Evidence of the color pattern variation in populations of Gymnotus pantanal ( Gymnotiformes ) from three streams in the upper Paraná River basin , Brazil

Programa de Pós-Graduação em Recursos Pesqueiros e Engenharia de Pesca, Universidade Estadual do Oeste do Paraná. Rua da Faculdade, 645, 85903-000 Toledo, PR, Brazil. frana_gerpel@yahoo.com.br (VAF) Universidade Estadual de Maringá, Centro de Ciências Biológicas, Departamento de Biologia, Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (Nupélia). Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil. weferson@nupelia.uem.br (WJG) (corresponding author) Centro de Engenharias e Ciências Exatas, Gerpel, Grupo de Pesquisas em Recursos Pesqueiros e Limnologia, Docente do Programa de PósGraduação em Recursos Pesqueiros e Engenharia de Pesca e do Programa de Pós-Graduação em Conservação e Manejo de Recursos Naturais, Universidade Estadual do Oeste do Paraná. Rua da Faculdade, 645, 85903-000 Toledo, PR, Brazil. egubiani@yahoo.com.br (ÉAG) Centro de Ciências Biológicas e da Saúde, Getech, Grupo de Pesquisa em Tecnologia de Produção e Conservação de Recursos Pesqueiros e Hídricos, Docente do Programa de Pós-Graduação em Recursos Pesqueiros e Engenharia de Pesca e do Programa de Pós-Graduação em Conservação e Manejo de Recursos Naturais, Universidade Estadual do Oeste do Paraná. Rua Universitária, 2069, 85819-110 Cascavel, PR, Brazil. vpmargarido@unioeste.br (VPM) Evidence of the color pattern variation in populations of Gymnotus pantanal


Introduction
The genus Gymnotus Linnaeus comprises 35 valid species of electric fishes, including aggressive and nocturnal gymnotiform eels of shallow freshwaters distributed from southern Mexico to Argentina (Cognato et al., 2007;Maxime & Albert, 2009;Richer-de-Forges et al., 2009).Gymnotus pantanal was described by Fernandes et al. (2005) in the Paraná-Paraguay system, in Brazil and Paraguay, and in the River Chapare-Mamoré, in Bolivia.Fernandes et al. (2005) used morphological, cytogenetic, and molecular data to describe that species; in their diagnosis color pattern was the main morphological trait: 'Gymnotus pantanal differs from other members of the G. pantherinus Steindachner speciesgroup (except G. anguillaris Hoedeman) in possessing a color pattern composed of thin obliquely-oriented pale pigment bands (about one third the width of the dark bands) with wavy margins restricted to the ventral portion of the body (rarely extending above the lateral line) on the anterior half of the body'.Later, Graça & Pavanelli (2007) and Margarido et al. (2007) reported this species for the upper Paraná River basin.
In samples collected recently in two streams in the urban area of Toledo, upper Paraná River basin, southern Brazil, some specimens of Gymnotus exhibited a different color pattern.Considering the importance of color in the taxonomy of Gymnotus, we analyzed those specimens based on morphological and cytogenetical data with the main objective to investigate if the specimens with different color patterns belongs to the species G. pantanal.

Study area
This study was carried out in three first-order streams (Strahler, 1957; Fig. 1) located within the urban perimeter of Toledo, State of Paraná, southern Brazil, within the sub-basin Paraná III.This sub-basin is composed of several microbasins with peculiar characteristics, and, among those, the micro-basin of the River São Francisco Verdadeiro stands out.This micro-basin is formed by several rivers and streams, and due to human activities, it became a highly-impacted area (Gubiani et al., 2010).As a result, the streams flowing in the city show different pollution levels.The micro-basin of the river São Francisco Verdadeiro flows through the cities of Cascavel, Toledo, Ouro Verde do Oeste, São José das Palmeiras, Entre Rios do Oeste, Marechal Cândido Rondon, and Pato Bragado, and it runs along ca.10,000 rural properties.
Three streams were selected in Toledo: Panambi, Pinheirinho, and Jacutinga.The headwaters of the stream Panambi are located downtown; its margins are totally occupied by residences, and it receives domestic and industrial sewage.The headwaters of the stream Pinheirinho are located outside the city and are affected by agriculture; in its medium portion the stream is also impacted by residences.The stream Jacutinga is less affected by residences; though it is affected by aquaculture and agriculture (see Gubiani et al., 2010).

