Fish fauna of headwater streams of Perobas Biological Reserve , a conservation unit in the Atlantic Forest of the Northwestern Paraná State , Brazil

organizations (Aquino et al. 2009). Although the freshwater fishes of the Upper Paraná River have been relatively well known (Agostinho et al. 1997; 2007), this information has been concentrated in a few parts of this large area (Langeani et al. 2007; Agostinho et al. 2008; Súarez, 2008). Studies conducted by Maier et al. (2008) in the Barra Bonita River and by Araújo et al. (2011) in Itiz stream, both tributaries of the Ivaí River, indicated the presence of additional new species. These findings demonstrated that by increasing sampling efforts, mainly in the small tributaries, additional and new species could still be recorded. The objective of the present study was to develop a taxonomic list of the fish fauna in streams that arise in or flow through the Perobas Biological Reserve, in order to provide a basis for the establishment of strategies for conservation of these aquatic systems.


Introduction
The Perobas Biological Reserve (Reserva Biológica das Perobas or "Rebio das Perobas") is a Federal Conservation Unit, and comprises the largest remnant forest in northwestern Paraná, Brazil.The reserve covers an area of 8,716 ha located in a region of contact between Semideciduous Seasonal Forest and Mixed Ombrophilous Forest, two physiognomies of the Atlantic Forest sensu lato existing in Paraná State (Joly et al. 1999).It is considered one of the priority areas for protection of these types of forest formations (ICMBio 2012).The Rebio das Perobas is also part of the Atlantic Forest Biosphere Reserve and part of the UNESCO Man and the Biosphere program, as a core zone, for which the main objective is conservation of biodiversity (Lino et al. 2009).With endangered status, the Atlantic Forest is one of the world's 25 biodiversity hotspots that support a very high species richness and endemism (Tabarelli et al. 2005;Abell et al. 2008;Nogueira et al. 2010).
The Rebio das Perobas is located at the junction of two major watersheds of the Paraná State (Ivaí and Piquiri), contributing to the formation of numerous small watercourses, with preserved headwaters and many protected springs.The rivers that drain these basins are among the main tributaries of the Paraná River, which is the second-largest drainage system in South America, with an area of 3.2 million km 2 (Lowe-McConnell 1999).In the state of Paraná, the drainage areas related to the Upper Paraná River basin occupies about 128,507 km 2 (Parolin et al. 2010), in which about 400 species of fishes are reported (Abilhoa and Duboc, 2004;Langeani et al. 2007;Cetra et al. 2012).
Biological inventories in Conservation Units are crucial for the planning and establishment of management strategies by governmental and non-governmental Abstract: The Perobas Biological Reserve is a Conservation Unit characterized by two physiognomies of the Atlantic Forest: Semideciduous Seasonal Forest and Mixed Ombrophilous Forest.The reserve is situated on a ridge watershed between the basins of the Piquiri and Ivaí rivers, Upper Paraná River and had its fish fauna surveyed for the first time.Fishes were collected quarterly from December 2010 to March 2012, using electrofishing in five stretches of 50 m along the four streams, and gillnets only at one site in December, 2010.A total of 2628 individual were collected, belonging to 34 species in 25 genera, 13 families and six orders.Ten taxa may be new species or part of a complex of species within each genus, and requiring more detailed taxonomic revisions.Nine species are the first records for the Piquiri river basin.located in the São João Mountains, and the river flows for 485 km until it discharges into the Paraná River.The most important tributaries of the right margin are the Goio-erê, Tricolor and Cantú rivers (Parolin et al. 2010).
The land in the vicinity of the Perobas Biological Reserve is mainly devoted to agriculture, where the most important crops are sugarcane, soybeans, cassava and corn (maize), as well as pastures.Potential environmental impacts are increasing soil erosion, siltation of water resources, and pesticide contamination.The main physical characteristics and geographical coordinates are shown in Table 1 and Figure 2.

