Check List ire tc > PENSUFT. NOTES ON GEOGRAPHIC DISTRIBUTION Check List 14 (6): 1053-1058 https://doi.org/10.15560/14.6.1053 Morphological and molecular identification of Geophagus sveni Lucinda, Lucena & Assis, 2010 (Cichlidae, Cichliformes) from the Parana river basin, Argentina Mauricio F. Benitez', Juan C. Cerutti?, Danilo R. Aichino?, Diego Baldo! 1 Laboratorio de Genética Evolutiva, Instituto de Biologia Subtropical (CONICET—UNaM), Félix de Azara 1552, N3300LQH, Posadas, Misiones, Argentina. 2 Proyecto Biologia Pesquera Regional, Instituto de Biologia Subtropical (CONICET—UNaM), Rivadavia 2370, N3300LDX, Posadas, Misiones, Argentina. Corresponding author: Mauricio F. Benitez, mauriciofbenitez@gmail.com Abstract During 2015, we collected several specimens of a cichlid tentatively assigned to Geophagus in Yacyreta reservoir in the Parana 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. Key words New record; freshwater fish; Argentine ichthyofauna; cytochrome oxidase 1; acara. Academic Editor: Felipe Polivanov Ottoni | Received 9 July 2018 | Accepted 29 September 2018 | Published 16 November 2018 Citation: Benitez MF, Cerutti JC, Aichino DR, Baldo D (2018) Morphological and molecular identification of Geophagus sveni Lucinda, Lucena & Assis, 2010 (Cichlidae, Cichliformes) from the Parana river basin , Argentina. Check List 14 (6): 1053-1058. https://doi.org/10.15560/14.6.1053 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, Mada- gascar, Iran, southern India, and Sri Lanka (Kullander 2003). Historically classified as part of the taxonomically conflictive Perciformes, recent phylogenetic works sug- gested 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 Neotropi- cal cichlids that belong to the subfamily Cichlinae. This genus was originally diagnosed to include large cichlids with an expanded anteroventral lamina on the first epi- branchial, lined with gill-rakers. Based on the number of supraneural bones, Gosse (1976) divided the genus into Gymnogeophagus Miranda Ribeiro, 1918 with 2 supra- neurals, Geophagus with 1, and Biotodoma Eigenmann & Kennedi, 1903 without a supraneural bone. Later on Kul- lander (1986) resurrected Satanoperca Gunther, 1862 and redefined Geophagus to include only species with a swim- bladder 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” stein- dachneri species groups. Molecular phylogenetic studies (Lopez-Fernandez et al. 2010, Ilves et al. 2018) also split species of Geophagus in these 3 different clades. Geopha- Copyright Benitez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PARAGUAY ATLANTIC OCEAN 500 km SI Figure 1. Distribution of Geophagus sveni. * = type locality of G. sveni.™= specimens identified at GenBank as G. proximus but whose genetic distance with G. sveni defined herein is very low (D < 0.2%). @ = localities reported in this work: 1 = Puerto Mani, 2 = Candelaria, 3 = Garupa, 4 = Posadas, 5 = Santa Tecla (all in Argentina). 