CompCytogen 14(3):437—451 (2020) COMPARATIVE A veerrerewet open-access oven doi: 10.3897/CompCytogen.v | 4i3.55279 Kan Cyto genetics http://compcytogen.pensoft.net International journal of Plant & Animal Cytogenetics, Karyosystematics, and Molecular Systematics Comparative cytogenetic of six species of Amazonian Peacock bass (Cichla, Cichlinae): intrachromosomal variations and genetic introgression among sympatric species Janice Quadros', Alex M. V. Ferreira', Patrik E Viana', Leandro Marajo', Ezequiel Oliveira?, Efrem Ferreira”, Eliana Feldberg' | Laboratorio de Genética Animal, Coordenagio de Biodiversidade, Instituto Nacional de Pesquisas da Amazonia, Av. André Araujo 2936, Petropolis, 69067-375, Manaus, AM, Brazil 2. Laboratério de Ecologia de peixes, Coordenacio de Biodiversidade, Instituto Nacional de Pesquisas da Amazonia, Av. André Araujo 2936, Petrépolis, 69067-375, Manaus, AM, Brazil 3 Departamento de Genética e Evolugdo, Universidade Federal de Sto Carlos, Sao Carlos, Séo Paulo 13565-905, Brazil Corresponding author: Eliana Feldberg (feldberg@inpa.gov.br) Academic editor: R. Noleto | Received 9 June 2020 | Accepted 2 September 2020 | Published 17 September 2020 http://zoobank. org/8EEA88F 2-82 18-48 C8-9F6C-F3A4CADB42CD Citation: Quadros J, Ferreira AMV, Viana PE, Marajé L, Oliveira E, Ferreira E, Feldberg E (2020) Comparative cytogenetic of six species of Amazonian Peacock bass (Cichla, Cichlinae): intrachromosomal variations and genetic introgression among sympatric species. Comparative Cytogenetics 14(3): 437-451. https://doi.org/10.3897/CompCytogen.v14i3.55279 Abstract Cytogenetic data for the genus Cichla Bloch et Schneider, 1801 are still very limited, with only four karyo- type descriptions to date. The sum of the available cytogenetic information for Cich/a species, points to a maintenance of the diploid number of 48 acrocentric chromosomes, considered a typical ancestral feature in cichlids. In the current study, we performed molecular and classical cytogenetic analyses of the karyo- type organization of six species of Cichla, the earliest-diverging genus of Neotropical cichlids. We cytoge- netically analysed Cichla kelberi Kullander et Ferreira, 2006, Cichla monoculus Agassiz, 1831, Cichla piquiti Kullander et Ferreira, 2006, Cichla temensis Humboldt, 1821, Cichla vazzoleri Kullander et Ferreira, 2006 and Cichla pinima Kullander et Ferreira, 2006, including three individuals that showed mixed morpho- logical characteristics, likely from different species, suggesting they were hybrid individuals. All individu- als analysed showed 2n = 48 acrocentric chromosomes, with centromeric heterochromatic blocks on all chromosomes and a terminal heterochromatic region on the g arm of the 2™ pair. Mapping 18S rDNA gave hybridization signals, correlated with the nucleolus organizer regions, on the 2" pair for all analyzed individuals. However, we found distinct patterns for 5S rDNA: interstitially at the proximal position on Copyright Janice Quadros 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. 438 Janice Quadros et al. / Comparative Cytogenetics 14(3): 437-451 (2020) 6" pair of four species (C. kelberi, C. pinima, C. piquiti and C. vazzoleri), and on the distal of the 4" pair in two (C. monoculus and C. temensis). Accordingly, we present here new data for the genus and discuss the evolutionary trends in the karyotype of this group of fish. In addition, we provide data that supports the occurrence of hybrid individuals in the Uatuma River region, mainly based on 5S rDNA mapping. Keywords 5S rDNA, FISH, Heterochromatin, Hybridization, karyotype Introduction The genus Cichla Bloch et Schneider, 1801 belongs to the subfamily Cichlinae that, joint- ly with Retroculus Eigenmann et Bray, 1894, makes up