Closely Related Syntopic Cytotypes of Astyanax taeniatus (Jenyns,1842) from the Upper Piranga River,Upper Doce Basin in Southeastern Brazil

Abstract

Astyanax taeniatus occurs in coastal areas of southeastern Brazil, and it is very abundant in the Upper Doce River Basin. Our objective was to study C-, argyrophilic nucleolar organizer region (Ag-NOR) and fluorescent in situ hybridization (FISH) banding patterns using 5S, 18S, CA₍₁₅₎, and GA₍₁₅₎ repetitive DNA probes on a population of A. taeniatus present in the Piranga River, in the Doce Basin. Two syntopic cytotypes were found, both with 2n = 50: cytotype A (14m + 12sm + 16st + 8t) and cytotype B (10m + 14sm + 18st + 8t). In both cytotypes, heterochromatic blocks occurred in all the chromosomes; Ag-NOR sites were multiple, ranging from four to eight. The 5S rDNA probe marked eight chromosomes in both cytotypes, a unique condition within Astyanax, suggesting a recent divergence between these cytotypes. The 18S rDNA probe differed between the cytotypes, marking 10 and 8 chromosomes in cytotypes A and B, respectively. CA₍₁₅₎ and GA₍₁₅₎ FISH patterns were mainly subtelomeric, but CA₍₁₅₎ showed centromeric markings that were diagnostic for each cytotype. Although overall cytogenetic evidence suggests that these cytotypes are closely related, morphological and molecular data in progress will provide further hypothesis test on their phylogenetic relationship.

Introduction

It is estimated that over 7,000 species of freshwater fish are present in the Neotropical region; of them, 1973 species belong to the order Characiformes. Within this taxon, 1345 species belong to the family Characidae.¹ Reflecting the complex systematics of this family, several of its genera are still included in incertae sedis condition, including all the species of the genus Astyanax.² The morphological^3,4 and molecular^5,6 data corroborate that some of the genera that were previously considered incertae sedis are nonmonophyletic.

With 154 valid species, Astyanax is the most species-rich genus in the order Characiformes. Most of them are widely distributed in the Neotropical region from southern United States to northern Argentina.⁷ Cytogenetic research shows high levels of variation in this genus, and it also suggests that some nominal species such as Astyanax altiparanae Garutti & Britski, 2000,^8,9 Astyanax fasciatus (Cuvier, 1819),^10,11 Astyanax scabripinnis (Jenyns, 1842),^12,13 and Astyanax hastatus Myers, 1928¹⁴ are actually species complex, and several good species have been validated within these complexes. Astyanax species are morphologically similar, and their taxonomic classification has been historically difficult; the last revision in this genus was made almost a hundred years ago.^15–18 Moreover, a complicating factor for understanding the group's systematics is the occurrence of hybridization in some of its taxons.^10,11,19

Astyanax taeniatus was described in 1842 by Jenyns from the type locality Socego, Rio de Janeiro State. This species also occurs in the Paraíba do Sul River Basin, in the drainage of coastal rivers of Rio de Janeiro and Espírito Santo,² in the Doce River Basin,²⁰ and in the São Mateus and Mucuri river basins.²¹ This species is the most abundant characin present during the annual reproductive migration in the Risoleta Neves Dam, in the Upper Doce River. Although the diploid number and chromosomal formula have been characterized from specimens of A. taeniatus belonging to the isolated coastal basins such as Jucu and Benevente,²² the cytogenetic banding and fluorescent in situ hybridization (FISH) patterns from Upper Doce River Basin population are still unknown.

