cytogenetic analysis in three species from genus astyanax (pisces; characiformes) with a new...

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Cytogenetic analysis in three species from genusAstyanax (Pisces; Characiformes) with a newoccurrence of B chromosome in Astyanax paranaeEdner Abelinia, Isabel Cristina Martins-Santosb & Carlos Alexandre Fernandesc

a Paraná State Secretariat of Education and Socioeducation Units Education Program(PROEDUSE), Maringá, Brazilb Department of Cell Biology and Genetics, State University of Maringá, Maringá, PR,Brazilc State University of Mato Grosso do Sul, Mundo Novo, MS, BrazilPublished online: 20 Aug 2014.

To cite this article: Edner Abelini, Isabel Cristina Martins-Santos & Carlos Alexandre Fernandes (2014) Cytogeneticanalysis in three species from genus Astyanax (Pisces; Characiformes) with a new occurrence of B chromosome inAstyanax paranae, Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics, 67:2, 160-171, DOI:10.1080/00087114.2014.931638

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Cytogenetic analysis in three species from genus Astyanax (Pisces; Characiformes) with a newoccurrence of B chromosome in Astyanax paranae

Edner Abelinia, Isabel Cristina Martins-Santosb and Carlos Alexandre Fernandesc*aParaná State Secretariat of Education and Socioeducation Units Education Program (PROEDUSE), Maringá, Brazil; bDepartmentof Cell Biology and Genetics, State University of Maringá, Maringá, PR, Brazil; cState University of Mato Grosso do Sul, MundoNovo, MS, Brazil

Cytogenetic studies were carried out in three species from genus Astyanax: Astyanax paranae (Tagaçaba Stream andTauá Stream), Astyanax altiparanae (Iguaçu River and Maringá Stream) and Astyanax fasciatus (Maringá Stream). Bothpopulations of A. paranae featured 2n = 50 chromosomes, although with different karyotype formulae and Ag-nucleolarorganizer regions systems. Using the FISH technique in the Tagaçaba population, it was possible to detect rDNA genesin 14 chromosomes and bitelomeric marking in one chromosome. The constitutive heterochromatin pattern also showeddifferences between the two populations. Moreover, the Tagaçaba population showed macrochromosome B in 50% ofanalyzed females. Both A. altiparanae populations featured 2n = 50 chromosomes, but also differed in karyotypeformula. Both populations featured multiple-NOR systems and quite similar constitutive heterochromatin patterns. Forthe A. fasciatus population from Maringá Stream, the diploid number was 2n = 46 chromosomes with karyotypeformulae 14m, 10sm, 12st and 10a (fundamental number = 82), with single-NOR system and heterochromatin patternshowing evident markers in the telomeric regions of several subtelocentric and acrocentric chromosomes. The datapresented for this species differ from some previously analyzed populations with regard to diploid number (2n = 48chromosomes) and karyotype formula for those with the same diploid number. The present data strengthen theaccentuated cytogenetical variability of the genus Astyanax.

Keywords: chromosomes; C-band; Ag-NOR; 18S rDNA; fishes

Introduction

The genus Astyanax has wide geographic distribution,and its representatives are easily found in Brazil’s waterbasins (Britski 1972); it consists of small fishes, compris-ing over 100 species and subspecies found in Neotropi-cal rivers (Froese and Pauly 2013). This genus revealsseveral almost-similar forms, forming a highly complexgroup, whose precise identification has been difficult(Melo 2001), representing the greater unity of the generaincertae sedis in Characidae (Lima et al. 2003).

Astyanax scabripinnis was proposed as a complex ofspecies based on morphological and karyotypic analyses(Moreira-Filho and Bertollo 1991). The A. scabripinnisspecies complex is composed of 15 species (Bertaco andLucena 2006). In the upper Paraná river basin, the onlyrecognized species of the A. scabripinnis complex is A.paranae, a very common species represented by severalisolated populations in headwater streams (Garutti andBritski 2000).

Astyanax feature diploid numbers ranging between2n = 36 chromosomes in A. schubarti (Daniel-Silva andAlmeida-Toledo 2001) and 2n = 50 chromosomes inA. altiparanae (Fernandes and Martins-Santos 2004),A. scabripinnis complex (Moreira-Filho and Bertollo1991), A. bockmanni (Fernandes et al. 2010; Hashimotoet al. 2011), and A. jacuhiensis (Pacheco et al. 2010),among others. Despite this wide chromosomal variability,

one trait has remained constant in the genus: thepresence of a metacentric-type pair of chromosomes,larger than the other chromosomes in the complement,except for A. fasciatus with 2n = 46 chromosomes(Fernandes et al. 2009).

Another form of variation in the diploid/haploidnumber in the genus is the occurrence of supernumerarychromosomes or B chromosomes, which have beendetected rather frequently in the A. scabripinnis complex,and may appear as microchromosomes (Mizoguchi andMartins-Santos 1997; Alves and Martins-Santos 2002),mid-sized chromosomes (Fernandes and Martins-Santos2005) and macrochromosomes (Salvador and Moreira-Filho 1992; Mizoguchi and Martins-Santos 1997; Néo,Bertollo, et al. 2000; Fernandes and Martins-Santos2005; Machado et al. 2012).

In the present study, three species of the genusAstyanax were karyotyped. The results obtained wereemployed to discuss some aspects of the chromosomeevolution in the genera incertae sedis in Characidae. Inaddition, a new occurrence of a B chromosome inAstyanax paranae is recorded.