the tribe Cichlini, and is the earli- est-diverging lineage of Neotropical cichlids (Leo Smith et al. 2008). This taxon is widely distributed within the Amazon, Tocantins, and Orinoco River basins, and in the smaller rivers draining the Guianas to the Atlantic Ocean. Most Cich/a species follow an allopatric distribution pattern, although some species are sympatric or even syntopic (Kullander and Ferreira 2006). However, some species, such as C: monoculus Agassiz, 1831, C. kelberi Kullander et Ferreira, 2006 and C. piquiti Kullander et Ferreira, 2006, have been intro- duced into other areas, where they are well established, due to their generalist habit. Cichla are very emblematic fish in South America, with high economic and ecological impor- tance, especially since they are predators in Amazonian rivers and used widely for sport fishing (Nascimento et al. 2001; dos Santos et al. 2016; Diamante et al. 2017). Representatives of the genus Cich/a are easily distinguished from all other Neotropical cichlids by the shape of the dorsal fin, and the presence of 1 to 4 dark vertical bars along the body. However, the species are very similar, and while their color patterns still provide the best species diagnostic characters, in some cases these may complicate accurate identifi- cation, since key characters may show ontogenetic changes (Kullander and Ferreira 2006). According to Kullander and Ferreira (2006), the genus comprises 15 morphologi- cally distinct species, and recently another species has been described (Cichla cataractae Sabaj et al. 2020) from the Essequibo River basin, where it is endemic). However, Willis et al. (2012), based on multilocus data, recognized only eight species. The species often have restricted natural distributions, but to variable extents. For example, while C. monoculus is found all over the Amazon River and low tributary course, C. temensis Humboldt, 1821 is found only in black water rivers, whereas C. piquiti and C. kelberi are restricted to the Tocantins River. Cytogenetic data concerning the family Cichlidae points to a remarkable trend in the maintenance of the diploid number 2n = 48, mostly in the acrocentric form (Thompson 1979). However, as more species were karyotyped, a huge chromosomal diversity was observed in the derived clades (ranging from 32 to 60 chromosomes), but with predominance of 2n = 48 in most lineages, which is considered an ancestral trait for this group (Feldberg et al. 2003; Gross et al. 2009; Poletto et al. 2010; da Costa et al. 2019). For the genus Cichla, only C. monoculus, C. temensis, C. kelberi and C. piquiti have had their karyotypes described, all exhibiting a diploid number com- Comparative cytogenetic analysis of Cichla species 439 posed of 48 acrocentric chromosomes, as the species from earliest-diverging Cichlinae tribes (Retroculini, Astronotini and Chaetrobranchini) (Feldberg et al. 2003; Alves- Brinn et al. 2004; Poletto et al. 2010; Mour4o et al. 2017). Interestingly, Alves-Brinn et al. (2004), based on cytogenetic data, reported the oc- currence of hybridization between C. monoculus and C. temensis in the Uatuma River (Balbina Hydroelectric Dam). In addition, interspecific hybridization and introgres- sion between species has been much discussed in relation to the adaptive advantages and increase of genetic variability (Willis et al. 2012). For some authors, hybridization may be related to diversification and speciation, or the extinction of populations or species (Mourao et al. 2017). Under either species concept, the phylogenetic breadth of introgression in this group is clear, with both sister species and species from different mtDNA clades exhibiting genetic introgression (Willis et al. 2012). In the current study, we used different classical