Fish sampling
Fish were sampled bimonthly from October 2007 to February 2009 in the selected streams at three different zones (headwater, middle and mouth) (Fig. 1).The length of the sampling transect in each site was 40 m, which is slightly longer than recommended (35 times the stream's width, Simonson & Lyons 1995).We used electrofishing, an efficient method for collecting small species (Severi et al., 1995), in lotic environments (Mazzoni et al., 2000).The electrofishing equipment was powered by a portable generator (HONDA, 2.5 kW, 220 V, 3-4 A) connected to a DC transformer and two electrified net rings (anode and cathode).Output voltage varied from 400 to 600 V.Each transect was sampled three times from downstream to upstream by four people in a constant fishing effort of 30 min, following Esteves & Lobón-Cerviá (2001).Both extremities of the sampled transect were blocked by a net (0.5 cm mesh) to prevent fish from getting in and out of the sampling site.Institutional acronyms are described on the website: http://research.calacademy.org/research/ichthyology/catalog/abtabr.html.Voucher specimens [NUP 9311 (5) and NUP 9312 (11)] were deposited in the fish collection of Nupélia (Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura), at Maringá State University, Brazil (http://www.nupelia.uem.br/colecao).(2005).Total length and head length expressed in mm.Anal-fin length, body depth, and body width as percentage total length.Other measurements are expressed as percentage head length.Gymnotus pantanal typical color pattern n = 25 and atypical color pattern n = 16.M is the mean and Range is the minimum and maximum value.

Morphological analysis
Counts and measurements followed Albert (2001) and Fernandes et al. (2005).When referring to Gymnotus pantanal sensu stricto or typical color pattern we followed Fernandes et al. (2005), who found a color pattern of obliquely-oriented thin pale pigment bands.The other specimens analyzed exhibited an atypical color pattern.Morphometric characters were summarized with a Principal Components Analysis (PCA; Pearson, 1901;Hotelling, 1933).Since the morphometric variables (Table 1) were strongly correlated, PCA was chosen as the most appropriate ordination technique to summarize variations.To choose the principal components for interpretation, we used the broken-stick model (Jackson, 1993).
To determine whether there were differences in the average scores of the axes retained for interpretation between fish with typical and atypical color patterns we used Student's ttest.The assumption of normality was evaluated with Shapiro-Wilk's test, whereas the assumption of homoscedasticity was evaluated with Levene's test.Mann-Whitney's U-test (similar non-parametric; Zar, 1999) was used when those assumptions   were not met.Student's t-test and Mann-Whitney's U-test were performed in Statistica TM 7.0.The significance level adopted for all analysis was p<0.05.

Cytogenetic analysis
Chromosome analysis was carried out in 15 specimens with atypical color pattern (eight males and seven females).Mitotic chromosomes were obtained from cephalic kidney cells (Bertollo et al., 1978;Foresti et al., 1993).Fish were anesthetized with clove oil according to Henyey et al. (2002).Chromosomes were classified according to arm ratio (Levan et al., 1964) into two groups: meta-submetacentric (m-sm) and acrocentric (a).