Karyotypic comparisons between species, associated with taxonomic and molecular biological studies, provide useful information for studying evolution, phylogeny, and understanding of mechanisms of speciation.²³ In this study, we aimed to analyze a population of A. taeniatus (Jenyns, 1842) present in the Piranga River, using various cytogenetic tools. The study area is located in the Doce River Basin. Like many other Neotropical drainages, the Doce River Basin is small and isolated and is characterized by low levels of species richness and high levels of endemism.²⁴

Materials and Methods

Specimen collection

The specimens of A. taeniatus were collected in the Hydroelectric Risoleta Neves Reservoir fish scale (20°11′41.92"S 42°51′04.95"W), in the Piranga River, Upper Doce River Basin, located in the municipalities of Rio Doce and Santa Cruz do Escalvado, Minas Gerais, Brazil. Collection permit of the Instituto Chico Mendes de Biodiversidade (ICMBio) (SISBIO14975-1) was issued to Jorge Abdala Dergam. Species identification followed Eigenmann.¹⁷ Voucher specimens were deposited in the scientific collections of the Museu de Zoologia João Moojen in Viçosa, Minas Gerais, Brazil (voucher MZUFV3992) and the Universidade Federal do Rio Grande do Sul, Rio Grande do Sul, Brazil.

Karyological analyses

The specimens of A. taeniatus were anesthetized with clove oil²⁵ as approved by the Universidade Federal de Viçosa Animal Welfare Committee (CEUA authorization 58/2013). Mitotic chromosomes were obtained from kidney following Bertollo et al.²⁶ The following cytogenetic techniques were used: Giemsa conventional staining for basic karyotypic analysis; C-banding²⁷ stained with fluorescent 4′,6-diamidino-2-phenylindole (DAPI) dye²⁸ to identify blocks of constitutive heterochromatin; argyrophilic nucleolar organizer region (Ag-NOR) banding for their identification²⁹; and FISH³⁰ using 5S and 18S rDNA probes and CA₍₁₅₎ and GA₍₁₅₎ repetitive DNA probes.

Ribosomal DNA probes 18S and 5S were labeled using PCR with digoxigenin-11-dUTP (Roche Applied Science) and the signal was detected with anti-digoxigenin-rhodamine (Roche Applied Science). DNA was denatured with 70%/2× SSC formamide solution at 72°C for 3 min; each slide was treated with 20 μL of hybridization mix containing the following: 200 ng of labeled probe, 50% formamide, 2× SSC, and 10% dextrane sulfate. This hybridization mix was heated for 10 min at 85°C; slides were kept in a moist chamber at 37°C overnight; and after the hybridization step, the slides were washed twice in 2× SSC for 5 min and incubated in a 3% NFDM 4× SSC solution for 10 min and placed in a moist chamber at 37°C for 1 h with a detection solution using anti-digoxigenin-rhodamine (Roche Applied Science).

Finally, the slides were washed thrice in a 4× SSC/0.5% Tween solution for 5 min at room temperature; they were dehydrated in an alcoholic series and counterstained with DAPI. As for the probes CA₍₁₅₎ and GA₍₁₅₎, they were synthesized and labeled with Cy3 fluorochrome at the 5′ end (Sigma). Digital images were captured in a BX53F Olympus microscope equipped with DP73 and MX10 Olympus camera for colored and fluorescent techniques, respectively, using the CellSens imaging software. The karyotypes were measured with the Image-Pro Plus^® software and classified according to arm ratios as metacentric (m), submetacentric (sm), subtelocentric (st), and telocentric (t).³¹

Results

A total of 77 individuals were cytogenetically analyzed, of which, 28 were males, 44 were females, and 5 were juveniles. The diploid chromosome number was 2n = 50, without sex chromosomes. Two karyotypes were found: 30 specimens showed cytotype A: 2n = 14m + 12sm + 16st + 8t (Fig. 1A), whereas 47 specimens showed cytotype B: 2n = 10m + 14sm + 18st + 8t (Fig. 1B).

FIG. 1.