and molecular cytogenetic markers to characterize Cichla species, from different river drainages within the Amazon basin and investigate the likely existence of hybrid individuals, where more than one species occurs, such as at Uatuma River (Balbina Hydroelectric Dam). Material and methods In the current study, we sampled 50 individuals of the genus Cich/a from five locations in the Brazilian Amazon basin (Table 1, Figs 1, 2) under ICMBIO (Instituto Chico Mendes de Conservacao da Biodiversidade) permit number: 28095-1. Voucher specimens were de- posited in the Fish Collection of the National Institute of Amazonian Research (Instituto Nacional de Pesquisas da Amazénia — INPA) (Table 1). Dr. Efrem Ferreira and Dr. Jansen Zuanon, following description of Kullander and Ferreira (2006), identified the Cich/a spe- cies included in the current study. However, three individuals had mixed characteristics of more than one species, and were thus considered possible hybrids by specialists. Chromosomal preparations were obtained from the kidney, following the protocol of Gold et al. (1990). The active nucleolus-organizing region (NOR) was detected with Table |. The Cichla species included in the current study, collecting localities, the number of indi- viduals analyzed, and Voucher number. & = male, 2 = female. AM = Amazonas State, PA = Para State, MT = Mato Grosso State. Species Number of Collecting localities Coordinates Voucher individuals C. kelberi 43 39 Araguaia River — Sao Félix, MT 11°39'03.9"S, 50°52'59.4"W MZUSP125273 C. monoculus 52 Anavilhanas (Negro River), AM (Black water) 2°33'28.4"S, 60°46'29.7"W INPA-ICT059045 C. monoculus 39 Uatuma River (Balbina Hydroelectric Dam) AM, Black water) 1°55'02.2"S, 59°28'23.7"W INPA-ICT059046 C. monoculus 192 Tapajés River — Santarém, PA (Clear water) 2°24'53.0"S, 54°46'48.3"W INPA-ICT059047 C. monoculus 44 19 Cataldo Lake, AM (Mix of white and black water) 3°10'30.8"S, 59°56'30.3"W INPA-ICT059044 C. pinima 73 69 Tapajés River (Mix of white and clear water) 24°21'16.4"S, 54°70'23.16"W INPA-ICT059045 C. piquiti 24 29 Araguaia River — Sao Félix, MT 11°38'01.7"S, 50°40'11.3"W MZUSP125272 C. temensis 24 29 Uatuma River (Balbina Hydroelectric Dam). AM, Black water) 1°55'02.2"S, 59°28'23.7"W INPA-ICT059043 C. vazzoleri 24 39 Uatuma River (Balbina Hydroelectric Dam, AM, Black water) 1°55'02.2"S, 59°28'23.7"W INPA-ICT059048 Hybrids 34 Uatuma River (Balbina Hydroelectric Dam, AM, Black water) 1°55'02.2"S, 59°28'23.7"W = INPA-CT059047 440 Janice Quadros et al. / Comparative Cytogenetics 14(3): 437-451 (2020) -Anavilhanas (Medium Negro River} - Catalio Lake (Negro River) -Uatuma River - Tapajos River - Santarém/PA - Araguaia River - Sao F élix/MT Figure |. Map showing the collection points of Cichla species analyzed in current study. silver nitrate impregnation (Ag-NOR), following Howell and Black (1980), while con- stitutive heterochromatin was detected following Sumner (1972). DNA was extracted using the Wizard Extraction Kit (Promega), following manufacturer’s recommenda- tions, and quantified using a NanoVue Plus spectrophotometer (GE Healthcare). Amplification of 18S and 5S rDNA used the Polymerase Chain Reaction (PCR) with primers 18S F(5’ -CCG CTT TGG TGA CTC TTG AT-3’) and R(5’ -CCG AGG ACC TCA CTA AAC CA-3’) (Gross et al. 2010), and the primers 5S F(5’ -TAC GCC CGA TCT CGT CCG ATC-3’) and R(S’ -CAG GCT GGT ATG GCC GTA AGC-3’) (Martins and Galetti 1999). All PCRs were performed with a final volume of 25 uL, containing genomic DNA of each species (200 ng), 10x buffer with 1.5 mM MgCl, DNA polymerase (5 U/uL), dNTPs (1 mM), primers (5 mM) and Milli-Q. The reaction profile for 18S rDNA was 1 min. at 95 °C, 35 cycles of 1 min. at 94 °C, 1 min. at 56 °C and 1 min. and 30 s at 72 °C, followed by 5 min. at 72 °C. The reaction profile for 5S rDNA amplifica- tion was 1 min. at 95 °C, followed by 30 cycles of 1 min. at 94 °C, 1 min. at 59 °C and 1 min. and 30 s at 72 °C. The final extension was 5 min. at 72 °C. PCR products were checked on 1% agarose gel, quantified on a NanoVue Plus spectrophotometer (GE Healthcare). PCR products were labeled with digoxigenin (Dig-Nick Translation mix; Roche) and biotin (Bio-Nick Translation mix; Roche), and used as probes for the fluorescent in situ hybridization technique (FISH). Hybridizations were performed according to the protocol described by Pinkel et al. (1986), with a stringency of 77% (2.5 ng/uL) for 18S rDNA, 5S rDNAr, 50% formamide, 10% dextran sulfate and 2xSSC at 37 °C for 18 h), post-hibridization washes were made with formamide 15% and 2xSSC Tween 0.5%. Chromosomes were counterstained with DAPI (2 mg/mL) using the Vectashield (Vetor) mounting medium. Telomeric segments were generated using non-templated PCR with primers (TTAGGG)5 and (CCCTAA)5 (Ijdo et al. 1991). Comparative cytogenetic analysis of Cichla species 44] Cichla kelberi Cichla monoculus (UHE Balbina - Uatuma River) Cichla pinima —<—S Cichla piquiti Cichla monoculus (Medium Negro River) Cichla temensis Cichla monoculus (Santarém -Tapajos River) Hybrids 10.0 cm Figure 2. Cichla species and individuals considered morphologically hybrid (A-C). C. kelberi SL = 170.0 mm; C. pinima SL = 190.5 mm; C. piquiti SL = 200.0 mm; C. temensis SL = 210.0 mm; C. vazzoleri SL = 250.0 mm; C. monoculus (Uatuma River) SL = 160.0 mm; C. monoculus (Catalao Lake, Negro River) SL= 150.0 mm; C: monoculus (Anavilhanas, Medium Negro River) SL= 180.0 mm; C. monoc- ulus (Santarém, Tapajés River) SL = 180.0 mm; Hybrid A SL = 280.0 mm; Hybrid B SL = 230.0 mm; Hybrid C SL = 320.0 mm. We analyzed at least 30 metaphase per individual to confirm the diploid number and karyotype structure. Images were captured using an Olympus BX51 epifluores- cence microscope, and processed using Image-PRO MC 6.0 softwares. Chromosomes 442 Janice Quadros et al. / Comparative Cytogenetics 14(3): 437-451 (2020) were measured using the Image J program, arranged in descending order of chromo- some size, and classified according to Levan et al. (1964). All methodological procedures in the current study were performed in accordance with the guidelines of the Ethics Committee of the National Institute of Amazonian Research (Instituto Nacional de Pesquisas da Amazénia — INPA), protocol: CEUA No. 009/2018. Results The six species analyzed (C. kelberi, C. monoculus, C. pinima, C. piquiti, C. temensis and C. vazzoleri) all had a diploid number equal to 48 acrocentric chromosomes, and a fundamental number (FN) equal to 48. The NORs (Ag-NORs and 18S rDNA) were located in a distal position on the g arms of pair n° 2 in all species (Figs 3, 4). Cichla monoculus was the sole species sampled in more than one location, and it showed no difference when compared to data in Alves-Brinn et al. (2004) and Schneider et al. (2013) (data not shown). The 5S rDNA site was located interstitially at the proximal position of pair n° 6 in four species (C. kelberi, C. pinima, C. piquiti and C. vazzoleri), and on distal portion of pair n° 4 in two (C. monoculus and C. temensis) (Fig. 4). The six species had centromeric heterochromatic blocks on all chromosomes and a terminal heterochromatic region on pair 2, which corresponds to the same position as the NORs. However, some blocks were species-specific: terminal blocks were ob- served on the g arm of C. kelberi pairs 1, 3 and 4; pair 3 of C. pinima and C. temensis; pairs 1 and 3 of C. piquiti; and in C. vagzoleri pairs 3 and 6 (Fig. 3). C. monoculus, which was sampled in four different locations, showed variable