Fish sampling
Fishes were collected using two methods: the electrofishing technique in five sampling reaches in four streams of third to fourth order, located inside and surrounding the Perobas Biological Reserve, quarterly from December 2010 to March 2012 (Table 1, Figures 1  and 2).We used electrofishing with three passes of 40 minute in extensions of 50 meters at each reach.The electrofishing equipment was powered by a portable generator (Honda, 2.5 kW, 220 V, 3 -4 A) connected to a DC transformer, with two electrified net rings (anode and cathode).Output voltage varied from 400 to 600 V.For both types of sampling we installed blocking nets (mesh size 0.5 mm) at the ends of each reach to prevent any fish escaping.In the Índios River, downstream reach (Table 1, Figures 1 and 2), that had depth greater than 1.5 m, we made a single sampling in December 2010 using gillnets with simple (2.4, 3, 4, 5, 6 and 7 cm between non-adjacent knots) and trammel nets (three layers; internals meshes of 6 and 7 cm), all 15 m long.Gill nets were set for 24 hours, with checks in the morning (08:00 h), afternoon (16:00 h) and evening (22:00 h).
After sampling, the collected specimens were sacrificed in a solution of clove oil (Eugenol, 2 drops per litre; cf.American Veterinary Medical Association 2001), and fixed in 10% formalin.Fish were collected under the license of the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) (Process numbers 24196-1/22/06/2010 -27252-1/02/03/2011). Specimens were identified in the laboratory according to Graça and Pavanelli (2007), and then preserved in 70% alcohol.Voucher specimens were deposited in the fish collection of Nupélia (Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura), of the State University of Maringá, Brazil.

