the availability of the resources for fish.Recent studies have pointed to the importance of allochthonous sources for fish feeding mainly coming from marginal areas of dendritic reservoirs and with riparian forests (Araújo et al., 2005;Mazzoni & Rezende, 2003;Silva et al., 2014).Besides spatio-temporal variations in food availability strongly modulate foraging behavior (López-Bao et al., 2011) and seasonal variation in the quantity and also quality of the diet (Junk, 1980) may be a consequence of the hydrological regime of reservoirs (Abelha et al., 2001).
The colonization of macrophytes in many reservoirs may enhance aspects of invertivory in fish diet (Pelicice & Agostinho, 2006) expanding or concentrating the area of available resources for feeding.Aquatic macrophytes are important habitats for many groups of organisms as immature stages of Diptera, Ephemeroptera and Trichoptera (Carpenter & Lodge, 1986;Lalonde & Downing, 1992;Dibble et al., 1996;Agostinho et al., 2003;Casatti et al., 2003) and many fish species are found in these habitats, benefiting from a diversity of food items as well as refuge from predation (Pelicice & Agostinho, 2006).Macrophytes have also been considered key components in aquatic environments, enhancing the spatial heterogeneity and increasing the number of niches (Margalef, 1983;Trivinho-Strixino & Strixino, 1993;Santos et al., 2008).
We employed stomach contents analysis to access the importance of habitat differences as spatial complexity for Astyanax feeding in two tropical reservoirs with different environmental features.This was investigated for Astyanax aff.bimaculatus Linnaeus, 1758 and Astyanax parahybae Eigenmann, 1908 (Characiformes: Characidae), which are sympatric species with frequent occurrence in both environments (Gomes et al., 2008;Uehara et al., 2015) and similar diets.Given the differences between these reservoirs, and based on aspects mentioned above we predicted that the environment where fishes inhabit will control the composition of the diet (niche width) influencing the trophic relation between the sympatric species (niche overlap) more than seasonality.

MATERIAL AND METHODS
Study area.This study was conducted in two reservoirs belonging to the complex of electrical power company Light SA (Piraí Municipality, Rio de Janeiro State, Brazil).The Ribeirão das Lajes Reservoir (Lajes Reservoir) (22°42'-22°50'S, 43°53'-44°05'W) situated at 415-430 m above sea level, has a surface area of 30.7 to 47.8 km 2 and an average depth of 15 m and a maximum of 40 m and retention time of 300 days.The reservoir presents oligo-mesotrophic waters, used for the domestic water provision of around 1.1 million people.The high water quality of the Lajes Reservoir has been associated to the presence of a rain forest in most of its surroundings and low human influence.The dendritic shape of the lake and no abrupt fluctuations in water level impose an important ecotone between the aquatic and terrestrial areas, subject to direct exploitation by aquatic fauna.
The Santana Reservoir is located 361-363 m above sea level (between 22°31'50"S and 43°49'15"W).It was built in 1945, has a surface area of approximately 5.23 km 2 , with an average depth of 3.3m and a maximum of 12 m and a retention time of one day.The reservoir is considered eutrophic and is characterized by a high degree of human impact, since its waters receives drainage from urban, industrial and agricultural activities.The surroundings are covered by pasture and the continued nutrient input has resulted in the proliferation of macrophytes throughout the lake.
The climate of the region where the reservoirs lie is classified as high-altitude tropical with average temperatures of 20.5° C ranging from 16.6° C in July to 23.9° C in February.The months with the highest rainfall are November, December and January and the months with the least rainfall are May, June and July.The Lajes Reservoir level is maintained artificially and the highest level is reached at the end of the rainy season (April) and the lowest at the beginning of this season (November).There is no influence of seasonality on the level of the Santana Reservoir, which is managed according to operational needs and daily variations of up to 1.5 meters can occur.
Sampling.Adult fish were collected at both reservoirs in dry (May 2005, June/July 2007) and rainy (March 2004, January 2008) seasons, by means of gill-nets with mesh sizes ranging from 1.5 to 4.0 cm.The samplings followed a previously established standardized effort in the two sites, considering the amount of nets, as well as the total hours of capture.Gill-nets were placed parallel to the shore or in closed meanders and capture time was 12 hours, from dusk to sunrise.Since fishes can modify their food habits during different life stages, we selected only adult individuals based on their standard length that ranges from 6.5 to 12.5 cm (Astyanax aff.bimaculatus) and 7.1 to 12.5 cm (Astyanax parahybae).The fish were weighed, measured (standard length), and killed by freezing immediately after collection.The fish stomach content was removed at the laboratory and fixed in 10% formalin.All voucher specimens were deposited in the Collection of the Museu Nacional (Universidade Federal do Rio de Janeiro -UFRJ), under the code MNRJREG20050418.