Giemsa-stained karyotype of Astyanax taeniatus: (A) cytotype A (14m + 12sm + 16st + 8t), (B) cytotype B (10m + 14sm + 18st + 8t). Mean values of arm ratios are in parenthesis. DAPI-stained C-banding pattern of A. taeniatus: (C) cytotype A, (D) cytotype B. Argyrophilic nucleolar organizer region (Ag-NOR) banding patterns in A. taeniatus: (E) cytotype A, (F) cytotype B. Fluorescent in situ hybridization (FISH) pattern with 5S rDNA (green probe) and 18S rDNA (red probe) in A. taeniatus: (G) cytotype A, (H) cytotype B.

In both cytotypes, all chromosomes showed centromeric heterochromatin blocks with additional pericentromeric marks in most submetacentric and subtelocentric chromosomes (Fig. 1C, D). The Ag-NOR banding showed multiple marks in both cytotypes varying within and among individuals from four to eight subtelocentric chromosomes located on their short arms; ranging from chromosome pairs 14 to 17 in cytotype A (Fig. 1E) and chromosome pairs 13 to 16 in cytotype B (Fig. 1F).

The 5S rDNA probe marked four chromosome pairs 19, 20, 21, and 25 in both cytotypes (Fig. 1G, H green probe), while the 18S rDNA probe marked five chromosome pairs 9, 14, 15, 16, and 17 in cytotype A, chromosome pair number nine lacked Ag-NOR markings, and only four chromosome pairs 13, 14, 15, and 16 in cytotype B (Fig. 1G, H red probe). Repetitive FISH patterns obtained with CA₍₁₅₎ varied between both cytotypes: cytotype A had three pairs of chromosomes with strong centromeric marks (two metacentric pairs and one submetacentric pair), while cytotype B had only two pairs with strong centromeric marks (one metacentric pair and one submetacentric pair) (Fig. 2A, B). Repetitive FISH patterns obtained with GA₍₁₅₎ probe marked the subtelomeric regions of all chromosomes in both cytotypes (Fig. 2C, D).

FIG. 2.

FISH patterns obtained with repetitive DNA probes in A. taeniatus: (A) CA₍₁₅₎ patterns in cytotype A, (B) CA₍₁₅₎ patterns in cytotype B, (C) GA₍₁₅₎ patterns in cytotype A, (D) GA₍₁₅₎ patterns in cytotype B.

Discussion

A. taeniatus has 50 chromosomes as well as most species of the genus Astyanax; a ploidy considered a primitive character state within the family Characidae.³² This is the first report of two distinct syntopic karyotypes in this species. These cytotypes differed from the Jucu and Benevente rivers population, with its karyotypic formula = 12m + 2sm + 24st + 12t.²² The Piranga River cytotypes also differed from each other by non-Robertsonian chromosome rearrangements, suggesting that chromosomal divergence involved pericentromeric inversions that changed the number of chromosomes assigned to each morphological class.^8,33

The C- and Ag-NOR bandings showed similar results in both cytotypes suggesting a recent karyotypic divergence between these lineages. Furthermore, all chromosomes showed conspicuous heterochromatic blocks that were apparently more intense than the ones observed in other Astyanax species^9,34–36 and provide further evidence of close phylogenetic relationships between cytotypes A and B. High level of intraindividual and intrapopulational variation of multiple Ag-NORs are frequently observed in the genus Astyanax.³⁷ Up to 10 Ag-NOR-bearing chromosomes have been reported in A. altiparanae and A. fasciatus species complex, with a maximum number of 15 in the A. scabripinnis complex (reviewed in Abelini et al.³⁸). In other Astyanax species, Ag-NORs cistrons occur in all chromosome morphological classes except telocentrics and are located on either the short or long chromosome's arms.^38,39

The 5S rDNA probe showed a conserved pattern in both cytotypes. Within the genus Astyanax, this feature can be considered a derived condition since all species already studied with this probe showed fewer markers than the ones observed in this study (revised in Kavalco et al.⁹), further suggesting a common origin of both cytotypes. The 18S rDNA probe also showed a large number of sites. When compared with the Ag-NOR banding, it suggests that most ribosomal sites are active in these cytotypes. Moreover, other species of the genus Astyanax also show small discrepancies between the number of Ag-NOR and 18S rDNA sites.^40–43

