Environ Biol Fish (2017) 100:69–84 DOI 10.1007/s10641-016-0556-z

Life history of Gymnotus refugio (; Gymnotidae): an endangered species of weakly

Aline Salvador Vanin & Julia Giora & Clarice Bernhardt Fialho

Received: 16 June 2016 /Accepted: 21 November 2016 /Published online: 28 November 2016 # Springer Science+Business Media Dordrecht 2016

Abstract The present study describes the life history of Keywords Electric-fish . Threatened species . Gymnotus refugio, a species classified as Endangered in Reproduction . Feeding the last published list of threatened species of the Brazilian fauna. The study was conducted at a conser- vation unity that protect one of the last remaining Introduction semideciduous forests in the region. The reproductive period was estimated as occurring from the end of The order Gymnotiformes is composed by freshwater winter to the last summer months. Gymnotus refugio weakly electric fishes, which are able to produce and exibited fractional spawning, the lowest relative fecun- detect electric fields with the function of localization and dity registered among the Gymnotifomes species stud- intra and interspecific communication (Crampton and ied at the present, and male parental care behavior. The Hopkins 2005). They can be found from southern analyses showed a seasonal pattern on the species diet, Mexico to Argentina, and in the Caribbean island of associating different food categories to winter, autumn, Trinidad (Albert and Crampton 2003). The and spring. According to food items analysis and esti- Gymnotus is the representative with the largest distribu- mated intestinal quotient, G. refugio was classified as tion among the order, including 40 valid species invertivorous, feeding mainly on autochthonous . (Eschmeyer and Fong 2016) occurring over their entire The results obtained herein suggest that the position of distribution area (Mago-Leccia 1994). Environmental G. refugio as an Endangered species might be influ- conditions (e.g., water flow, temperature, dissolved ox- enced by its territoriality, habitat specificity, parental ygen, conductivity and vegetation) are important factors care behavior, and low fecundity, reinforcing the impor- influencing the distribution of electric fishes (Crampton tance of swampy forest environment conservation as the 1998). These fishes also have several strategies which only means of the species maintenance. facilitate adaptations to environments with a large range of abiotic and biotic factors (Hopkins 1974;Kirschbaum 1975, 1979, 2000;Crampton1998;Albertand Electronic supplementary material The online version of this Crampton 2005), including air breathing adaptations to article (doi:10.1007/s10641-016-0556-z) contains supplementary material, which is available to authorized users. cope with prolonged periods of hypoxia or anoxia : : (Hopkins 1974; Crampton 1998; Kirschbaum 1975, A. S. Vanin J. Giora (*) C. B. Fialho 1979, 2000; Albert and Crampton 2005). Laboratório de Ictiologia, Departamento de Zoologia, Instituto de The recently described species, Gymnotus refugio Biociências, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43435, Porto Alegre, RS CEP (Giora and Malabarba 2016), belongs to the species 91501-970, Brazil group Gymnotus pantherinus currently represented by e-mail: [email protected] 15 species. Gymnotus refugio was formerly 70 Environ Biol Fish (2017) 100:69–84 misidentified as either the species G. pantherinus effective ones requires knowledge on the species’ (Corrêa et al. 2015)orG. aff. pantherinus (Malabarba life history traits. Therefore, the present study aims et al. 2013) in the state of Rio Grande do Sul. Gymnotus to understand the particular biological predictors refugio is found inhabiting the interior of wetlands and related to the low distribution and abundance of edges of swampy forests composed by dense and float- G. refugio, which lead the species to the ing riparian vegetation (Ferrer et al. 2015), and is dis- Endangered category in southern Brazil. The re- tributed from the coastal rivers of the southern Rio sults concerning reproductive biology, gonadal Grande do Sul state to the southern Santa Catarina state maturation, and feeding habits unveiled subsidizing (Giora and Malabarba 2016). According to the last future conservation actions focusing the species published list of threatened fauna species of the Rio and its habitat. Grande do Sul state, G. refugio (quoted as Gymnotus pantherinus) is labelled as EN (Endangered) according to the UCN criteria (FZB 2014). This classification is Material and method based on the species distribution in the state, which covers an estimated area of only 24 km2,dueitshabitat Study area specificity, and restriction to streams and fragmented areas exposed to anthropic actions. Up until now, The sampling area is located in the Refugio da Vida G. refugio is easily found and noted in populations with Silvestre Banhado dos Pachecos (RVSBP) (30°09′57″S; higher number of specimens only in two conservation 50°84′99 W) in Viamão municipality, state of Rio areas: Refúgio da Vida Silvestre Banhado dos Pachecos Grande do Sul, Brazil. The RVSBP is a conservation (RVSBP) and Parque Estadual de Itapeva, being rare unit created in 2002, covering 2.560 ha inserted in the and scarce in all the other places where it has been Gravataí river basin, where the medium and low seg- registered (Giora and Malabarba 2016). ments are extremely impacted by human activities Characters related to measures of habitat condi- (IBGE 2010). According to Oliveira et al. (2005), the tions, behavior, and trophic requirements have fragments of swampy forest surrounding the Gravataí been useful for relating the distribution of fresh- river are the last remaining of semideciduous forest water fishes to environmental variables (Olden permanently submitted to the fluvial influence of the et al. 2006). On the other hand, life history traits river basin. The stream where G. refugio was sampled is are strong predictors of the vulnerability of fish characterized by murky water, abundant floating vege- populations as they determine how resilient a spe- tation, muddy substrate, and dense riparian vegetation cies is to disturbances such as habitat loss and the known as Mata Paludosa (= swampy forest). adverse effects of invasive species (Hamidan and Britton 2015). Reproduction represents one of the Sampling most important aspects of the biology of a species, the maintenance of viable populations depending The specimens of G. refugio were monthly sampled on its success (Suzuki and Agostinho 1997). from March/2011 to February/2012 between 9:00 and Feeding ecology is thoroughly linked to population 17:00 h. Fishes were collected using a dip net under dynamics and contributes to the understanding of floating vegetation and an electric fish finder (Crampton subjects such as resource partitioning, habitat pref- et al. 2007). In the field, the specimens sampled were erence, prey selection, predation, competition, tro- euthanized by immersion in 10% eugenol solution and phic ecology, and evolution (Braga et al. 2012). then fixed in 10% formalin solution. Water and air Since a disturbed ecosystem can affect directly the temperature, water pH, dissolved oxygen, and conduc- dynamic, seasonality, and behaviour of species, tivity were recorded at the time and place of sampling. developing conservation efforts can require com- The monthly amounts of rainfall (in millimeters) and prehension of patterns that shape the biological time of sunshine (in hours) were obtained at the 8th cycles and the ecological interaction between these District of Meteorology of Porto Alegre (available at organisms and their habitat. On account of the Meteorological databank for Education and Research of conservation status of G. refugio populations, con- the National Institute of Meteorology, Brazil, station servation measures are needed and the design of 83967). Environ Biol Fish (2017) 100:69–84 71

