Morphological and molecular identification of Geophagus sveni Lucinda , Lucena & Assis , 2010 ( Cichlidae , Cichliformes ) from the Paraná river basin , Argentina

During 2015, we collected several specimens of a cichlid tentatively assigned to Geophagus in Yacyretá reservoir in the Paraná river basin (Argentina). By means of morphological and molecular evidence, we identified these specimens as Gephagus sveni, a species known from middle portion of the Tocantins River. Here we report the presence of the genus Geophagus (sensu stricto) in Argentina for the first time.


Introduction
The family Cichlidae is a very diverse fish group with 1711 species (Fricke et al. 2018) distributed in fresh-and brackish waters of North, Central, and South America, Africa, the Jordan Valley in the Middle East, Madagascar, Iran, southern India, and Sri Lanka (Kullander 2003).Historically classified as part of the taxonomically conflictive Perciformes, recent phylogenetic works suggested that together with monotypic Pholidichthyidae, they belong to the order Cichliformes (Betancur-R. et al. 2013, Mirande 2017, Betancur-R. et al. 2017, Ilves et al. 2018).
The genus Geophagus Heckel, 1840 are Neotropical cichlids that belong to the subfamily Cichlinae.This genus was originally diagnosed to include large cichlids with an expanded anteroventral lamina on the first epibranchial, lined with gill-rakers.Based on the number of supraneural bones, Gosse (1976) divided the genus into Gymnogeophagus Miranda Ribeiro, 1918 with 2 supraneurals, Geophagus with 1, and Biotodoma Eigenmann & Kennedi, 1903 without a supraneural bone.Later on Kullander (1986) resurrected Satanoperca Günther, 1862 and redefined Geophagus to include only species with a swimbladder prolongation into the caudal region, which is lined by 6-12 epihemal ribs and also more caudal than precaudal vertebrae.Those characters define the Geophagus sensu stricto species group and are absent in the species from the "Geophagus" brasiliensis and "Geophagus" steindachneri species groups.Molecular phylogenetic studies (López-Fernández et al. 2010, Ilves et al. 2018) also split species of Geophagus in these 3 different clades.Geopha-gus sensu stricto and "Geophagus" steindachneri groups are related to Gymnogeophagus (Geophagines), whereas "Geophagus" brasiliensis group is more closely related to Mikrogeophagus (Mikrogeophagines) and Biotodoma (Ilves et al. 2018).
Geophagus sensu stricto is currently composed of 20 valid species widely distributed in South America and adjacent Central America (Deprá et al. 2014).Although the Amazon native Geophagus proximus (Castelnau, 1855) has been recorded from reservoirs of the Upper Paraná river basin in Brazil since the early 2000s (Graça and Pavanelli 2007, Moretto et al. 2008, Gois et al. 2015), there were no records of the genus from the Argentine stretch until now (Mirande and Koerber 2015).In a recently published update to Graça and Pavanelli (2007), Ota et al. (2018) determined as Geophagus sveni Lucinda, Lucena & Assis, 2010 specimens previously assigned to Geophagus cf.proximus.In this paper, we corroborate the presence of G. sveni in Argentina on the basis of morphological analyses and corrected pairwise genetic distances of a fragment of mitochondrial gene Cytochrome oxidase subunit I (COI); and also discuss the presence of G. proximus in the Paraná river basin.