Our study is the first that used CA₍₁₅₎ and GA₍₁₅₎ repetitive DNA probes to characterize an Astyanax species. In other species, such as Hoplias malabaricus⁴⁴ and Mystus bocourti,⁴⁵ these probes hybridized on subtelomeric chromosome regions. In Eremias velox,⁴⁶ Leporinus species,⁴⁷ and Triportheus trifurcatus,⁴⁸ these probes showed additional interstitial marks in their sex chromosomes. In this study, the CA₍₁₅₎ and GA₍₁₅₎ probes were mainly subtelomeric, but the CA₍₁₅₎ pattern included additional centromeric markings that were specific for each cytotype.

Our results indicate that the species A. taeniatus collected in the Upper Doce River exhibits two syntopic cytotypes. If ongoing molecular data confirm the specific status of specimens bearing different karyotypes, it would be interesting to understand the reproductive isolation mechanism between these cytotypes. In A. scabripinnis, large water bodies apparently act as physical barriers for the species. This species is restricted to headwaters or small springs, which hinders sympatry of different lineages, precluding hybridization between them.^12,13 However, A. taeniatus occurs in the river channel and is a very active and opportunistic species with regard to habitat and feeding aspects,⁴⁹ suggesting reproductive barriers that may be supported by some behavioral/genetic mechanisms.

In the A. fasciatus species complex, karyotypic variations have been interpreted as evidence for the existence of viable hybrids that withstand a set of chromosomal combinations,^10,11 while this condition may not apply to other sympatric populations of this species complex.^35,36,50 Another widely studied species complex is A. scabripinnis that shows high levels of karyotypic diversity among allopatric⁵¹ and sympatric⁵² populations; in this case, however, no intermediate hybrid forms have ever been found.^12,13

Although sympatric speciation cannot be ruled out, its occurrence is difficult to test.^53,54 Within the Doce River Basin, vicariance between the river bed and a quaternary lacustrine system may have caused speciation between the small predator species Oligosarcus argenteus and Oligosarcus solitarius (Dergam, unpublished) and has also caused deep genetic differentiation within Geophagus brasiliensis populations.⁵⁵ Even if the two cytotypes of A. taeniatus are proven sister species, they may have evolved separately by allopatric speciation and became sympatric as a secondary contact.

Footnotes

Acknowledgments

The authors thank “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),” “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)” and “Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)” for their financial support.

Disclosure Statement

No competing financial interests exist.

References

Albert

, Reis

: Historical Biogeography of Neotropical Freshwater Fishes. University of California Press, Berkeley, CA, 2011.

Lima

FCT

, Malabarba

, Buckup

, Pezzi da Silva

, Vari

, Harold

, et al. Genera incertae sedis in Characidae. In: Check List of the Freshwater Fishes of South and Central America. Reis

, Kullander

, Ferraris

(eds), pp. 106–169, Porto Alegre, Edipucrs, Brasil, 2003.

Mirande

. Weighted parsimony phylogeny of the family Characidae (Teleostei: Characiformes). Cladistics, 2009; 25:574–613.

Mirande

. Phylogeny of the family Characidae (Teleostei: Characiformes): from characters to taxonomy. Neotrop Ichthyol, 2010; 8:385–568.

Javonillo

, Malabarba

, Weitzman

, Burns

. Relationships among major lineages of characid fishes (Teleostei: Ostariophysi: Characiformes), based on molecular sequence data. Mol Phylogenet Evol, 2010; 54:498–511.

Oliveira

, Avelino

, Abe

, Mariguela

, Benine

, Orti

, et al. Phylogenetic relationships within the speciose family Characidae (Teleostei: Ostariophysi: Characiformes) based on multilocus analysis and extensive ingroup sampling. Evol Biol, 2011; 11:275–300.

Eschmeyer

. Catalog of Fishes. California Academy of Sciences. http://research.calacademy.org/research/ichthyology/catalog/fishcatmain.asp (Accessed date August 22, 2015).