Data analysis randomly removed from each selected gonad and the largest possible oocyte diameter was obtained by exam- In the laboratory, fishes were transferred to 70% ethanol ination under a stereomicroscope equipped with a solution and, afterward, the total length in millimeter millimetered ocular (Vazzoler 1996). and total weight in grams were measured. Individuals The sex ratio was determined by the distribution of were dissected to register gonad and stomach weight, male and female frequency during the sample period. and intestine length. Stomach repletion index (RI) and The χ2 test (α <0.05) was applied to determine the gonadosomatic index (GSI) were estimated following existence of significant differences between the propor- the formula adapted from Santos (1978). These indexes tion of males and females in the population. The same represent the percentage organ weight related to fish test was also applied to the distribution of relative fre- total weight: RI = Ws ×100/Wt and GSI = Wg ×100/Wt. quencies of males and females in different total length Ws corresponds to stomach weight, Wg to gonad classes in order to test sexual dimorphism related to total weight, and Wt to total weight. The intestinal quotient length. (IQ) represents the ratio of the intestine length related to Stomach content analysis was performed with the the fish total length: IQ = Li/Lt. Li corresponds to the help of a stereomicroscope and the items of stomach intestine length and Lt to the total length. contents were identified to the lowest possible taxonom- The reproductive period for males and females was ic level (Mugnai et al. 2010). The alimentary items were established through the analyses of monthly variation of analyzed by the frequency of occurrence method the mean GSI values. The analysis of variance (Hyslop 1980), including all food items, where the (ANOVA) with Tukey’s post-test was applied to verify number of times that each item has occurred was treated possible differences between the monthly values of both as the percent of total occurrence number of all items. GSI and RI of males and females separately. A simple For statistical analyses, the food items were grouped in linear regression was applied to verify possible correla- eight broad categories according to their ecological tion between the RI and GSI of males and females. A characteristics and origin: allochthonous insects (Al), multiple linear regression was applied to verify possible autochthonous insects (Au), digested organic matter correlation between the GSI and RI of females and (DOM), fish (Fis), crustacea (Crus), plant material males with the abiotic factors (photoperiod, rainfall, (PM), sediment (Sed), and others (Other). For the PM water and air temperature, water conductivity, pH, and category, all items of vegetal origin such as aquatic dissolved O2). plants (leaves and roots) and algae were considered; To corroborate the macroscopic characterization and whereas Sed included substrate components such as define the gonadal maturation phases, 30 male and 36 rock fragments, earth, sand and mud. female gonads were selected for histological analysis The factors sex, seasonality, and length classes were being dehydrated in ethanol series, and infiltrated and tested on the diet composition of the species through the embedded in glycolmethacrylate resin. Sections of 3 μm Permutational Multivariate Analysis of Variance were performed on a Leica RM2245 microtome with (PERMANOVA) (p ≤ 0.05) (Anderson 2001)withthe glass knifes, and were stained with Toluidine blue. The Bray-Curtis dissimilarity matrix (Borcard et al. 2011). In slides were photographed under a Nikon AZ 100 mm order to test significant variations in the diet of G. microscope. The phases of gonadal maturation were refugio, the months of the year were grouped into sea- classified according to Brown-Peterson et al. (2011). sons: autumn from March to May; winter from June to For monthly variation of the gonadal maturation phases August; spring from September to November; summer of females and males the developing subphases were not from December to February. The length classes were included. Absolute fecundity was estimated by counting determined according to the Sturges rule (Vieira 1991). all vitellogenic oocytes present in the ovaries of seven The Principal Coordinate’s Analysis (PCoA) was ap- females with the highest GSI values recorded. The rel- plied in order to visualize possible variations in the ative fecundity was determined by the number of categories consumed due to the factors considered. vitellogenic oocytes counted per female milligram of The Indicator Value Index (IndVal) was applied to test total weight (Adebisi 1987). The same gonads selected which alimentary categories might be the most influ- for fecundity analysis were used for the determination of enced by seasonality, sex, and length class. The IndVal the spawning type. A sub-sample of 150 oocytes was results were also expressed in terms of specificity (A) 72 Environ Biol Fish (2017) 100:69–84 and fidelity (B), both ranging [0–1] and representing of variance (ANOVA) with Tukey’s post-test, male respectively the abundance of a category over all and female mean GSI values differ significantly bet- groups, and the presence or absence of a category within ween the months of sampling (F = 5.34, p <0.05for the site group. All analyses were performed through the males; F = 3.75, p < 0.05 for females). For females, software R Project for Statistical Computing 3.0.1. mean GSI value obtained in October differed from April to July and from January. For males, mean GSI value obtained in October differed significantly from Results May to July and December differed from August, October and November. The simple linear regression Reproduction indicated a positive relationship between the GSI of both females (F = 1.33; p < 0.05) and males (F = 11.1; A total of 123 specimens of G. refugio, 62 females p < 0.05) and their respective RI. Although the (55.8 mm – 200.8 mm) and 61 males (63 mm – monthly data for dissolved oxygen, pH, and water 240.7 mm), were sampled. The estimated reproduc- temperature (Table 1) were not completely recorded tive period lasted from August to March, with GSI for each month, the multiple linear regression indica- peak occurring in October for females and August and ted no correlation between these abiotic factors and October for males (Fig. 1). According to the analysis the GSI of both females (F = 0.62; p = 0.72) and males

