Cytogenetics of Gymnogeophagus setequeda s ( Cichlidae : Geophaginae ) , with comments on its geographical distribution

We provide cytogenetic data for the threatened species Gymnogeophagus setequedas, and the first record of that species collected in the Iguaçu River, within the Iguaçu National Park’s area of environmental preservation, which is an unexpected occurrence for that species. We verified a diploid number of 2n = 48 chromosomes (4sm + 24st + 20a) and the presence of heterochromatin in centromeric and pericentromeric regions, which are conserved characters in the Geophagini. The multiple nucleolar organizer regions observed in G. setequedas are considered to be apomorphic characters in the Geophagini, whereas the simple 5S rDNA cistrons located interstitially on the long arm of subtelocentric chromosomes represent a plesiomorphic character. Because G. setequedas is a threatened species that occurs in lotic waters, we recommend the maintenance of undammed environments within its known area of distribution.


Introduction
Neotropical cichlids are arranged in seven tribes (Sparks, Smith, 2004;Smith et al., 2008), including Geophagini.Within that tribe, Gymnogeophagus Miranda Ribeiro, 1918 can be diagnosed by two apomorphic traits, the absence of a supraneural bone and the presence of a forward-facing spine of the first dorsal pterygiophore (Reis, Malabarba, 1987).Gymnogeophagus currently comprises 17 species, distributed in the Paraná, Paraguay and Uruguay River basins, as well as the coastal basins of southern Brazil and Uruguay (Kullander, 2003;González-Bergonzoni et al., 2009;Malabarba et al., 2015;Loureiro et al., 2016).Gymnogeophagus setequedas is the only species of that genus considered threatened in all the Brazilian red lists.Reis et al. (1992) described Gymnogeophagus setequedas based on 91 specimens collected in Paraguayan rivers and six from a tributary on the Brazilian margin of the Itaipu Reservoir.Since this description, there have been a number of unsuccessful attempts to collect new specimens from within the known geographical range of the species (Agostinho et al., 2004;Pavanelli, Reis, 2008).However, during a recent evaluation of the material deposited in the ichthyological collection of the Museu de Ciências e Tecnologia of the Pontifícia Universidade Católica do Rio Grande do Sul (MCP) (Porto Alegre, Brazil), eight specimens of G. setequedas were identified.These specimens were collected in the Iguaçu River, approximately 200 km from the type locality (Iguazú River at Juan E. O'Leary, Paraguay).More recently, surveys conducted both up and downstream from 2 e160035 [2] the Iguaçu Falls in the Iguaçu National Park resulted in the collection of 15 specimens of G. setequedas.
The fish fauna of the Iguaçu River is highly endemic, due to the presence of the Iguaçu Falls, which form a natural barrier to the dispersal of fish, and includes areas of endemism within the river's lower basin (Vera-Alcaraz et al., 2009b).In this case, the occurrence of G. setequedas upstream from the falls was unexpected.For instance, Baumgartner et al. (2012), who published a catalog of the fish of the lower Iguaçu River basin, did not register the occurrence of G. setequedas in that region.Abilhoa, Duboc (2004) included Gymnogeophagus setequedas among the threatened species of the Brazilian state of Paraná, using its restricted distribution and reduction in its available habitat as justification.Pavanelli, Reis (2008) subsequently included G. setequedas in the Brazilian red list of endangered species, and it has been listed as Vulnerable (VU) by the International Union for Conservation of Nature (IUCN).In a subsequent review of the Brazilian list (decree #445 in the Official Federal Gazette of December 17, 2014), the Brazilian Environmental Ministry raised the status of the species to Endangered (EN).Vera-Alcaraz et al. (2009a) also listed G. setequedas as Endangered (EN) in Paraguay.
In this study, we present the first basic and molecular cytogenetic data for G. setequedas, as well as the 5S and 18S rDNA-FISH for the genus as a whole.We also discuss the recent expansion of the known geographical distribution of G. setequedas and the implications of that discovery on its current conservation status.