Fernandes

, Martins-Santos

. Cytogenetic studies in two populations of Astyanax altiparanae (Pisces, Characiformes). Hereditas, 2004; 141:328–332.

Kavalco

, Pazza

, Brandão

KDO

, Garcia

, Almeida-Toledo

. Comparative cytogenetics and molecular phylogeography in the group Astyanax altiparanae–Astyanax aff. bimaculatus (Teleostei, Characidae). Cytogenet Genome Res, 2011; 134:108–119.

10.

Artoni

, Shibatta

, Gross

, Schneider

, Almeida

, Vicari

, et al. Astyanax aff. fasciatus Cuvier, 1819 (Teleostei; Characidae): evidences of a species complex in the upper rio Tibagi basin (Paraná, Brazil). Neotrop Ichthyol, 2006; 4:197–202.

11.

Pazza

, Kavalco

, Bertollo

LAC

. Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae). 1. Karyotypic analysis, Ag-NORs and mapping of the 18S and 5S ribosomal genes in sympatric karyotypes and their possible hybrid forms. Cytogenet Genome Res, 2006; 112:313–319.

12.

Moreira-Filho

, Bertollo

LAC

. Astyanax scabripinnis (Pisces, Characidae): a species complex. Braz J Genet, 1991; 14:331–357.

13.

Castro

, Moura

, Moreira-Filho

, Shibatta

, Santos

, Nogaroto

, et al. Diversity of the Astyanax scabripinnis species complex (Teleostei: Characidae) in the Atlantic Forest, Brazil: species limits and evolutionary inferences. Rev Fish Biol Fisher, 2015; 25:231–244.

14.

Kavalco

, Brandão

, Pazza

, Almeida-Toledo

. Astyanax hastatus Myers, 1928 (Teleostei, Characidae): a new species complex within the genus Astyanax?. Genet Mol Biol, 2009; 32:477–483.

15.

Eigenmann

. The American Characidae. Mem Mus Comp Zoo, 1917; 43:1–102.

16.

Eigenmann

. The American Characidae. Mem Mus Comp Zoo, 1918; 43:103–208.

17.

Eigenmann

. The American Characidae. Mem Mus Comp Zoo, 1921; 43:209–310.

18.

Eigenmann

. The American Characidae. Mem Mus Comp Zoo, 1927; 43:311–428.

19.

Peres

WAM

, Bertollo

LAC

, Buckup

, Blanco

, Kantek

DLZ

, Moreira-Filho

. Invasion, dispersion and hybridization of fish associated to river transposition: karyotypic evidence in Astyanax “bimaculatus group” (Characiformes: Characidae). Rev Fish Biol Fisher, 2012; 22:519–526.

20.

Vieira

. A ictiofauna do rio Santo Antônio, bacia do Rio Doce, MG: proposta de conservação. Universidade Federal de Minas Gerais, Minas Gerais, Brazil, 2006.

21.

Pompeu

. Os peixes do rio Mucuri. In: Relatório do Instituto Estadual de Florestas–MG. pp. 36–43, MG.BIOTA, Belo Horizonte, 2010. Instituto Estadual De Floresta (ed) Online Access: http://www.ief.mg.gov.br/images/stories/MGBIOTA/mgbiota11/mgbiot_%20v.2n.5.pdf (Accessed August 22, 2015).

22.

Stange

EAR

, Silva

, Dutra

. Citogenética no gênero Astyanax (Pisces–Characidae) das bacias dos rios Benevente e Jucu. Proc Simp Citog Evol e Aplic de Peixes Neotropicais, 1986; 1:58.

23.

Artoni

, Vicari

, Bertollo

LAC

. Citogenética de peixes neotropicais: métodos, resultados e perspectivas. Biol Health Sci, 2000; 6:43–60.

24.