Fig. 1 Monthly variation of mean gonadosomatic index (GSI) of Gymnotus refugio females (a) and males (b). Vertical bars represent the standard deviation. Numbers above the bars correspond to the numbers of specimens included in the analysis Environ Biol Fish (2017) 100:69–84 73

Table 1 Monthly variation of the water conductivity (μS/cm), February/2012 in the Refugio da Vida Silvestre Banhado dos dissolved oxygen (mg/l), pH, water and air temperature (°C), Pachecos, Rio Grande do Sul, Brazil photoperiod (hours), and rainfall (mm) from March/2011 to

2 Conductivity Diss. O pH Temp. (H20) Temp. (air) Photoperiod Rainfall

Mar 43.5 23 215 83.1 Apr 36.4 4.63 23.7 25.5 162.7 172.7 May 19.7 4.1 4.96 22.6 24 122.4 50.1 Jun 18.7 5.6 4.9 14.7 17.5 107.5 109.6 Jul 26.5 3.5 4.8 18.6 24 101.3 225.7 Aug 18.5 3.5 5.4 19.3 29 118.6 182.3 Sep 41.1 3.4 5.4 19.2 16.7 180.3 53 Oct 43.5 2.8 5.25 25.5 27 200 123.7 Nov 22.1 23.5 34 262 13.7 Dec 48.6 3.6 31.3 22.8 233.2 52.1 Jan 47.2 2.3 4.68 23 26 298.4 166 Feb 28 5.7 4.63 27.4 226.5 139.5