Results and Discussion
At the six sites sampled, 2628 individual fish were collected, belonging to 34 species in 25 genera, 13 families and six orders (Table 2).Only Poecilia reticulata Peters 1859 and Erythrinus erythrinus (Bloch and Schneider 1801) were considered non-native species, and 10 species are probably undescribed.Based on species number, the most important order was Siluriformes, with 14 species (41.2%), followed by Characiformes with 11 species (32.4%).Loricariidae and Characidae were the families with the largest number of species (Table 2).The predominance of Characiformes and Siluriformes is a common pattern in the Neotropics (Lowe-McConnell 1999; Buckup et al. 2007), and has also been found in several surveys of the fish fauna in streams of different basins in Paraná (Maier et al. 2008;Cunico et al. 2009;Gubiani et al. 2010a;Araújo et al. 2011).
This survey found a higher proportion of Siluriformes, especially due to the presence of several species of armored catfishes (Loricariidae) and small catfishes (Heptapteridae).The relatively high richness of Siluriformes is correlated with the characteristics of the habitat.The higher numbers of environments with riffles and rocks bottoms, as well as streamside vegetation (grasses) submerged in some reaches, favors the occurrence of individuals belonging to this taxon (Súarez et al. 2007;Araújo et al. 2011;Pagotto et al. 2011).The other orders recorded (Gymnotiformes, Cyprinodontiformes, Perciformes and Synbranchiformes) are widely distributed in Brazilian hydrographic basins, and generally show lower species diversity (Langeani et al. 2007;Oliveira et al. 2009;Cetra et al. 2012;Volcan et al. 2012).
Surveys in the main channel of the Piquiri River were conducted by Agostinho et al. (1997;2004), who recorded 57 fish species; and by Gubiani et al. (2010b), who found 69 species.In our study, were caught nine species that did not appear in three previous surveys.Thus, are new records for the river basin Piquiri the following species:  only electrofishing, and our samples were taken only in the headwater streams.Thus, in this context, our study constitutes the first listing for streams in the Piquiri River basin, one of the least-inventoried basins in the state and more threatened by future construction of dams (ANEEL 2009).
With respect to the three constant species in our list belonging to the genus Gymnotus, two of them were probably previously recorded as Gymnotus carapo Linnaeus 1758.According to Graça and Pavanelli (2007), specimens of Gymnotus inaequilabiatus (Valenciennes 1839) and Gymnotus sylvius Albert and Fernandes-Matioli 1999 may have been erroneously identified as G. carapo.Thus, the occurrence of these species in the region may be natural.In any event, Graça and Pavanelli (2007) also did not discard the hypotheses that the occurrence of these two species, as with Gymnotus pantanal Fernandes, Albert, Daniel-Silva, Lopes, Crampton and Almeida-Toledo 2005, may be the result of inter-basin movement, through the use of specimens as live bait or also through invasion from the Upper Paraná River after the Itaipu Reservoir was formed.A similar situation with respect to identification occurs in the case of genus Hoplias.According to Pazza and Júlio Júnior (2003), three cytotypes occur in the upper Paraná River region, and specimens of these were all previously identified as Hoplias aff.Malabaricus (Bloch 1794).In our study, we differentiated them as Hoplias sp. 2 and Hoplias sp. 3, according to Graça and Pavanelli (2007).
The fish fauna was characterized by small-sized specimens; about 75% had a mean standard length (SL) lower than 150 mm.Only Crenicichla britskii Kullander 1982, Gymnotus inaequilabiatus, G. pantanal, G. sylvius, Hypostomus ancistroides (Ihering 1911), Hoplias sp. 2, Hoplias sp. 3, Rhamdia quelen (Quoy and Gaimard 1824) and Symbranchus marmoratus reached greater lengths.Castro (1999) stated that the predominance of small-sized species is the only general pattern found for the ichthyofauna of streams in the Neotropical region.The species generally show a strong dependence on riparian vegetation, and have little or no commercial value (Cetra et al. 2012).However, they are important in the trophic relationships of these ecosystems.
Of the total species collected, only four were common to all the sampling reaches: Astyanax altiparanae Garutti    Reis et al. (2003).(Graça and Pavanelli 2007) and that the many individuals caught (191 in a single collection) showed mature gonads (personal observation), we can suggest that the Índios River is a spawning ground for this species.Thus, it is appropriate to emphasize the importance of this river as a biodiversity corridor, interconnecting the water drainage net on the north and east sides of the Rebio das Perobas with the Ivaí River.This information becomes even more important when one considers that projects to construct small electrical-energy production centers (PCHs) exist on this river.On the other hand, 10 taxa were recorded only at the points belonging to affluent streams in the Piquiri River basin, which represents about 30% of the total richness.These results indicate differences in the species composition among the streams of the basins of the Piquiri and Ivaí rivers.Notably, of the 34 species collected, 10 have unresolved taxonomy (i.e., are still undescribed and lack a specific epithet), and may be new species or part of a species complex within each genus, and therefore require moredetailed taxonomic revisions.These results confirm the statements of Langeani et al. (2007) and Maier et al. (2008) that, although the fish fauna of the Upper Paraná River is among those most intensively investigated, the number of presently known species is far from representing the reality, especially in stream and headwater environments.This lacuna, allied to the status of the Atlantic Forest as one of the two recognized biodiversity hotspots in Brazil, with a large number of hydrographic basins subject to harmful human actions, emphasizes the importance of surveys and studies of distribution in Conservation Units.Nogueira et al. (2010) suggested that the number of threatened species of freshwater fishes in Brazil is at least four times higher than presently indicated by the world and national red lists.
The number of species in this checklist may well undergo changes with respect to the valid species names.Nevertheless, this is the first list of the fish fauna from this Conservation Unit, one of the few areas with native vegetation remaining in northern and northeastern Paraná.We hope that the checklist will serve as a reference to aid in the management of the Perobas Biological Reserve, in environmental education programs for the surrounding communities, and as a basis for comparisons for present and future studies of the ecology of fishes of streams.

Table 2 .
List of fish and their respective occurrences at sampling reaches of the Rebio das Perobas, Ivaí and Piquiri River basin, state of Paraná, Brazil.Systematic positions were based on