Material and Methods
Fifteen specimens of Gymnogeophagus setequedas were analyzed for cytogenetic characters, comprising six females, six males and three specimens of unidentified sex.The sex determination was made by microscopic analysis of the gonads.Specimens were captured in the Iguaçu River upstream (25°37'13.20″S/54°23'29.20″W:NUP 14913 (6) and NUP 15962 (5)) and downstream from the Iguaçu Falls (25°38'18.72″S/54°28'4.The study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, approved by the Committee on the Ethics of Animal Experiments of the Universidade Estadual do Oeste do Paraná (License Number: Protocol 13/09 -CEEAAP/Unioeste).Individuals were sacrificed with an overdose of clove oil, following Griffiths (2000).Mitotic chromosomes were obtained from kidney cells by direct method of air-drying according to Bertollo et al. (1978).Methodologies for detection of NORs by silver impregnation (AgNORs) and heterochromatic regions by technique of barium hydroxide (C-band) followed the protocols of Howell, Black (1980) and Sumner (1972), respectively, with modifications in the step coloration suggested by Lui et al. (2012).Physical mapping of 5S rDNA and 18S rDNA sequences was carried out by fluorescence in situ hybridization (FISH) in accordance with Pinkel et al. (1986) and modifications suggested by Margarido, Moreira-Filho (2008), with probes obtained from Megaporinus elongatus (Valenciennes, 1850) (Martins, Galetti, 1999) and Prochilodus argenteus Spix & Agassiz, 1829 (Hatanaka, Galetti, 2004), respectively.The 5S rDNA probe was labeled with digoxigenin-11-dUTP (Dig Nick Translation Kit -Roche®, Basel, BS, Switzerland) and the 18S rDNA probe was labeled with biotin-16-dUTP (Biotin Nick Translation Kit -Roche®).Each probe was dissolved at a concentration of 3 ng/µl in a hybridization mixture (50% formamide, 2xSSC, 10% dextran sulfate).Hybridization was carried out in a wet chamber at 37ºC for approximately 16 hours.To detect the signal, the protocol used amplified antidigoxigenina-rhodamine (Roche®) for the 5S rDNA probe and avidin-FITC with anti-avidin biotinylated (Sigma-Aldrich, Buchs, SG, Switzerland) for the 18S rDNA probe.Chromosomes were subsequently counterstained with 4',6-diamidino-2-phenylindol (DAPI) at a concentration of 50 µg/ml.The software DP Controller 3.2.1.276was used with an Olympus DP 71 digital camera attached to an epifluorescence microscope BX 61 (Olympus America Inc., Center Valley, PA, United States of America) to photograph the slides.To organize the karyotype, we calculated the relation of the arms as proposed by Levan et al. (1964).
We also compiled chromosomal data for Cichlidae from this and prior studies, and present those data in a phylogenetic context at the level of genera and in tabular format at the level of species (Tab.1).Chromosomal data presented for the genera were chosen preferentially for the species included in Smith et al. (2008), when chromosomal data for these species were available.Otherwise, we chose a species with the most common chromosomal pattern to represent the genus.