constitutive hetero- chromatin patterning, where individuals from the Uatuma River (Balbina Hydro- electric Dam) also had terminal blocks on the g arms of the chromosomal pairs 1, 6, 9, 12, 15, 19. Catalao Lake individuals appeared to have terminal pale blocks on all pairs, with conspicuous ones on 1, 5, 6, 8, 10 chromosomal pairs. This also occurred for individuals from Anavilhanas, but in these, the blocks were more conspicuous in practically all chromosomes, and still had interstitial markings on pairs 1, 3 and 6. Individuals from the Tapajés River had terminal blocks on pairs 1, 3, 5, 11, 14, 15, and interstitials on pairs 14 and 15 (Fig. 5). The three individuals morphologically considered hybrids also had 2n = 48 acro- centric chromosomes and FN = 48, Ag-NOR and 18S rDNA on the second pair at terminal position on the g arm, collocated with a conspicuous heterochromatic por- tion (Figs 6, 7). Constitutive heterochromatin was present in the centromeric region of all chromosomes in the three individuals and the first pair had an interstitial block. Additionally, individual A (Fig. 6b) had terminal blocks on pairs 1 and 5; individual B (Fig. 6e) had terminal blocks on most chromosomes and interstitials on pairs 3 and 6; individual C (Fig. Gh) had terminal pale blocks on pairs 3 and 10. 5S rDNA was detected interstitially on one pair 4 homolog, and on one pair 6 homolog in two hybrid individuals (A and C). Individual B showed 5S rDNA sites on both pair 4 chromosomes (Fig. 7). Comparative cytogenetic analysis of Cichla species 443 C. kelberi a) os Chashateaanane b) DG OG HA 05 a ce tees ®) aa stia eaaeenns aneseser stla ap aene 90 60 ce pean 11 13 14 15 10 11 12 13 14 15 16 YT TY YYTe TTT yee 9a be 28 62 @8 @e Be ee 17 18 19 20 21 22 23 £24 17 18 19 20 21 22 23 «424 C. pinima d CLOG AN GetPeonees °) RUAG EA ts cennecan As stia oe dneonoagns ante sta] RO RARRRARm BAAS OO 11 12 13 14 15 16 9 10 11 12 13 14 15 16 $0 DADAREIGAGO as SrAGREAARABREAAAE 17 18 19 21 22 23 24 17 18 19 20 21 22 23 24 C. piquiti D)/AGasteasesconnecn "| S000 Oban eeneasan [20 12 38 4 5 6 Ff 8 if 2 *, ere - | 2 sta] AA AG O26 88 6460 OR ee sta| 88 68888 22 SR AA BE 2 10 Be ie oe oe 8 9 10 1 #4212 #13 #14 «15~«16 S82 fH 26 6646 Ae bene 864486880 6468 oe of 17 18-19 200 21 22 2324 17 18 19 20 21 22 23 «24 C. temensis ; Dipaceaabaseteteae ” bene betes oe ae eee As 1 2 3 4 5 6 7 8 2 sta| #8 eee Oe ee be te an stla abeceons ae oe eens 9 10 11 12 13 14 15 16 9 1 26 1s 44 15 Ae se ee aeeaee th oe be sees 4866 @@ eeecse 17 18 19 20 21 22 23 24 17 18 19 20 21 22 23 24 C. vazzoleri ™ | A0OAAD GC babe Hae ")/ AGAA GABE On An AA Ae °) Ae 1 2 ~: | 4 5 6 ri 8 1 2 3 4 5 stia| AO RRAABOARAAREAA opal AR me AA BABA aa aa - 9 10 1 #12 #13 14 «15 16 9 10 1t 12 48 (4 #415 46 86 66808 BAAAKE BE GA 4 AA GA mem 04 me Am &A 7 18 1 20 24 22 2 -24 17 18 19 20 2158) 22 23.24 10.0 pm Figure 3. Karyotypes analyzed by conventional Giemsa staining, C banding and Ag-NOR: Cichla kelberi (a, b, c) C. pinima (d, e, f) C. piquiti (g, h, i) C. temensis (j,k, 1) C. vazzoleri (m, n, 0). For all analysed species, hybridization with telomeric probes showed, as expected, only markings on the terminal portions of both arms (data not shown). Discussion For Cichlidae species, a diploid number equal to 48 acrocentric-like chromosomes is considered an ancestral feature (Thompson 1979), and chromosomal evolution in this family was thought to be conserved from the karyotype macrostructure point of view (Feldberg et al. 2003). However, as more Cichlidae species were cytogenetically stud- ied and more accurate techniques were applied (e.g. mapping of different molecular 444 Janice Quadros et al. / Comparative Cytogenetics 14(3): 437-451 (2020) 1118S rDNA I 5s rDNA C. monoculus AGRRARhAGE C. kelberi BGARGE RR AA Se ee 26 1 2 3 4 5 6 7 8 val gee@ean ; | 664660006068060 80608 9 10 10 11 12 13 14 15 16 6666866666666 4a 66 20 1718 19 21 22 23 24 C. pinima C. piquiti c) d) PAGE GA AR ROG. 88 An Aa daae San b6 be 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 sia} AA BG 66 Aw AA OO SA AA sta '4 66 'éa ea aRaeeé 9 12 13 14 15 16 12 10 11 9 10 1 13 14 88 @A wA 8A AA AA GA BA $8664 68 468 6 na. 20 23 17 18 19 21 22 24 17 18 19 20 21 22 C. temensis C. vazzoleri PR ASCO M+ ave Be TELL 1 2 3 4 5 6 7 4 2 3 ® $8 ee 9 Bie eneaeaase 11 13 14 15 16 .