(F = 0.81; p = 0.61), and the RI of both females characterized by the presence of blood vessels, oogo- (F = 1.7; p = 0.31) and males (F = 1.01; p =0.52). nias, and primary growth oocytes (Fig. 4d), indicating a The monthly variation of gonadal maturation phases reorganization in the structure for a new beginning of of females and males (Fig. 2) and the histological anal- the gonad maturation process. ysis of female gonads (Figs. 3 and 4)demonstratedall The histological analysis of male gonads showed gonadalmaturationphasesasproposedbyBrown- the following phases of gonadal maturation: devel- Peterson et al. (2011). For females, immature individ- oping, spawning capable, regressing and regenerating uals occurred only in October, ovaries showing predom- (Fig. 5). Males in the developing phase (Fig. 5a, b) inance of oogonia and primary growth oocytes (Fig. 3a). were captured throughout most of the sampling Females in the developing phase were captured during period, except by November and February. the whole sampling period except by March, being more Histologically, these gonads showed active spermato- predominant from May to September. During this peri- genesis and presence of great amount of primary and od, the female gonads presented primary growth oo- secondary spermatocytes, crescent number of sperma- cytes, more abundant in the early developing subphase tids and spermatozoa, and scarce spermatogonia. (Fig. 3b), with primary and secondary vitellogenic oo- Spawning capable individuals were sampled in cytes increasing in number as the gonad maturation August and from October to February showing high reached the mid and late developing subphase (Fig. 3c amount of spermatozoa in lumen of sperm ducts, the and 3d, respectivelly). The spawning capable phase was other spermatogenic cells being poorly scatter in the observed in August, and from October to February gonad (Fig. 5c). Regressing individuals occurred in (Fig. 4a); whereas actively spawning females were col- August and December, showing depleted stores of lected in October, November, and March (Fig. 4b). spermatozoa in sperm ducts and the lumen of the These two phases differed from each other due the lobules, cessation of spermatogenesis, and an increas- presence of postovulatory follicle complex in the active- ing number of spermatogonias (Fig. 5d). Fishes re- ly spawning phase indicating oocytes released. main in the regressing phase for a relatively short Regressing individuals were sampled in April, January, time, moving to the regenerating phase, which was and February, when the histological analysis demon- observed in April, July, and September. This phase strated unorganized gonads with abundant blood ves- is characterized by the massive presence of sper- sels, postovulatory follicle complexes, and enlarged matogonia and residual spermatozoa in sperm ducts ovarian lamellae (Fig. 4c). The regenerating phase ob- (Fig. 5e). Fish in regenerating phase are sexually served in April, October, and February was mature but reproductively inactive. There are no 74 Environ Biol Fish (2017) 100:69–84

Fig. 2 Monthly variation of the gonadal maturation phases of females (a)andmales(b)of Gymnotus refugio histologically analysed

results for males in March/2011 and January/2012, length class established (Fig. 7). According to the same since only females were sampled in these months. test (p < 0.05), the sex ratio established for G. refugio The absolute fecundity of G. refugio had an average was 1:1 in each sampled month, and also in the total value of 419 oocytes ranging from 0.15 mm −2.85 mm sample. of diameter for females with total length varying from 121.3 mm to 196.8 mm (Table 2). Average relative Feeding fecundity was estimated as 0.05 oocytes per mg total weight. Analyses of absolute frequency distribution of A total of 22 food items were identified in the 118 oocytes diameters show that the specie has oocyte de- stomach analysed. Five empty stomachs were regis- velopment synchronic in more than two groups, tered, one stomach in April, June, and January, and iteroparity, and fractional spawning (Fig. 6). two stomachs in September. Empty stomachs were ex- The chi-square test (p < 0.05) did not show signifi- cluded from the analysis. The most frequent items ob- cant differences in total length related to sexual dimor- served were larvae of diptera, odonata and trichoptera, phism for G. refugio however, males reached the largest plant material, and digested organic matter (Table 3). Environ Biol Fish (2017) 100:69–84 75