Results
We verified a diploid number of 48 chromosomes (4sm + 24st + 20a) for Gymnogeophagus setequedas and observed no differences in karyotype macrostructure either between sexes or between up-and downstream populations (Fig. 2a).The silver impregnation revealed multiple NORs located terminally on the long arm of the subtelocentric chromosome pair 8 and on one of the acrocentric chromosomes of pair 20 (Fig. 2a, in box).Heterochromatin was observed in centromeric/pericentromeric positions in most chromosomes, where it coincided with rDNA sites (Fig. 2b).The 18S rDNA-FISH confirmed the findings of the silver impregnation in both homologous chromosomes of pairs 8 and 20, in addition to highlighting cistrons in the terminal region of the long arm of one of the acrocentric chromosomes of pair 24 (Fig. 2c).The 5S rDNA-FISH revealed cistrons in the interstitial region of the long arm of the subtelocentric chromosome pair 4 (Fig. 2c).7) Vicari et al. (2006).Classification used follows Smith et al. (2008).
The karyotype macrostructure of G. setequedas and its congeners is similar to this ancestral model, but with more subtelo-acrocentric chromosome pairs than metasubmetacentric ones, which appears to be a conserved character in the geophagins.
Silver nitrate impregnation revealed multiple NORs in G. setequedas, a pattern also found in one population of G. gymnogenys (Pires et al., 2010), but with considerable differences in the NORs distribution.In G. setequedas, the NORs are present in a terminal position on the long arm of the subtelo-acrocentric pair 8 and in one acrocentric chromosome of pair 20.In G. gymnogenys, by contrast, the NORs are found in an interstitial position on the long arm of the subtelo-acrocentric chromosome pair 3 and a terminal position on the short arm of the subtelo-acrocentric pair 9 (Pires et al., 2010).By contrast, simple NORs were observed in an interstitial position on the short arm of the metasubmetacentric chromosome pair in G. balzanii (Roncati et al., 2007), G. labiatus (Pires et al., 2010) and B. cupido (Valente et al., 2012), and in a terminal position on the short arm of the subtelo-acrocentric chromosome in Geophagus brasiliensis (Vicari et al., 2006) and Satanoperca jurupari (Poletto et al., 2010a).The presence of simple NORs in the first chromosome pair (meta-submetacentric) is probably a plesiomorphic condition in Neotropical cichlids (Feldberg, Bertollo, 1985b).In this case, multiple NORs in a terminal position of the long arm of the subtelo-acrocentric pair in G. setequedas can be considered apomorphic in the Geophagini (Fig. 3), where the location of NORs on different chromosome pairs must be the result of chromosomal rearrangements such as translocations.
The specific implications of the 5S and 18S rDNA-FISH data of G. setequedas in comparison with other cichlids.Our FISH data are the first reported for Gymnogeophagus, and revealed multiple 18S rDNA cistrons similar to the Neotropical heroins Mesonauta festivus and Uaru amphiacanthoides, and the African species Haplochromis obliquidens and Oreochromis niloticus (Pseudocrenilabrinae).However, the number and position of these cistrons vary considerably among species.In G. setequedas, they are found in a terminal position on the long arms of the five subtelo-acrocentric chromosomes, whereas in H. obliquidens, M. festivus and O. niloticus these cistrons are located in a terminal position of the short arms of the subtelo-acrocentric chromosomes, in four, five and six pairs, respectively (Poletto et al., 2010a).In the remaining species of the Etroplinae, Pseudocrenilabrinae and Cichlinae, analyses have shown simple 18S rDNA cistrons (Poletto et al., 2010a), which vary in their position (terminal or interstitial) and the chromosome pairs (metasubmetacentric or subtelo-acrocentric) that bear them.Cichla monoculus (Cichlini) and Retroculus lapidifer (Retroculini) belong to basal cichline genera (Smith et al., 2008), and differ in the location of the 18S rDNA cistrons, which are found in a terminal position on the long arm of the subtelo-acrocentric chromosomes in C. monoculus (Schneider et al., 2013) and in a terminal position on the short arm of the subtelo-acrocentric chromosomes in R. lapidifer (Poletto et al., 2010a).In Pseudetroplus maculatus (Bloch, 1795) (Etroplinae) and Hemichromis bimaculatus (Pseudocrenilabrinae), considered here as outgroups, simple 18S rDNA cistrons are located on the short arm of the subtelo-acrocentric chromosomes and represent a plesiomorphic character in the Cichlidae (Fig. 3).In G. setequedas, by comparison, multiple cistrons are found in a terminal position on the long arm, indicating that this is an apomorphic character in this species.
Remarks on the conservation status of G. setequedas.Since its discovery, the conservation status of Gymnogeophagus setequedas has invariably been assigned to a threatened category.The threat has been attributed to lentic waters of the Itaipu Reservoir, which have isolated populations of this rheophilic species, which formerly occurred on tributaries of both banks, in Paraguay and Brazil, and probably in the lower Paraná River proper.After the construction of the reservoir, these populations disappeared.The recent new records of G. setequedas in the Iguaçu River extend the geographical distribution of the species from its original description.
Prior to their formal publication herein, these data were also considered in the most recent evaluation of the conservation status of the Brazilian populations, which led to reclassification of G. setequedas as endangered in accordance with IUCN (2014) criterion B. Its previously known distribution in the Iguaçu River spanned about 100 km from above the Iguaçu Falls to the middle of the Itaipu Reservoir.That value yields an estimated extent of occurrence (EOO) of just 200 km 2 by multiplying the linear extent of the river where the species occurs by two, according to the IUCN recommendation for the rough calculation of the area of a drainage basin (personal communication with M. F. Tognelli, Program Officer, IUCN | CI Biodiversity Assessment Unit, Global Species Programme, in 2012, when the species was reassessed).The inclusion of the newly discovered lot from Porto Vorá would add a river extension of 225 km 2 .Multiplying 225 km 2 by two would result in an addition of 450 km 2 to the extent of occurrence (EOO) of the species, totaling 650 km 2 , which is still far below the IUCN threshold of 5,000 km 2 for classifying the species as endangered.The reservoir's severe fragmentation of the population and the continued decline in original extent of occurrence (EOO), area of occupancy (AOO), and area, extent and quality of habitat caused by deforestation, silting and pollution by pesticides, consequences of agriculture and livestock satisfy subcriteria B.1.a(i,ii,iii) and B.1.b(i,ii,iii).
The fact that the known healthy populations of this species occurs only in stretches of the Iguaçu River free of impoundment reinforces the requirement of rapid waters for its survival.Its sporadic capture only twice after building reservoirs, such as Itaipu and Salto Caxias, and its later disappearance detected by monitoring those two large reservoirs confirm its dependence on rapid environments.As the construction of a new hydroelectric power station (Baixo Iguaçu) is already under way between the Caxias Reservoir and the Iguaçu Falls, we strongly recommend keeping the tributaries and the area downstream from the reservoir free of additional dams, to guarantee the long-term survival of the species.

Fig. 1 .
Fig. 1.Partial map of southern South America showing distribution of Gymnogeophagus setequedas.Yellow symbols represent lots of specimens from type-material and additional material housed in Swedish Museum of Natural History.The yellow star marks the type-locality (río Iguazú Reservoir, at Juan E. O'Leary, Paraná River basin, Paraguay).Red squares represent the new specimens discovered from the Iguaçu River basin, Brazil.White lines indicate physical barriers to dispersal.