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 (Ives et al. 2018). Geophagus sensu stricto is currently composed of 20 valid species widely distributed in South America and adjacent Central America (Depra et al. 2014). Although the Amazon native Geophagus proximus (Castelnau, 1855) has been recorded from reservoirs of the Upper Parana river basin in Brazil since the early 2000s (Graca 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 Graca 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 (COZ); and also discuss the presence of G. proximus in the Parana river basin. Methods Specimens were collected by means of gillnets at dif- ferent upstream points of the Yacyreta Reservoir in the Parana River, Corrientes and Misiones provinces, Argentina (Fig. 1). Fishes were euthanized by overdose in benzocaine anesthetic solution (Close et al. 1996, Neiffer and Stamper 2009), fixed in a 10% formalin solu- tion, preserved in 70% ethylic alcohol, and deposited Check List 14 (6) at the “Coleccion Ictiol6gica, Laboratorio de Génetica Evolutiva, Universidad Nacional de Misiones” (LGEP). Measurements and counts were performed in 11 speci- mens (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 pre- served 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 ul reactions using 0.2 ul Taq (Genbiotech). A 650-bp DNA sequence from the 5’ region of mitochondrial gene Cytochrome oxidase subunit I (COI), was amplified using the cocktail prim- ers: VF2 tl; FishF2_ tl; FishR2 tl; Frid_tl (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 anneal- ing, and 72 °C (1 min) for extension followed by a final extension step at 72 °C (10 min) (Ward et al. 2005). PCR- amplified 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 Table 1. Morphological measurements and counts of Geophagus sveni from Parana River in Misiones, Argentina. Measurements n Range Mean SD SL (mm) 11 96.1-163.25 i ‘i Percents of SL Head length 11 29.02-31.16 30.31 0.64 Body depth 11 42.72-48.48 45.53 2.11 Body depth sin dorsal 11 39.26-46.62 42.75 1.98 Caudal peduncledepth 11 11.84-14.61 13.48 0.90 Caudal peduncle length 11 15.8-22.95 21.23 2.03 Pectoral fin length 11 36.03-43.42 39.80 2.34 Pelvic fin length 11 39.43-68.09 52.98 9.40 Last D spine length 8 17.4-19.42 18.37 0.68 Percents of HL Snout length 11 48.78-59.41 54.14 3.01 Orbital diameter 11 21.91-27.59 25.06 1.77 Head depth 11 110.89-136.50 119.60 6.55 Head width 11 46.81-56.41 52.55 273 Interorbital width 11 25.59-32.76 28.55 2.01 preorbital depth 11 37.15-46.66 42.37 3.03 Counts Range Modal value SD E1 scales 9 32-35 34 0.95 H scales 10 6-7 7 0.32 ULL scales 10 21-23 21.22 0.67 LLL scales 11 15-18 18 1.08 Dorsal fin rays 10 (XVII-XVIII)+(1 1-12) XVII+12 0.60 Pectoral fin rays 11 14-15 15 0.40 Pelvic fin rays 11 +5 I+5 0 Anal fin rays 10 I+7-Il1+8 IIl+8 0:32 Benitez et al. | First record of Geophagus sveni for Argentina GenBank under the accession numbers MH780911. For comparison, CO/ sequence of a Geophagus sveni from Tocantins river basin was used (voucher material LBP- 17378 is stored at Laboratorio de Biologia e Genética de Peixes, Universidade Estadual Paulista “Julio de Mesquita Filho”, Campus de Botucatu. Sao 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). Results New records. All specimens collected by Aichino DR, Cerutti JC and Massin SA. Argentina. Misiones, Garupa, Garupa stream (27°29'34" S, 055°49'07" W): October 30, 2015 (7 specimens, LGEP 364); December 17, 2015 (2 specimens, LGEP 366); February 12, 2015 (1 specimen, LGEP 367); March 19, 2015 (1 specimen, LGEP 395); April 15, 2015 (1 specimen, LGEP 396); April 15, 2015 (1 specimen, LGEP 397); February 12, 2015 (1 speci- men, LGEP 400). From Corrientes, Santa Tecla, Parana River (27°26'17" S, 056°22'31" W): February 17, 2015 (1 specimen, LGEP 394). Misiones, Corpus, Parana River (27°06'21" S, 055°30'51" W): May 20, 2015 (3 specimens, LGEP 398). Misiones, Candelaria, Parana River (27°26'53" S, 055°43'53" W): June 12, 2010 (1 1055 specimen, LGEP 399). Misiones, Posadas, Parana River (27°21'16.4" S, 055°54'14.0" W) June 28, 2016 (1 speci- men, LGEP 452) collected by Torres J. 