Fig. 3 Histological sections of Gymnotus refugio ovaries in dif- primary growth oocytes, ca cortical alveolar oocytes, vtg1 primary ferent gonadal maturation phases. (a) Immature. (b) Early devel- vitellogenic oocytes, vtg2 secundary vitellogenic oocytes, vtg3 oping. (c) Mid developing. (d) Late developing. * oogonias, pg tertiray vitellogenic oocytes

The distribution of mean values of RI (Fig. 8)show associated to any alimentary category in particular. In significant differences throughout the sampling period addition, the terms A and B (IndVal) indicate the occur- only for males (F = 10.35; p < 0.05), December differing rence of autochthonous insects in the stomachs of all of all the other months except of May, and February specimens sampled on winter, although the category is differing of August and September. There was no sig- not season exclusive (A = 0.3; B = 1). The same is shown nificant difference between the mean RI values of males for digested organic matter found in almost all stomachs and females and the abiotic factors analyzed. in the spring (A = 0.36; B = 0.9). Allochthonous insects The PERMANOVA results indicate a seasonal pattern were consumed mainly in autumn, not occurring in all in the species diet (F = 16.5 p = 0.001). The food stomachs analysed in the season (A = 0.76 B = 0.2). The categories were associated with neither ontogeny intestinal quotient for G. refugio was estimated as 0.29, (F = 0.72 p = 0.49) nor sex (F = 2.05 p = 0.12). The with a standard deviation of 0.05. PCoA demonstrate a seasonal pattern in the distribution of alimentary categories from autumn to spring (Fig. 9). In addition, the IndVal values show significant ingestion Discussion of autochthonous insects during winter (Stat: 0.55 p = 0.038), allochthonous insects during autumn (Stat: Similarly to other species of gymnotiforms inhabiting 0.39 p = 0.025), and ingestion of digested organic matter the southern South America (Cognato and Fialho 2006; during spring (Stat: 0.57 p = 0.001). Summer was not Giora and Fialho 2009;Schaanetal.2009; Giora et al. 76 Environ Biol Fish (2017) 100:69–84

Fig. 4 Histological sections of Gymnotus refugio ovaries in dif- vitellogenic oocytes, vtg2 secundary vitellogenic oocytes, vtg3 ferent gonadal maturation phases. (a) Spawning capable. (b) Ac- tertiray vitellogenic oocytes, dt ovarian duct, bv blood vessels, af tively spawning. (c) Regressing. (d) Regenerating. * oogonias, pg atresia; pof postovulatory follicle complexes primary growth oocytes, ca cortical alveolar oocytes, vtg1 primary

2014), G. refugio presented a long reproductive period. described for other gymnotiforms inhabiting southern In addition, the higher means of males GSI in the first Brazil and Uruguay, which shown reproduction posi- month of the established reproductive period suggests tively associated with both temperature and photoperiod that males were ready for reproduction before females. (Silva et al. 1999; Silva et al. 2002; Ardanaz et al. 2001; The increasing size of gonads during the reproductive Giora et al. 2012; Giora et al. 2014); therefore, period occupies larger space in the body cavity and may G. refugio fits the pattern proposed for all gymnotiforms reduce the space available for other organs such as the from high latitudes (Giora et al. 2014). stomach, interfering on the feeding behavior (Vazzoler The histological analysis of both males and females 1996). However, both males and females of G. refugio gonads indicated individuals capable of spawning in exhibited a positive relation between the GSI and RI the same months defined as the species reproductive values, suggesting that the species might increase feed- period. Even though the reproductive period was well ing activities during the reproductive cycle in order to defined for males, the histology of the testes demon- cope with the energetic demand. Although no correla- strated a fast and continuous spermatogenesis process tion was observed between the species GSI and the throughout the sampling period, impeding the differ- abiotic factors, the reproductive period matched months entiation of the actively spawning subphase integrating with the highest photoperiod and water temperature the spawning capable phase (Brown-Peterson et al. registered (INMET 2015). Similar results were 2011). This fast and continuous spermatogenesis can Environ Biol Fish (2017) 100:69–84 77

Fig. 5 Histological sections of Gymnotus refugio testes in differ- Regenerating. g spermatogonia, c1 primary spermatocytes, c2 ent gonadal maturation phases. (a) Mid developing. (b) Late secondary spermatocytes, t spermatids, ez spermatozoas, bv blood developing. (c) Sapawning Capable. (d) Regressing. (e) vessels be proven by the presence of spermatozoa in almost all 60 mm, pointing to an earlier development of the male phases of the maturation cycle. The smallest male juveniles. Although the samples were performed with (63 mm) and female (59 mm) collected were both in specific equipment for the capture of electric fishes, different phases of gonadal development, with the larvae agglomeration and juveniles were neither ob- female being immature and the male in its mid- served nor sampled in this study. Therefore, it is development phase. This observation suggests that possible to presume that the species uses the flooded the process of gonadal maturation for G. refugio may interior of the riparian forest, inaccessible for sam- start when individuals reach lengths shorter than pling, as a nursery for breeding and early development 78 Environ Biol Fish (2017) 100:69–84