& e 4, © 23 24 10.0 um Figure 4. Karyotypes analyzed with molecular chromosome markers. Double FISH with 18S (red) and 5S (green) rDNA probes. Cichla monoculus (a) C. kelberi (b) C. pinima (€) C. piquiti (d) C. temensis (e) C. vazzoleri (f). a) C. monoculus - Uatuma River b) C. monoculus - Catalao lake am bl : ; fa ane A B seeaa a , L , f >, & wo © ! sa & © + Le “sae Of _s & fe & - 2 a = i " ‘ 8 1 Z 3 4 5 6 Y 8 st/a sees F sean . : seen “ ™ 6 st/a >se eae eR SR Ree eR ee ee e° « 9 10 11 12 13 14 15 16 9 7 = 44 12 13 14 15 16 ~_ * Ae ese tt eo ee Se Se » * .* « . @ - : ~~ = - . - Pe ee ee ed eee ae i? 8 © 2 2f 22 8 24 C. monoculus - middle Negro River d) C. monoculus - Tapajos River c a8 A SA eae ee aaee te 4 . i @ a Sa b&b ae OD — an is $f a4 ee Se f a 4 + = ; az , - A | m } - = ° ° ' 1 2 re 4 5 6 7 8 1 2 3 4 5 6 ri 8 sval\@AR RAAB AARBRBGARGE tal ROSEARDE OARS BEEF 9 10 11 12 13 14 15 16 9 10 11 12 13 14 15 16 : }RAARRARReAEE BAARERRARA EA CaS 17 18 19 20 24 22 23 24 17 18 19 20 «21 22 23 24 10.0 pm Figure 5. Cichla monoculus karyotype from different locations with conventional Giemsa staining, C. band- ing: a Uatuma River b Catalao Lake (Negro River) ¢ Anavilhanas (middle Negro River) d Tapajés River. Comparative cytogenetic analysis of Cichla species hybrid A a ) $0 680000 00 08 on 00 sva/0@ as WITTY aa6666 11 13 14 15 «16 ca rs sea i se 66 6a Ga 17 18 19 20 21 22 23 24 hybrid B d) DOCREA BAER Oe Ae Aa 1 2 3 4 5 6 7 8 sal PA BR RBSR BAGO GOR8 9 10 11 12 13 14 15 16 SERS AR BRERA B AACS 17 18 19 20 21 22 23 24 hybrid C g) 1hAh G0 db on 00 dee volo MO 0008 008000 as 14 15 16 ae 86 ae re pape aaeea 445 b) AR 86 BA aa Al. /RAAN NA OA no as oe onan as a ha So 10 1 1 1 4 4S 46 BX AA OA AARE GA AA AA 7 18 12 20 21.22 23 oo sta °) Ah ARGS aato as been" an aol bP AD OS ETT TTY 9 11 12 13 14 15 16 apan 6a 8G0a GaGa a 7 18 #19 20 21 2 23 oe ) ‘ SRAGURCRAREACARS ta 1 2 a ae 5 6 7 8 2 sal ®@OAGRGAREG BARROS oS 10 Ht 42 43 #3 4S) 16 aasaceaseseaan OA aa wm 19 20. 24 22 23 24 17 18 #19 20 21 22 23 «24 10.0 pm Figure 6. Karyotype of hybrid individuals A, B and C (as in Fig. 2) respectively, with conventional Giemsa staining (a, d, g), C-banding (b, e, h) and Ag-NOR (, f, i). chromosomal markers), several karyotypic formulas and configurations have been found (Gross et al. 2009; Poletto et al. 2010; Schneider et al. 2013), suggesting that this fish group experienced multiple non-robertsonian chromosomal rearrangements during its evolution, since the 2n = 48 is retained in most Cichlinae lineages. In the current study, analyzes focused on the genus Cichla, which represents one of the most basal lineages of Neotropical cichlids (Leo Smith et al. 2008). To date, all species karyotyped possess a complement of 48 acrocentric-like chromosomes, with very similar karyotypes between species, including the NOR pattern, which is usu- ally found on the 2" pair (Alves Brinn et al. 2004; Schneider et al. 2013; Mourao et al. 2017; current study). In addition, some studies examining morphological-mito- chondrial divergences (Andrade et al. 2001; Willis et al. 2010), as well as chromosome features (Alves Brinn et al. 2004; Oliveira et al. 2006), and electrophoretic esterase comparisons (Teixeira and