Results
Thirty-three individuals were caught.Seventeen specimens were classified as typical, similar to the holotype (Fig. 2) and 16 as atypical (Fig. 3).Specimens of G. pantanal with atypical color pattern were observed in two sampling sites (Jacutinga and Pinheirinho).Total length varied from 113.4 to 248.0 mm (Table 1).The atypical color pattern was represented by a faint pigment band in the middle of the ventral region and another pigment band in the anal fin base (Fig. 3).
Mean values (min-max) of morphometric characters are presented in Table 1.Results from principal component analysis are summarized in the matrix of morphometric characters.Two axes were chosen for interpretation (cumulative explained variance = 48.73%;Table 2).
There was no significant difference between the scores of the axes retained for interpretation (Fig. 4).Thus, there were no morphometric differences between the groups of specimens analyzed.
The cytogenetic results for all examined individuals showed that the diploid number was 40 for females and 39 for males.Females had 7 meta-submetacentric chromosome pairs and 13 acrocentric chromosome pairs; males had 7 meta-submetacentric chromosome pairs plus a large metacentric chromosome (chromosome Y), and 11 acrocentric chromosome pairs plus two non-homologous medium sized acrocentric chromosomes (chromosomes X 1 and X 2 , Fig. 5).

Discussion
Color pattern is often the most important character for the diagnosis of Gymnotus species (e.g., Albert et al., 1999;Albert & Crampton, 2001, 2003;Fernandes et al., 2005;Cognato et al., 2007;Maxime & Albert, 2009;Richer-de-Forges et al., 2009).Therefore, we thought at first that the atypical color pattern might suggest the existence of a new species.However, cytogenetic and morphological analyses did not corroborate this hypothesis.Moreover, many species of Gymnotus exhibit some variation in body shape and color patterns within and between populations (see Mago-Leccia, 1994;Albert & Miller, 1995;Albert et al., 1999).Recently, Silva et al. (2010) observed variation in color pattern in specimens of Trichomycterus iheringi (Eigenmann, 1917) from the Rivers Itatinga and Claro, State of São Paulo, southeastern Brazil.These authors found two clearly distinct color patterns in this species and related this variation to body size and microhabitat preference.
Usually, morphological variations are associated with environmental, sexual, ontogenetic, and behavioral factors.This is very interesting, since we found Gymnotus pantanal specimens with typical and atypical color pattern syntopically in the streams Jacutinga and Pinheirinho, and only the typical color pattern in the stream Panambi.Daga (unpublished data) studied the effects of limnological and morphometric variables on the composition and structure of fish assemblage along a longitudinal gradient in the streams Pinheirinho, Jacutinga and Panambi, and observed a strong influence of urbanization in the distribution of the ichthyofauna.Variables such as total phosphorus, dissolved oxygen and conductivity were responsible for spatial changes in the fish assemblage.Moreover, the same author together with Gubiani et al. (2010) state that the stream Panambi is strongly affected by human impacts, as it receives both domestic and industrial sewage.With this, at the moment we cannot explain why color pattern varied only in the populations from the streams Pinheirinho and Jacutinga.
Additionally, the karyotype observed is identical to that of G. pantanal specimens studied by Fernandes et al. (2005) and by Silva & Margarido (2005).The occurrence of G. pantanal in the upper Paraná River basin was already described by Margarido et al. (2007) and by Graça & Pavanelli (2007).The formers used cytogenetic analysis to identify this invasive species, and pointed out the usefulness of this analysis for taxonomic diagnosis in this fish group due to its specific karyotype.
Therefore, our results corroborate the importance of integrating morphological and cytogenetic data to study the taxonomy of Gymnotus.External morphological data, mainly color pattern, may be misleading, as sometimes they result from mistakes in the identification of Gymnotus species.

Fig. 1 .
Fig. 1.Partial map of São Francisco Verdadeiro River, Toledo, State of Paraná, showing the location of sites studied.

Table 1 .
Morphometric and meristics data of G. pantanal.The data from G. pantanal sensu stricto follows Fernandes et al.

Table 2 .
Results of Principal Component Analysis (PCA).It is given, for each axis, the eigenvalues, the percent of variance explained and the broken-stick eigenvalues.For each variable is listed the eigenvector (loading or correlation).Number of specimens analyzed was: 25 of G. pantanal sensu stricto typical color pattern and 16 to G. pantanal atypical color pattern.