Table 2 Total length (Lt), total weight (Wt), gonadosomatic index (GSI), absolute fecundity (Fa), and relative fecundity (Fr) of 7 females of Gymnotus refugio

Lt (mm) Wt (g) IGS Fa Fr

121.32 4.64 11.1 286 0.06 135.33 5.56 12.4 330 0.05 148.91 8.06 11.4 409 0.05 164.84 9.87 8.2 452 0.04 165.74 8.16 9 238 0.02 170.36 11.06 7.7 518 0.04 196.81 9.13 10.9 698 0.07 Mean 157.62 8.07 10.1 419 0.05 Fig. 7 Distribution of relative frequency (%) of Gymnotus refugio females and male for total length classes (mm). Numbers above the column represent the number of specimens in each length class of the juveniles. Although the production of sperm demands some energetic investment (Nakatsuru and relative fecundity much lower than all these values Kramer 1982) that would be much lower than female registered for gymnotiforms up until now. According energetic investment to produce oocytes. In that case, to Vazzoler (1996), fecundity depends on the availabil- the results suggest that G. refugio males benefits of the ity of female body cavity and on the size of oocytes continuous spermatogenesis and early maturation, be- produced. In line with these suggestions, the maximum ing ready for mate spawning capable females through- diameter of a vitellogenic oocyte of G. refugio was out a long period of time. estimated as 2.85 mm, whereas Cognato and Fialho According to the obtained data, G. refugio has very (2006) estimated for Gymnotus aff. carapo amaximum low relative fecundity. Available information diameter of vitellogenic oocytes of 1.7 mm. concerning relative fecundity of gymnotiforms points Parental care is a behavior that improves offspring out the species low fecundity value, Brachyhypopomus fitness and is often related to low fecundity values, since gauderio reaching 0.2 (Giora et al. 2014), greater parental investment in individual progeny im- Brachyhypopomus draco 0.17 (Schaan et al. 2009), prove juvenile survivorship (Winemiller 1987;Menezes Brachyhypopomus bombilla 0.21 (Giora et al. 2012), and Vazzoler 1992). This behavior is largely described and Eigenmannia virescens 0.27 (Kirschbaum 1979), as among gymnotiform species, with larvae guarding and well as other Gymnotus species such as Gymnotus aff. nest building by males documented for different species carapo reaching 0.20 oocytes/mg body weight (Assunção and Schwassmann 1995; Vazzoler 1996; (Cognato and Fialho 2006). However, G. refugio shows Kirschbaum and Schugardt 2002;Quintanaetal. 2004; Crampton and Hopkins 2005; Cognato and Fialho 2006; Giora and Fialho 2009). Although direct evidence of parental care (i.e detection of larval agglom- eration protected by an adult, and/or nests) were not record in the field, the species low fecundity indicate that the same behavior might be performed by G. refugio. Moreover, as similarly associated by Cognato and Fialho (2006)forG .aff.carapo, the de- crease in the RI of G. refugio males after periods of high GSI, might be another indicator of the existence of parental caring. The species spawning analysis demonstrated the ex- istence of an actively spawning subphase in spawning Fig. 6 Distribution of relative frequency of 150 oocyte diameters of the seven females of Gymnotus refugio with the highest regis- capable females, classifying the species as fractional tered GSI values spawner. Moreover, the distribution of vitellogenic Environ Biol Fish (2017) 100:69–84 79

Table 3 Frequency of occurrence (%) of alimentary items identified for Gymnotus refugio