Oliveira 2005) have inferred hybridization in natural and in artificial or disturbed environments/populations. For constitutive heterochromatin, the distribution pattern can often be used as a spe- cies-specific or population marker (Feldberg et al. 2003; Vicari et al. 2006; Benzaquem et al. 2008; Perazzo et al. 2011). In our analyses, for instance, we found four different heterochromatin patterns for C. monoculus (Fig. 5). This is one of the most widely- distributed species in the Amazon River basins (Kullander and Ferreira 2006), and the 446 Janice Quadros et al. / Comparative Cytogenetics 14(3): 437-451 (2020) (18S rDNA IM 5S rDNA a) hybrid A 17 18 hybrid B hybrid C Teh Figure 7. Karyotypes of hybrid individuals A, B and C (as shown in Fig. 2) respectively with molecular chromosomal markers. Double FISH with 18S (red) and 5S (green) rDNA probes. commonly introduced into dam resevoirs throughout Brazil (dos Santos et al. 2016; Di- amante et al. 2017). Heterochromatin is known to play important roles in the chromo- somal architecture and karyotype organization, such as assisting in chromosomal segre- gation, nuclear organization and expression of gene regulation, associated with responses to environmental changes (Grewal and Jia 2007; Varriale et al. 2008; Buhler 2009; Ribeiro et al. 2017; Viana Ferreira et al. 2019). This seems to be the case for the differ- ent C. monoculus populations analyzed in our study, where individuals from black and acid waters (Negro and Uatuma rivers), white and black mixed waters (confluence of Negro and Solimées rivers), and in the confluence of white and clear waters (confluence of Amazonas and Tapajés rivers), showed intraspecific variability in their heterochro- matic patterns, possibly reflecting chromatin adaptation and/or epigenomic responses to changes in the specific environment inhabited by these different populations. Comparative cytogenetic analysis of Cichla species 447 Interestingly, the heterochromatic patterns of the three probable hybrids was very similar and much closer to the pattern described for the Negro River (C. monoculus from Anavilhanas) with some interstitial blocks. Could it be heterochromatinization? Such heterochromatin variability can also be explained by stressors, such as environ- mental changes, or even hybridization processes (Richards et al. 2010; Ribeiro et al. 2017), which would explain the heterochromatin distribution differences found in C. monoculus and in the probable hybrids. Besides the conservation of the karyotype macrostructure, Schneider et al. (2013) reported that 12 out of 13 Cichlinae species analyzed in their study had only one chro- mosome pair harboring 5S rDNA sites, but at different karyotypic positions, indicating that 5S rDNA sites are a robust molecular chromosomal marker in cichlid species. 5S rDNA is an important cytotaxonomic and evolutionary marker, since it helps provide a better understanding of fish chromosomal diversity (Bellafronte et al. 2005; Teixeira et al. 2009; Vicari et al. 2010). For instance, a study by Ferreira et al. (2016), mapping of 5S rDNA sequences in Bunocephalus coracoideus Cope, 1874, revealed an association between this rDNA site and a multiple sex chromosome system previously unknown in Siluriformes (X,X,X,X,/X,Y X.Y,). Repetitive 5S and 18S rDNA sequences are the most well-studied in fish, and have been gaining prominence mainly in studies of between-species evolutionary relationships, population characterization and genome structure (Martins et al. 2004; Terencio et al. 2012; Schneider et al. 2013). In the current study, individuals of all six species, including the hybrids, had 18S rDNA on terminal position of the g arm of the 2"! chromosomal pair (same position as NORs). Meanwhile, 5S rDNA