Godinho

, Vieira

. Ictiofauna. In: Biodiversidade em Minas Gerais: um atlas para sua conservação. Costa

CMR

, Hermann

, Martins

(eds), pp. 44–46, Fundação Biodiversitas, Belo Horizonte, Minas Gerais, Brazil, 1998. http://www.biodiversitas.org.br/atlas/ (Accessed date October 25, 2011).

25.

Lucena

CAS

, Calegari

, Pereira

EHL

, Dallegrave

. O uso de óleo de cravo na eutanásia de peixes. Bol Soc Bras Ictiol, 2013; 105:20–24.

26.

Bertollo

LAC

, Takahashi

, Moreira-Filho

. Cytotaxonomic considerations on Hoplias lacerdae (Pisces, Erythrinidae). Rev Bras Genet, 1978; 1:103–120.

27.

Sumner

. A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res, 1972; 75:304–306.

28.

Schweizer

. Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma, 1976; 58:307–324.

29.

Howell

, Black

. Controlled silver-staining o nucleolus organizer regions with a protective colloidal developer: as 1-step method. Experientia, 1980; 36:1014–1015.

30.

Pinkel

, Straume

, Gray

. Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc Natl Acad Sci U S A, 1986; 83:2934–2938.

31.

Levan

, Fredga

, Sandberg

. Nomenclature for centromeric position on chromosomes. Hereditas, 1964; 52:201–220.

32.

Oliveira

, Toledo

LFA

, Foresti

, Britski

, Toledo-Filho

. Chromosome formulae of Neotropical freshwater fishes. Rev Bras Gen, 1988; 11:577–624.

33.

Pazza

, Kavalco

. Chromosomal evolution in the Neotropical characin Astyanax (Teleostei, Characidae). Nucleus, 2007; 50:519–543.

34.

Santos

, Ferreira-Neto

, Artoni

, Vicari

, Bakkali

, Oliveira

, et al. A comparative structural cytogenetic study in three allopatric populations of Astyanax scabripinnis (Teleostei: Characidae). Zoologia, 2012; 29:159–166.

35.

Peres

WAM

, Buckup

, Kantek

DLZ

, Bertollo

LAC

, Moreira-Filho

. Chromosomal evidence of downstream dispersal of Astyanax fasciatus (Characiformes, Characidae) associated with river shed interconnection. Genetica, 2009; 137:305–311.

36.

Pansonato-Alves

, Hisdorf

AWS

, Utsunomia

, Silva

DMZA

, Oliveira

, Foresti

. Chromosomal mapping of repetitive DNA and cytochrome c oxidase I sequence analysis reveal differentiation among sympatric samples of Astyanax fasciatus (Characiformes, Characidae). Cytogenet Genome Res, 2013; 141:133–142.

37.

Kavalco

, Moreira-Filho

. Cytogenetical analyses in four species of the genus Astyanax (Pisces, Characidae) from Paraíba do Sul River Basin. Caryologia, 2003; 56:453–461.

38.

Abelini

, Martins-Santos

, Fernandes

. Cytogenetic analysis in three species from genus Astyanax (Pisces; Characiformes) with a new occurrence of B chromosome in Astyanax paranae. Caryologia, 2014; 67:160–171.

39.

Medrado

, Figueiredo

AVA

, Waldschmidt

, Affonso

PRAM

, Carneiro

PLS

. Cytogenetic and morphological diversity in populations of Astyanax fasciatus (Teleostei, Characidae) from Brazilian northeastern river basins. Genet Mol Biol, 2008; 31:208–214.

40.

Peres

WAM

, Bertollo

LAC

, Moreira-Filho

. Physical mapping of the 18S and 5S ribosomal genes in nine Characidae species (Teleostei, Characiformes). Genet Mol Biol, 2008; 31:222–226.

41.

Pacheco

, Rosa

, Giuliano-Caetano

, Júlio

Jr. , Dias

. Cytogenetic comparison between two allopatric populations of Astyanax altiparanae Garutti & Britski, 2000 (Teleostei, Characidae), with emphasis on the localization of 18S and 5S rDNA. Comp Cytogenet, 2011; 5:237–246.