Alimentary items Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb

Allochthonous 100 11.1 30 0 0 0 25 0 7.1 30 20 9 Acari 0 0 0 0 0 0 12.5 0 14.3 10 0 27.3 Coleopetra(adult)011.1200000001000 Hymenoptera(adult)10000000007009 Unidentifiedpartsofinsect00000025000200 Autochthous insect 100 100 100 100 100 100 100 93.7 100 90 100 81.8 Coleoptera (larvae) 0 0 10 0 0 0 37.5 0 0 0 70 0 Diptera (larvae) 100 88.8 100 100 100 100 87.5 93.7 100 100 100 63.6 Diptera (pupae) 0 0 20 11 20 50 0 12.5 7 0 20 0 Ephemeroptera (larvae) 0 44.5 10 0 0 0 25 6.25 35.7 20 0 27.3 Hemiptera(larvae)00000012.5000100 Odonata (larvae) 100 66.6 70 88.8 60 70 37.5 50 57 30 10 18.2 Plecoptera(larvae)01101100000000 Trichoptera (larvae) 100 33.3 80 66.6 60 40 25 12.5 7 30 70 45.5 Unidentifiedpartsofinsect000000000105718.2 Crustaceae 0 0 10 11 20 10 12.5 18.8 14.3 0 0 18.2 Amphipoda 0 0 0 11 20 10 11 18.8 0 0 0 18.2 Cladocera 0000000014.3000 Copepoda 0010000000000 DOM 100 44.4 30 22.2 0 90 87 81.3 100 70 70 91 Fish 0000000001000 Other 0 0 10 20 0 0 12.5 6.3 14.3 0 10 27.3 Fishegg 00000012.56.300100 Insectegg 00102000000000 Plant material 0 55.5 20 22.2 30 60 75 56.3 43 50 20 9 Sediment 01100000002000 Total of stomachs 1 9 10 9 10 10 8 16 14 10 10 11

oocytes in different maturation classes indicates that may solve competition issues for spawning sites bet- oocytes are released in different moments until the end ween the females of the same population (Nikolskii of reproductive period, whereas store oocytes (primary 1969). Since the swampy forest is a habitat extremely growth oocytes) and vitellogenic oocyte were continu- unstable, with seasonal great variance in water deep and ously observed through the maturation phases (Brown- consequently micro-habitats availability, the fractional Peterson et al. 2011). The same spawning strategy is spawning can cope with G. refugio population mainte- described for all gymnotiform species studied at the nance. Additionally, gymnotiform species have greatly present time (Barbieri and Barbieri 1982; Assunção reduced coelomatic cavity, and fractional spawning and Schwassmann 1995; Kirschbaum and Schugardt strategy can help to enhance fecundity under a space 2002; Quintana et al. 2004; Crampton and Hopkins restriction. Therefore, the quoted strategies (i.e fraction- 2005; Cognato and Fialho 2006; Giora and Fialho al spawning, long reproductive period, parental care and 2009;Schaanetal.2009; Giora et al. 2012;Giora low fecundity) agree with the life history characters of et al. 2014). Although fractional spawning can require ‘K-strategists’ species as formerly proposed by Pianka a higher reproductive effort than a single spawning (Burt (1970). The sex ratio 1:1 defined for G. refugio is also et al. 1988), species with this strategy tend to be better commonly observed in natural population of fishes and adapted to unfavorable environmental conditions and may diverge when one of the sexes has particular 80 Environ Biol Fish (2017) 100:69–84

Fig. 8 Monthly variation of mean repletion index (RI) for Gymnotus refugio females (a)and males (b). Vertical bars represent the standard deviation. Numbers above the bars correspond to the numbers of specimens included in the analysis

advantage (Reay 1984). On the other hand, when com- intense in the studied region (Nimer 1990). paring the body length of G. refugio males and females, Consequently, the water level might rise facilitat- the highest length classes were reached by males. ing the predation on the most available items, Similarly, species of different Gymnotiformes genus, which may include Diptera larvae and other im- such as Egeinmannia (Kirschbaum 1979; Giora and mature invertebrates highly abundant in streams Fialho 2009)andBrachyhypopomus (Giora et al. (Lowe-McConnel 1987; Higuti and Takeda 2002; 2012; Giora et al. 2014) had the same pattern of sex Motta and Uieda 2004;HahnandFugi2007; ratio and total length distribution described for both Uieda and Motta 2007). Moreover, the continuous males and females. distribution of riparian forests surrounding the Even though the direct influence of abiotic fac- streams contributes indirectly with the allochtho- tors on the species diet was not significant, the nous items in fishes diet, serving as shelter and seasonal variations on the diet of G. refugio might food resource for the invertebrates preyed (Soares be a result of environmental changes defining each 1979). Further, the frequent plant material found in season. For example, the increasing RI of both the stomachs could integrate the species diet, but it males and females from May to August, matches is also possible that this could be an item eventu- the period in which the rainfall tend to be more ally consumed in association with the capture of Environ Biol Fish (2017) 100:69–84 81