Methods
Specimens were collected by means of gillnets at different upstream points of the Yacyretá Reservoir in the Paraná River, Corrientes and Misiones provinces, Argentina (Fig. 1).Fishes were euthanized by overdose in benzocaine anesthetic solution (Close et al. 1996, Neiffer andStamper 2009), fixed in a 10% formalin solution, preserved in 70% ethylic alcohol, and deposited at the "Colección Ictiológica, Laboratorio de Génetica Evolutiva, Universidad Nacional de Misiones" (LGEP).Measurements and counts were performed in 11 specimens (Table 1) following Kullander and Nijssen (1989) and Kullander et al. (1992).Specimen LGEP367 was cleared and stained according to Taylor and Van Dyke (1985) for osteological analysis.
DNA sequence analysis.Muscle tissue samples for molecular studies were obtained post-mortem and preserved in 100% ethylic alcohol.Total genomic DNA was extracted from ethanol-preserved muscle tissue of specimen LGEP452, using the Qiagen DNeasy kit.PCR amplifications were carried out in 30 µl reactions using 0.2 µl Taq (Genbiotech).A 650-bp DNA sequence from the 5ʹ region of mitochondrial gene Cytochrome oxidase subunit I (COI), was amplified using the cocktail primers: VF2_t1; FishF2_t1; FishR2_t1; Fr1d_t1 (Ivanova et al. 2007).The PCR protocol consisted of an initial denaturation step at 95 °C (2 min), 30 cycles consisting of 94 °C (30 s) for denaturation, 54 °C (30 s) for annealing, and 72 °C (1 min) for extension followed by a final extension step at 72 °C (10 min) (Ward et al. 2005).PCRamplified products were cleaned using AccuPrep PCR Purification Kit.The products were sequenced with an automated sequencer (Macrogen, Korea) and all samples were sequenced in both directions to check for potential errors.Chromatograms obtained from the automated sequencer were processed and edited using ChromasPro Version 2.1.2(Technelysium Pty Ltd) and deposited in  = localities reported in this work: 1 = Puerto Maní, 2 = Candelaria, 3 = Garupá, 4 = Posadas, 5 = Santa Tecla (all in Argentina).
GenBank under the accession numbers MH780911.For comparison, COI sequence of a Geophagus sveni from Tocantins river basin was used (voucher material LBP-17378 is stored at Laboratório de Biologia e Genética de Peixes, Universidade Estadual Paulista "Júlio de Mesquita Filho", Campus de Botucatu.São Paulo).Sequences from various species of Geophagus, available at GenBank (http://www.ncbi.nlm.nih.gov/Genbank) and BOLD (http://www.barcodinglife.org)were also used.Employed sequences and references are shown in Table 1.Pairwise genetic distances were calculated for fragments of 543 pb using MEGA, version 6 (Tamura et al. 2013) to estimate genetic divergence (K2P distances) between our specimen, G. sveni from Tocantins and other species of Geophagus (Table 2).Identification.The presence of paired caudal extensions of the swimbladder lined by 6-12 epihemal ribs and more caudal than precaudal vertebrae allowed us to assign the studied specimens to genus Geophagus sensu stricto.

New
Correspondence of specimens to the nominal species Geophagus sveni (Fig. 2) was based on the absence of a suborbital stripe, the lack of a preopercular mark and the possession of 5 faint, vertical, parallel, solid bars on the flank.
The absence of a head stripe distinguish G. sveni from all the species outside the  Besides, scarce (≤ 0.2%) or no differences were found between these, and the sequences GU701783, GU701785, GU701786, and JN988869 stored in GenBank as G.
proximus.The remaining sequences from GenBank identified as belonging to G. proximus were found to be more related to 'G'.brasiliensis species group (D ≥ 15%).Thus, they probably do not represent specimens of Geophagus sensu stricto.

Discussion
The finding of Geophagus sveni in the Paraná River in Argentina constitutes the first report of the genus Geophagus sensu stricto from the country; although the presence of the genus in the Paraná river basin has been suggested since 2007, with reports of G. proximus (Graça and Pavanelli 2007, Moretto et al. 2008, Gois et al. 2015).The first report of G. sveni in the Upper Paraná river basin is an updated checklist of fishes from the Upper Paraná floodplain by Ota et al. (2018).These authors reassigned specimens previously reported as Geophagus cf.proximus (Graça and Pavanelli 2007) to G. sveni.Additionally, G. proximus was reported from 3 reservoirs of middle and lower Tietê (Moretto et al. 2008) and Upper Paraná River (Gois et al. 2015).Those authors found a correlation between the population growth of G. proximus and decreased abundance of Satanoperca pappaterra Heckel, 1840.We were not able to estimate if there is an impact on fish assemblage caused by G. sveni, but Graça and Pavanelli (2007) suggested that the species establishment was recent but successful.
Analysis of genetic distances revealed that the specimen here analyzed (LGEP452) has no difference with the specimen LBP-17378 from Tocantins River; as well as scarce or no differences were found between these and COI sequences stored in GenBank as Geophagus proximus (Table 3).These G. proximus sequences belong to specimens captured in the Upper Paraná river basin, but considering the usual problems of GenBank with misidentifications, we feel it pertinent that a careful revision of voucher material be made to accurately determine which species those sequences belong to.That will help establish a better understanding of the distribution of G. sveni and G. proximus.The correct determination of these cichlid species and the monitoring of fish assemblages are necessary to determine if we are seeing an invasive species expanding southwards.If so, measures can be taken aimed at containing or controlling its spread.
2010 and G. mirabilis Deprá, Kullander, Pavanelli & da Graça by having 5 faint, vertical, parallel, solid bars on the body flank and absence of head marks.Only G. parnaibae Staeck & Schindler, 2006 has 5 bars but second and third are medially bisected unlike that of G. sveni, which are solid.Also caudal fin color pattern distinguished G. sveni (alternating vertical white and dark bars) from G. parnaibae (alternating horizontal white and dark bars).Molecular identification by means of mitochondrial gene COI supports the phenotypical determination.The pairwise analysis of COI sequence distances, revealed no intraspecific variation among specimen LGEP 452 from Paraná River and topotypic specimen LBP-17378.

Table 1 .
Morphological measurements and counts of Geophagus sveni from Paraná River in Misiones, Argentina.

Table 2 .
List of COI sequences employed for genetic distance estimation.