42.

Silva

LLL

, Giuliano-Caetano

, Dias

. Chromosome studies of Astyanax jacuhiensis Cope, 1894 (Characidae) from the Tramandai River Basin, Brazil, using in situ hybridization with the 18S rDNA probe, DAPI and CMA3 staining. Folia Biol-Krakow, 2012; 60:135–140.

43.

Tenório

RCCO

, Vitorino

, Souza

, Oliveira

, Venere

. Comparative cytogenetics in Astyanax (Characiformes: Characidae) with focus on the cytotaxonomy of the group. Neotrop Ichthyol, 2013; 11:553–564.

44.

Cioffi

, Kejnovsky

, Bertollo

LAC

. The chromosomal distribution of microsatellite repeats in the genome of the wolf fish Hoplias malabaricus, focusing on the sex chromosomes. Cytogenet Genome Res, 2011; 132:289–296.

45.

Supiwong

, Liehr

, Cioffi

, Chaveerach

, Kosyakova

, Pinthong

, et al. Karyotype and cytogenetic mapping of 9 classes of repetitive DNAs in the genome of the naked catfish Mystus bocourti (Siluriformes, Bagridae). Mol Cytogenet, 2013; 6:51.

46.

Pokorna

, Kratochvíl

, Kejnovský

. Microsatellite distribution on sex chromosomes at different stages of heteromorphism and heterochromatinization in two lizard species (Squamata: Eublepharidae: Coleonyx elegans and Lacertidae: Eremias velox). Genetics, 2011; 12:90.

47.

Poltronieri

, Marquioni

, Bertollo

LAC

, Kejnovsky

, Molina

, Liehr

, et al. Comparative chromosomal mapping of microsatellites in Leporinus species (Characiformes, Anostomidae): unequal accumulation on the W chromosomes. Cytogenet Genome Res, 2014; 142:40–45.

48.

Yano

, Poltronieri

, Bertollo

LAC

, Artoni

, Liehr

, Cioffi

. Chromosomal mapping of repetitive DNAs in Triportheus trifurcatus (Characidae, Characiformes): insights into the differentiation of the Z and W chromosomes. PLoS One, 2014; 9:90946.

49.

Manna

, Rezende

, Mazzoni

. Ecologia trófica de Astyanax taeniatus (Characidae) de um riacho costeiro da Mata Atlântica–Saquarema–RJ. Anais do IX Congresso de Ecologia do Brasil, 13 a 17 de Setembro de 2009, São Lourenço, Minas Gerais, Brazil, 2009.

50.

Ferreira-Neto

, Artoni

, Vicari

, Moreira-Filho

, Camanho

JPM

, Bakkali

, et al. Three sympatric karyomorphs in the fish Astyanax fasciatus (Teleostei, Characidae) do not seem to hybridize in natural populations. Comp Cytogenet, 2012; 6:29–40.

51.

Souza

, Moreira-Filho

. Cytogenetic diversity in the Astyanax scabripinnis species complex (Pisces, Characidae). I. Allopatric distribution in a small stream. Cytologia, 1995; 60:1–11.

52.

Souza

, Moreira-Filho

, Bertollo

LAC

. Cytogenetic diversity in the Astyanax scabripinnis (Pisces, Characidae) complex. II. Different cytotypes living in sympatry. Cytologia, 1995; 60:273–281.

53.

Bush

. Sympatric speciation in animals: new wine in old bottles. Trends Ecol Evol, 1994; 9:285–288.

54.

Futuyma

, Mayer

. Non-allopatric speciation in animals. Syst Zool, 1980; 29:254–271.

55.

Alves-Silva

, Dergam

. Cryptic speciation within the Neotropical cichlid Geophagus brasiliensis (Quoy & Gaimard, 1824) (Teleostei Cichlidae): a new paradigm in karyotypical and molecular evolution. Zebrafish, 2015; 12:91–101.