Data analysis.The stomach content analysis was performed according to methodology proposed by (Windell, 1971;Hyslop, 1980;Branco et al., 1997).The food items were grouped into categories [Algae, Egeria (aquatic macrophyte), Terrestrial Plant, Zooplankton, Ostracoda, Nematoda, Acarina, Aranae, Gastropoda, Aquatic Insect, Terrestrial Insect, Fish Scale, Organic Matter and Sand].The taxonomic identification of food items was done according to appropriated literature and the insects were identified to family level.Based on the data of frequency of occurrence (% F) and volume percentage (V%), the Alimentary Importance Index -IAi was calculated (Kawakami & Vazzoller, 1980) -IAi = (%F x %V)/ å (%F x %V).To examine spatial patterns in fish diet, the volumes of the diet items were ordered using non-metric multidimensional scaling (NMDS) with a Bray-Curtis dissimilarity index.A parametric one-way ANOVA was performed to test diet differences, also a similarity percentage analysis (SIMPER) was used to examine the contribution of each food item to the average dissimilarity between species in each reservoir.All the analyses were performed using the program R (R Core Team, 2013).

RESULTS
We analyzed the stomach contents of sixty-five specimens of Astyanax aff.bimaculatus and seventy of A. parahybae.The food items were gathered in 14 food items categories shown in Tab.I. Insects and organic matter were the most important category items for both species.Some changes at the importance of some categories were found between rainy and dry seasons The ordering produced by NMDS analysis indicated separation in the diet of these species between reservoirs (Stress= 8.28%), and a slight influence of seasonality (Fig. 1).This points out a higher importance of environment conditions in the composition of the diet of fishes than the seasonality.Additionally, analysis of variance revealed no significant differences in food items between the species studied (Anova one-way F(1, 133)=1.9569,p=0.164) and between rainy and dry seasons (Anova one-way F=(1,132)=0.061;p= 0.805).However, we found a significative difference in the volume of food items between the reservoirs (one-way Anova F(1, 132)=4.4446;p= 0.037).
The SIMPER analysis showed which categories items were the most important in the differentiation of diets.On Lajes Reservoir, organic matter and insects, mainly terrestrial insect had greater importance for Astyanax aff.bimaculatus and aquatic insect for A. parahybae diet.On Santana Reservoir, organic matter and terrestrial insect were important itens for both species.The secundary items consumed by the fish were responsible for diet differences in this reservoir.Astyanax parahybae fed on algae and on aquatic and terrestrial vegetation probably near macrophytes patches.Astyanax aff.bimaculatus preyed on invertebrates associated to terrestrial vegetation, e.g., gastropods (Tab.II) showing different strategies on food acquision.
We also analyzed the niche width for both species that was considered low, between 0.17 and 0.35, for possible values between 0 and 1 (Fig. 2).The highest value of niche width was found for Astyanax aff.bimaculatus in Santana Reservoir and the lowest for the same species in Lajes Reservoir.The niche overlap for both species was higher for Lajes Reservoir (Fig. 2).

DISCUSSION
Also classified as opportunistic species (Dias et al., 2005), Astyanax display a great ability to change their prey according to environmental variations.This study confirmed the insectivorous-omnivorous habit and feeding plasticity for both Astyanax species, as indicated by several other authors (e.g.Nomura, 1975;Arcifa et al., 1991;Uieda et al., 1997;Bennemann et al., 2006;Abilhoa, 2007) and also highlighted the insetivorous feeding habit, likewise displayed by other Astyanax species specially in streams and rivers (see Vilella et al., 2002 for A. eigenmanniorum;Vidotto-Magnoni &Carvalho, 2009 for A. altiparanae andFerreira et al., 2012 for A. paranae).At this context, diet dissimilarities between the Astyanax species were revealed mainly due to the source of itens consumed.Feeding strategies may differ among environments and omnivory may evolve as a response to high environmental heterogeneity (Chubaty et al., 2014) or food availability.