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. 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 G. surinamensis complex [G. brasiliensis (Quoy & Gaimard, 1824); G. obscurus (Castelnau, 1855); G. crassilabris Steindachner, 1876; G. iporangensis Haseman, 1911; G. itapicuruensis Hase- man, 1911; G. pellegrini Regan, 1912; G. steindachneri Eigenmann, 1922; G. harreri Goose, 1976; G. argyrost- ictus Kullander, 1991; G. grammepareius Kullander & Taphorn, 1992; G. taeniopareius Kullander & Royero, 1992; G. gottwaldi Schindler & Staeck, 2006; G. diaman- tinensis Mattos, Costa & Santos, 2015; G. rufomarginatus Mattos & Costa, 2018; G. multiocellatus Mattos & Costa, 2018 and G. santosi Mattos & Costa, 2018], the absence of a dark preopercular mark noticeable in live and alcohol-preserved specimens distinguish it from G. proximus, G. brachybranchus Kullander & Nijssen, 1989; G. dicrozoster Lopez-Fernandez & Taphorn, 2004; G. winemilleri Lopez-Fernandez & Taphorn, 2004 and G. crocatus Hauser & Lopez-Fernandez, 2013. Geophagus Table 2. List of CO/ sequences employed for genetic distance estimation. Species GenBank Specimen or BOLD Id catalogue G. sveni MH78911 LGEP452 G. sveni MK12088 LBP-17378 G. proximus GU701783 LBP-37221 G. proximus GU701784 LBP-37220 G. proximus GU701785 LBP-37223 G. proximus GU701786 LBP-37222 G. proximus JN988869 LBPV-37219 G. dicrozoster DSFRE170-08 Not provided G. dicrozoster DSFRE171-08 Not provided G. surinamensis JN026710 GESU-Petshop-1 G. surinamensis KU568829 ES12-AT028 G. argyrostictus PARO178-08 Not provided G. argyrostictus PARO177-08 Not provided G. harreri DSFRE369-08 Not provided G. steindachneri UDEA115-18 CIUA-8855 G. steindachneri UDEA116-18 CIUVA-8868 G. pellegrini MG936927 stri-6733 G. pellegrini MG936928 stri-1764 G. crassilabris MG936924 stri-12254 G. crassilabris MG936925 stri-3618 G. brasiliensis JN988864 LBPV-40176 G. brasiliensis KP218743 CT2506 G. proximus HM064993 LBP-16081 G. proximus HM064991 LBP-16084 GenSeq EE aS in| Latitude Longitude genseq-4 27°21'16.4"S 055°54'14.0” W genseq-4 10°07'59.6"S 048°18'53.0” W genseq-4 21°14'44.2”S 048°17'50.3”W genseq-4 25°25'1.153? S 054°32'08.2” W genseq-4 21°14'44.2"S 048°17'50.3”W genseq-4 21°14'44.2”"S 048°17'50.3”W genseq-4 25°25 13-5 054°32'08.2” W x x x x x x genseq-4 * * genseq-4 7 *, x x x x x x x x x genseq-4 5°30'03.9”N 074°41'13.9"W genseq-4 5°30'03.9”N 074°41'13.9"W genseq-4 8°37'33.2" N 077°49'01.2”W genseq-4 8°50'45.2”N 077°41'17.5"W genseq-4 9°16'25.7"N 078°40'52.7”W genseq-4 8°58'45.8"N 078°30'20.2” W genseq-4 22°22'42.2"S 047°12'37.8"W genseq-4 19°30'00.0”"S 042°22’48.0" W genseq-4 22°00'00.0”S 041°19'58.8" W genseq-4 22°00'00.0"S 041°19'58.8"W 1056 Figure 2. Geophagus sveni. A. Live coloration. B. Cleared and stained specimen. C. Ethanol-preserved specimen. A and C