trophic category and intestine length. According to the results obtained concerning the diet composition of G. refugio, the species can be classified as invertivorous with tendency to insectivory. In comparison to other studies regarding feeding habits of gymnotiform species (Winemiller and Adite 1997; Penczak et al. 2000;Giora et al. 2005; Giora et al. 2012, 2014; Luz-Agostinho et al. 2006) it is possible to conclude that G. refugio integrates the order pattern of diet as it was proposed by Giora et al. (2014). Anthropic activities, such as pollution, habitat degradation, and the expansion of urban areas have contributed to the depletion of fish populations worldwide (Olden et al. 2007; Olden et al. 2010; Vitule et al. 2009; Abilhoa et al. 2011), increasing Fig. 9 Diagram from principal coordinate analysis (PCoA) for the the need to discover our biodiversity in order to seasonal distribution of the following food categories identified for preserve it (Braga et al. 2012). Rapid expansion of Gymnotus refugio. Al allochthonous insects, Au autochthonous agriculture over the past century has left an indelible insects, Crus Crustacea, DOM digested organic matter, Fis fish, Other other items, PM plant material, Sed sediment mark on the world as croplands, and livestock pas- tures now cover an area larger than many of Earth’s natural biomes (Foley et al. 2005). Due to its pres- other preys. Nonetheless, the dryness in the sam- ence restricted to areas of hydromorphic soil, the pling area during summer months might have hin- swampy forests are considered to be ecosystems dered the species locomotion through the water naturally fragmented, with a blotch distribution column affecting the foraging on specific preys, merged to grasslands and other forest formation which might explain the association of summer (Teixeira and Assis 2009). Although gymnotiform with no particular food category. species show diverse characteristics to cope with Gymnotiform fishes are characterized by a crepuscu- different environmental conditions, the results obtain- lar and nocturnal behavior, hunting actively at night ed herein are pointing to a very strong association (Mago-Leccia 1994; Albert and Crampton 2005). between G. refugio population and the swampy for- Given all sampling occurred at the daylight, part of the est habitat. The surrounding riparian forest can di- items was already digested at the moment the specimens rectly influence the species ecology, providing ener- were fixed, compromising the identification of food getic resources and serving, in its flooded interior, as items, and explaining the high frequency of digested a nursery and shelter during dry periods. The aquatic organic matter observed. Furthermore, although previ- environment profile, with excessive aquatic vegeta- ous studies (Albert and Crampton 2003) claim that tion and low water deep also prevent the occurrence species of the genus Gymnotus can prey on small fishes, of great size carnivorous fish (pers. obs.), benefiting there is no direct evidence of this behavior in studies K-strategist species as G. refugio. The largest num- regarding the feeding biology of the species of the genus ber of threatened freshwater species in Rio Grande (Winemiller and Adite 1997; Mérigoux and Ponton do Sul State (27 species; 67.5%) belong to the 1998; Penczak et al. 2000; Giora et al. 2005; Luz- family Rivulidae (Bertaco et al. 2016), which include Agostinho et al. 2006). For G. refugio, the only sign of the species known as annual fishes and live in piscivory observed is related to a single specimen temporary pools and swamps. These species are all among all the individuals sampled, suggesting an op- considered threatened due to their restricted area of portunistic predation on this alimentary item. occurrence and advanced degree of loss and frag- The low intestinal quotient estimated for G. refugio is mentation of habitats (Reis et al. 2003;FZB2014). in line with that proposed for species consuming ali- Therefore, the extreme dependence of G. refugio on mentary resources of origin according to the its specific habitat can explain the status of scale proposed by Fryer and Iles (1972) to compare Endangered [EN] of the species, and can ensure that 82 Environ Biol Fish (2017) 100:69–84 conservation actions focused on the reduction of area in South America —a case study of the current knowl- – anthropisation of its fragmented habitats in southern edge of Neotropical fish diversity. Zootaxa 4138(3):401 440 Borcard D, Gillet F, Legendre P (2011) Numerical ecology with R. Brazil are of extreme importance for maintenance of Springer, New York the fish fauna. Braga RR, Bornatowski H, Vitule JRS (2012) Feeding ecology of fishes: an overview of worldwide publications. Rev Fish Biol – Acknowledgements We are grateful to André Osorio Rosa, Fish 22(4):915 929 director of Refúgio da Vida Silvestre Banho dos Pachecos conser- Brown-Peterson NJ, Wyanski DM, Saborido-Rey F, Macewicz vation unity for supporting the collect expedition in the area. This BJ, Lowerre-Barbieri SK (2011) A standardized terminology project was funded by Programa Nacional de Pós for describing reproductive development in fishes. 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