Given the habitat differences between these reservoirs, different spatial complexity and presence of macrophyte patches, we expected dissimilar diet patterns.Our results showed that Astyanax aff.bimaculatus diet consisted of insects, terrestrial and aquatic insects, as well as the low consumption of zooplankton, which suggest feeding habit on the water column surface and near marginal area in both reservoirs.Meanwhile, aquatic insects and zooplankton had an inexpressive importance at Santana Reservoir diet, but algae had high significance.On the other hand, A. parahybae exhibited different preferences in each reservoir, aquatic  Tab.I. Food items found in the diet of Astyanax aff .bimaculatus Linnaeus, 1758 andA. parahybae Eigenmann, 1908 in the Lajes (Lajes) and Santana reservoirs, state of Rio de Janeiro, Brazil in rainy and dry seasons (N, number of individuals analysed; IAi, Alimentary Importance Index; SL, range of standard length).
The diet of Astyanax aff.bimaculatus in the environment with less impacted surrounding vegetation (Lajes Reservoir) showed that the individuals consumed a large proportion of allochtonous items, especially insects.This high consumption of allochthonous items must be associated with preserved surrounding vegetation, essential for the input of several items in the aquatic environment by rain, wind or simply falling into the water.Astyanax parahybae diet, also at Lajes reservoir, is more focused on autochthonous items such as aquatic insects.Besides the contribution of allochthonous items, riparian vegetation also provides the permanence of aquatic insect larvae that feed, directly or indirectly, on allochthonous organic material imported from riparian vegetation (Castro, 1999;Abilhoa, 2007).This may emphasize the importance of ecotone landwater zone for fish forraging behavior ( Dias et al., 2005;Barreto & Aranha, 2006;Casatti, 2010).
Dropping the riparian vegetation, as found in Santana Reservoir, would lead to a reduction of allochthonous resources.However, Astyanax aff.bimaculatus and A. parahybae diets revealed ingestion of large amounts of allochthonous items, such as terrestrial insects in this reservoir.Besides, we found a great proportion of algae, macrophytes and gastropods in fish diets in such reservoir.The presence of macrophytes patches apparently enhances the diversity and richness of animal groups at littoral zone and interfering with the dynamics of communities at the lake ecosystem as a whole (Margalef, 1983;Trivinho-Strixino & Strixino, 1993;Duncan & Kubecka, 1995;Peiró & Alves, 2006;Vidotto-Magnoni & Carvalho, 2009).For example, Ephemeroptera, the order of insects most consumed by A. parahybae in this reservoir, according to Casatti et al. (2003) can be associated to aquatic macrophytes.Thus, we can infer that the macrophytes pacthes probably influences the items used by Astyanax species at Santana Reservoir, even as the operational actions with daily level variations (Gomes et al., 2008) that enhance the input of allochtonous items at fishes diet (Abujanra et al., 2009).
Our results revelead similarities between Astyanax species food resources.The niche overlap can be considered high for both environments, which would be associated with the high abundance of the same resources.Despite the availability of resources was not measured in the present study, we assume that the preyed items were abundant based in high dominance by few resources (Novakowski et al., 2008).Other factors such as the combination of different microhabitats, periods of activity and strategies of foraging can allow coexistence when species present high niche overlap.This resource sharing represent a situation commonly found in tropical streams (Aranha et al., 1998;Casatti, 2002).A previous study at Lajes Reservoir also revealed high trophic niche overlap of Astyanax species and suggested ecomorphological differences as a way to probably decrease competition (Silva-Camacho et al., 2014).
Although the two species of Astyanax had have a high niche overlap, A. aff.bimaculatus showed a narrow niche breath at Lajes Reservoir.These findings may indicate that A. aff.bimaculatus are better adapted to lentic environments (Silva-Camacho et al., 2014).Also, this can show some strategy to avoid diret competition between these species at Lajes Reservoir.On Santana Reservoir, an extremely variable flood system under more antropic influence, they had to explore different habitat and feeding resources resulting in a wider niche breath.Besides the similar basic diet for these two species and high niche overlap, they drew a slight different diet for both reservoirs.These findings corroborate the importance of trophic plasticity in Astyanax species leading to a reduced competition in these environments (Araújo-Lima et al., 1995).
In conclusion, this study showed that these sympatric fishes despite presenting a similar diet can coexist with a low feeding competition over the reservoirs studied.Whether competition for food occurs, it is minimized by trophic plasticity and spatial segregation.Habitat heterogeneity plays an important role on food resource consumed by these Astyanax species.However, further studies are needed to allow the detection of possible changes over time, mainly to explore temporal effects of anthropogenic habitat modifications, as frequently found at reservoir environments.
Tab. II.SIMPER analyses of item category contributing to differences in Astyanax aff.bimaculatus Linnaeus, 1758 and A. parahybae Eigenmann, 1908 diet structure in each reservoir, State of Rio de Janeiro, Brazil.