correspond to voucher LGEP398 (161.7 mm) and B to LGEP367 (130.7 mm). Scale bars = 20 mm. sveni 1s distinguished from G. megasema Heckel, 1840; G. camopiensis Pellegrin, 1903; G. altifrons Heckel, 1840; G. surinamensis Bloch, 1791; G. abalios Lopez- Fernandez & Taphorn, 2004; G. brokopondo Kullander & Nijssen, 1989; G. neambi Lucinda, Lucena & Assis, 2010 and G. mirabilis Depra, Kullander, Pavanelli & da Graca 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 dis- tinguished 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 CO/J sequence distances, revealed no intraspecific variation among specimen LGEP 452 from Parana River and topotypic specimen LBP-17378. Besides, scarce (< 0.2%) or no differences were found between these, and the sequences GU701783, GU701785, GU701786, and JN988869 stored in GenBank as G. Check List 14 (6) 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 Parana River in Argentina constitutes the first report of the genus Geophagus sensu stricto from the country; although the presence of the genus in the Parana river basin has been suggested since 2007, with reports of G. proximus (Graca and Pavanelli 2007, Moretto et al. 2008, Gois et al. 2015). The first report of G. sveni in the Upper Parana river basin is an updated checklist of fishes from the Upper Parana floodplain by Ota et al. (2018). These authors reassigned specimens previously reported as Geophagus cf. proximus (Gracga and Pavanelli 2007) to G. sveni. Additionally, G. proximus was reported from 3 reservoirs of middle and lower Tieté (Moretto et al. 2008) and Upper Parana 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 Gracga and Pavanelli (2007) suggested that the species establishment was recent but successful. Analysis of genetic distances revealed that the speci- men 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 proxi- mus (Table 3). These G. proximus sequences belong to specimens captured in the Upper Parana river basin, but considering the usual problems of GenBank with mis- identifications, 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 assem- blages 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. Acknowledgments We thank the Biologia Pesquera staff for assistance during our fieldwork, IBS and UNaM for permanent support. MB is supported by a doctoral grant given by CONICET. JCC and DRA received financial support from the “Entidad Binacional Yacyreta”. We are also grateful to R. Britzke and C. Oliveira for its generous contribution with the sequences of topotypic Geophagus sveni. We are thankful to the reviewers whose contribu- tions helped to improve this manuscript. Benitez et al. | First record of Geophagus sveni for Argentina Table 3. CO/ gen K2P distance matrix of Geophagus. MH78911 0.000 0.000 0.002 MK12088 0.000 0.002 GU701783 0.002 GU701784 0.002 0.000 0.000 0.000 0.051 0.000 0.000 0.000 0.051 0.000 0.000 0.000 0.051 GU701785 0.000 0.000 0.051 0.002 GU701786 0.000 0.051 0.002 JN988869 0.051 0.053 DSFRE170-08 DSFRE171-08 JNO26710 KU568829 0.051 0.053 0.051 0.051 0.051 0.000 0.048 0.048 0.085 0.051 0.051 0.038 0.040 0.038 0.038 0.038 0.048 0.040 0.048 0.072 0.085 0.038 0.038 0.000 0.070 0.038 0.038 0.038 0.038 0.038 0.038 0.070 0.070 0.070 0.070 0.070 0.070 0.070 PARO178-08 0.070 0.072 0.070 0.070 0.070 0.081 0.081 