mapping in Cich/a species showed two patterns: on the 4" pair (C. monoculus and C. temensis), and on the 6" pair (C. kelberi, C. pinima, C. piquiti and C. vazzoleri). However, in the individuals morphologically considered hybrids, we found two distinct patterns: two of them (hybrids A and C) having 5S rDNA in one homologue of the 4" pair and one homologue of the 6" pair, while the other hybrid (individual B) had 5S rDNA on both homologues of the 4" pair. Since these probable hybrids were captured in the Uatuma River (Balbina Hydroelec- tric Dam), where C. monoculus, C. temensis and C. vazzoleri all occur (Kullander and Ferreira 2006), we believe that these species might be hybridizing. Interestingly, the karyotypes of C. pinima and C. vazzoleri (current study) are very similar, except for a heterochromatic terminal block on the g arms of the 6" pair in C. vagzoleri. It is notable that C. pinima was sampled in the Tapajés River and C. vazzoleri in the Uatuma River, very distant locations with no history of sympatry or migration (Ferreira, personal communication). However, according to Willis (2017), C. pinima sensu lato includes C. pinima, C. vazzoleri, C. jariina, Kullander et Ferreira, 2006 and C. thyrorus Kullander et Ferreira, 2006 (sensu Kullander and Ferreira 2006), and reports that the evolutionary relationships in this group are more complex than previously thought. Willis (2017) suggest that this separation into four species does not correspond to its evolutionary history and contemporary dynamics of the genus. In addition, Willis et al. (2012) reported that genetic introgression is a common phenomenon in Cich/a species. Introgression can be defined as the movement of DNA from the genetic pool of one species into that of another species by repeated backcross- 448 Janice Quadros et al. / Comparative Cytogenetics 14(3): 437-451 (2020) ing of hybrid individuals with one or both parent species. Such hybridization events are expected to occur most commonly in modified habitats, but interestingly, most of the hybridization cases known for Cichla species, were found in undisturbed natural environ- ments (Willis et al. 2012), suggesting that introgression forms a natural part of the evolu- tion of many tropical species, so increasing genetic diversity. In this sense, we cannot rule out hybridization and genetic introgression among the likely parental species, especially taking in account that all three probable hybrid individuals used here had male gonads. Conclusions Our data supports the tendency in the maintenance of the 2n = 48 chromosomes for Cichla species, as well as the conservation of the karyotypic formula and simple NOR, but reveals 5S rDNA to be an important cytogenetic marker for this group. In additon, here we provide, for the first time, the karyotype for C. pinima and C. vaz- zoleri. Furthermore, our data shows that the heterochromatin pattern may differenti- ate populations of C. monoculus, suggesting that this variation might be the result of epigenetic events triggered by different water types. Acknowledgements The authors are grateful to Jansen Zuanon for identifying the individual fish. Brazil- ian institutions: INPA/BADPI the post-graduate program in Freshwater Biology and Continental Fisheries, the Laboratory of Animal Genetics (LGA/INPA). This study was financed by the Centro de Estudos de Adaptacao as Mudancas Ambientais da Amazonia (INCT ADAPTA II, FAPEAM/CNPgq 573976/2008-2), Conselho Nacional de Desenvolvimento Cientifico e Tecnolégico and the Fundacéo de Amparo a Pesquisa do Estado do Amazonas. JMQ received a study grant from the Conselho Nacional de Desenvolvimento Cientifico e Tecnolégico. Dr. Adrian A. Barnett reviewed the English. 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