0.070 0.070 0.004 0.118 0.162 0.157 0.162 0.142 0.160 0.160 0.172 0.177 0.159 0.149 0.070 0.070 PARO177-08 0.123 0.167 0.157 0.162 0.142 0.160 0.160 0.167 0.177 0.164 0.154 0.111 0.111 0.111 0.111 0.107 0.175 0.170 0.178 0.153 0.165 0.165 0.162 0.185 0.157 0.162 0.107 0.175 0.170 0.178 0.153 0.165 0.165 0.162 0.185 0.157 0.162 0.107 0.175 0.170 0.178 0.153 0.165 0.165 0.162 0.185 0.157 0.162 0.109 0.177 0.172 0.181 0.155 0.168 0.168 0.164 0.188 0.159 0.164 0.107 0.175 0.170 0.178 0.153 0.165 0.165 0.162 0.185 0.157 0.162 0.107 0.175 0.170 0.178 0.153 0.165 0.165 0.162 0.185 0.157 0.162 0.107 0.175 0.170 0.178 0.153 0.165 0.165 0.162 0.185 0.157 0.162 DSFRE369-08 UDEA115-18 0.172 0.177 0.183 0.178 0.175 0.175 0.196 0.201 0.180 0.175 0.183 0.168 0.175 0.175 0.175 0.167 0.164 0.164 0.180 0.175 0.183 0.168 0.175 0.175 0.175 0.167 0.164 0.164 0.180 0.178 0.181 0.176 0.173 0.173 0.167 0.185 0.167 0.172 0.180 0.178 0.181 0.176 0.173 0.173 0.167 0.185 0.167 0.172 0.016 UDEA116-18 0.040 0.044 0.040 0.040 0.146 0.151 0.040 0.042 MG936927 0.020 MG936928 0.022 0.014 0.040 0.040 0.161 0.172 0.164 0.154 MG936924 0.000 0.152 0.159 0.162 0.157 0.022 0.014 MG936925 JN988864 KP218743 0.152 0.159 0.162 0.157 0.149 0.167 0.157 0.152 0.154 0.167 0.164 0.159 0.057 0.068 0.064 0.024 0.154 0.144 0.180 0.180 HM064993 HM064991 0.012 0.024 1057 Author’s contributions Conceptualization: MFB, DRA, JCC, DB; fieldwork: DRA, JCC; formal identification and analysis: MFB; founding acquisition: DB; writing of the original draft manuscript: MFB; revision of the manuscript: MFB, DB, DRA, JCC. References Betancur-R R, Broughton RE, Wiley EO, Carpenter K, Lopez JA, Li C, Holcroft NI, Arcila D, Sanciangco M, Cureton Ii JC, Zhang F, Buser T, Campbell MA, Ballesteros JA, Roa-Varon A, Willis S, Borden WC, Rowley T, Reneau PC, Hough DJ, Lu G, Grande T, Arratia G, Orti G (2013) The tree of life and a new classification of bony fishes. PLoS Currents Tree of Life 1. https://doi.org/10.1371/ currents.tol.53ba26640df0ccaee75bb165c8c26288 Betancur-R R, Wiley EO, Arratia G, Acero A, Bailly N, Miya M, Lecoin- tre G Orti G (2017) Phylogenetic classification of bony fishes. BMC evolutionary biology 17 (1): 162. https://doi.org/10.1186/ $12862-017-0958-3 Close B, Banister K, Baumans V, Bernoth EM, Bromage N, Bunyan J, Herhardt W, Flecknell P, Gregory N, Hackbarth H, Morton D, War- wick C (1996) Recommendations for euthanasia of experimental animals: part 1. Laboratory Animals 30 (4): 293-316. Depra GC, Kullander SO, Pavanelli CS, Graga WJ (2014) A new color- ful species of Geophagus, endemic to rio Aripuana in the Amazon basin of Brazil. Neotropical Ichthyology 12: 737—746. https://doi. org/10.1590/1982-0224-20140038 Fricke R, Eschmeyer WN, Fong JD (2018) Species by Family/Subfam- ily in the Catalog of Fishes. http://researcharchive.calacademy.org/ research/ichthyology/catalog/SpeciesByFamily.asp. Accessed on: 2018-09-23, Gois KS, Pelicice FM, Gomes LC, Agostinho AA (2015) Invasion of an Amazonian cichlid in the Upper Parana River: facilitation by dams and decline of a phylogenetically related species. Hydrobiologia 746: 401-413. https://doi.org/10.1007/s10750-014-2061-8 Gosse JP (1976) Révision du genre Geophagus (Pisces Cichlidae). Académie Royal des Sciences d’Outre-Mer, Classe des Sciences naturelles et médicales 19 (3): 1-173. Gracga WJ, Pavanelli CS (2007) Peixes da planicie de inunda¢ao do alto rio Parana e areas adjacentes. EDUEM, Maringa, Brasil, 241 pp. Ilves KL, Torti D, Lopez-Fernandez H (2018) Exon-based phylogenom- ics strengthens the phylogeny of Neotropical cichlids and identifies remaining conflicting clades (Cichliformes: Cichlidae: Cichlinae). Molecular Phylogenetics and Evolution 118: 232—243. https://doi. org/10.1016/j.ympev.2017.10.008 Ivanova NV, Zemlak TS, Hanner RH, Hebert PD (2007) Universal primer cocktails for fish DNA barcoding. Molecular Ecology Notes 7: 544-548. https://doi.org/10.1111/).1471-8286.2007.01748.x Kullander SO (1986) Cichlid Fishes of the Amazon River Drainage of Peru. Swedish Museum of Natural History, Stockholm, 431 pp. Kullander SO (2003) Family Cichlidae. In: Reis RE, Kullander SO, Ferraris CJ (Eds) Check List of the Freshwater Fishes of South and Central America. Edipucrs, Porto Alegre, Brazil, 605-654. Kullander SO, Nissen H (1989) The Cichlids of Surinam (Teleostei: Labroidei). EJ Brill, Leiden, 256 pp. Kullander SO, Royero R, Taphorn DC (1992) Two new species of Geophagus (Teleoste1: Cichlidae) from the Rio Orinoco drainage in Venezuela. Ichthyological Exploration of Freshwaters 3: 359-375. Lopez-Fernandez H, Winemiller KO, Honeycutt RL (2010) Multilocus phylogeny and rapid radiations in Neotropical cichlid fishes (Per- ciformes: Cichlidae: Cichlinae). Molecular Phylogenetic Evolution 55: 1070-1086. https://doi.org/10.1016/j.ympev.2010.02.020 Lucinda PH, Lucena CA, Assis NC (2010) Two new species of cichlid fish genus Geophagus Heckel from the Rio Tocantins drainage (Perciformes: Cichlidae). Zootaxa 2429: 29-42. 1058 Mirande JM (2017) Combined phylogeny of ray-finned fishes (Actinop- terygil) and the use of morphological characters in large-scale anal- yses. Cladistics 33 (4): 333-350. https://doi.org/10.1111/cla.12171 Mirande JM, Koerber S (2015) Checklist of the freshwater fishes of Argentina (CLOFFAR). Ichthyological Contributions of Pece- sCriollos 36: 1-68. Moretto EM, Marciano FT, Velludo MR, Fenerich Verani N, Espindola ELC, Rocha O (2008) The recent occurrence, establishment and potential impact of Geophagus proximus (Cichlidae: Perciformes) in the Tieté River reservoirs: an Amazonian fish species introduced in the Parana Basin (Brazil). Biodiversity and Conservation 17: 3013-3025. https://doi.org/10.1007/s10531-008-9413-5 Neiffer DL, Stamper MA (2009) Fish sedation, anaesthesia, analgesia, and euthanasia: considerations, methods, and types of drugs. ILAR Journal 50 (4): 343-360. Check List 14 (6) Ota RR, Depra GDC, Graga WJD, Pavanelli. CS (2018) Peixes da pla- nicie de inunda¢ao do alto rio Parana e areas adjacentes: revised, annotated and updated. Neotropical Ichthyology 16 (2): 1-111. https://doi.org/10.1590/1982-0224-20170094 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30: 2725-2729. https://doi.org/10.1093/ molbev/mst197 Taylor WR, Van Dyke GC (1985) Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. Cybium 9 (2): 107-119. Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PD (2005) DNA barcoding Australia’s fish species. Philosophical Transactions of the Royal Society of London B: Biological Sciences 360 (1462): 1847-1857. https://doi.org/10.1098/rstb.2005.1716