Material and methods

Two populations of A. paranae were analyzed: one fromTagaçaba Stream (23°30′44.29″ S, 52°01′42.69″ W), a

*Corresponding author. Email: [email protected]

© 2014 Dipartimento di Biologia Evoluzionistica, Università di Firenze

Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics, 2014Vol. 67, No. 2, 160–171, http://dx.doi.org/10.1080/00087114.2014.931638

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tributary of the Ivaí River (12 females and four males);and another from Tauá Stream (23°22′23.41″ S, 51º50′47.70″ W), a tributary of the Pirapó River (eight malesand two females), both in the city of Maringá, ParanáState, Brazil. One of the A. altiparanae populations, with13 individuals (five males and eight females) was col-lected from Maringá Stream (23°19′16.52″ S, 51°54′48.65″ W), a tributary of the Pirapó River; the other,with 10 individuals (four males and six females) wascollected at the Governador José Richa Reservoir (SaltoCaxias), in the Iguaçu River (25°31′41.85″ S, 53°29′28.85″W), Paraná State. Due to the location of the col-lection site, these A. altiparanae populations areregarded as geographically isolated. The A. fasciatuspopulation, with nine individuals (three males and sixfemales), was collected at the same site as the populationof A. altiparanae from Maringá Stream.

Metaphasic chromosomes were obtained from ante-rior kidney cells using the air-drying techniquedescribed by Bertollo et al. (1978). The technique usedto characterize the nucleolar organizer regions (NORs)was silver nitrate impregnation as described by Howelland Black (1980), while the fluorescence in situ hybrid-ization (FISH) technique was applied as per Pinkelet al. (1986), with slight modifications. The 18S and 5Sprobes were obtained from A. scabripinnis genomic

DNA and PCR amplified using the NS1 (5′-GTAGTCA-TATGCTTGTCTC-3′) and NS8 (5′-TCCGCAGGTTC-ACCTACGGA-3′) primers (White 1990). The techniqueused to observe the constitutive heterochromatin patternwas that described by Sumner (1972). The chromo-somes were classified as metacentric (m), submetacentric(sm), subtelocentric (st) and acrocentric (a) according totheir arm ratio (Levan et al. 1964). For the determina-tion of the fundamental number (FN), or number ofchromosome arms, the (m), (sm) and (st) chromosomeswere considered as bearing two arms and the acrocen-tric chromosomes only one arm.

At least 20 metaphases per specimen were analyzedand photographed under a Zeiss Axioskop 2 Plus fluo-rescence microscope. The images were captured using adigital camera (AxioCam HR Zeiss, Germany) attachedto the microscope, with the aid of Axiovision software.

Results and discussion

Astyanax paranae

Both populations showed the same diploid number,2n = 50 chromosomes, but differed with regard tokaryotype formula, FN, presence/absence of B chromo-some, NORs and C-banding pattern.

Figure 1. Conventional Giemsa karyotypes of Astyanax paranae (a) Tagaçaba Stream population, showing B chromosome;(b) Tauá Stream population.

Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics 161

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Table 1. Cytogenetic data of the Astyanax scabripinnis complex.

Location Basin 2n FN m sm st a Bs NORs Ref.a

Tagaçaba Stream A 50 88 10 22 6 12 0–1 2 1Tauá Stream A 50 86 10 18 8 14 0 4 1Macacos River D 50 86 8 20 8 14 0 7 16Tatupeba Stream A 50 84 6 22 6 16 1 7 26Sarandi Stream A 50 86 6 26 4 14 0 1–5 4, 5Yucatan River A 50 90 6 30 4 10 0–1 1–3 3, 4, 5Centenário Stream A 50 84 6 20 8 16 0 1–4 6, 7São Pedro Stream A 50 88 6 24 8 12 0 1–6 8Verde River A 50 86 8 18 10 14 0 2–7 2Cascatinha Stream A 50 90 8 22 10 10 0–1 1–4 8, 9, 10Viveiro das Mudas Stream B 50 94 6 30 8 6 0 1–7 6Jucu River C 50 64 6 8 0 36 0–4 1–15 14Grande Stream D 50 88 6 22 10 12 0 2–9 11, 12, 13Pequeno River D 50 86 6 20 10 14 0 2 6Tamanduá Stream A 50 86 6 26 4 14 0 1–4 15Grande Stream A 50 90 6 26 8 10 0 4–8 8Pardo River A 50 88 6 26 6 12 0 1–3 8Claro River A 50 90 6 28 6 10 0 1–4 8Claro River A 50 88 6 24 8 12 0 1–3 6,Capivara River A 50 90 8 22 10 10 0 1–5 8, 17Lavapés River A 50 90 6 26 8 10 0 1–4 8Araquá River A 50 88 4 30 4 12 0–1 1–3 8, 17, 18Bicudos Stream A 50 92 8 20 14 8 0 – 19Canta Galo Stream A 50 84 4 26 4 16 0 1–6 20Monjolinho Stream A 50 88 6 24 8 12 0 2–8 6,Perdizes Stream A 50 88 6 22 10 12 0–2 2 21Pedras Stream A 50 88 6 22 10 12 0–2 2 21, 22Casquilho Stream A 50 88 6 22 10 12 0–1 2 21Capivari Stream A 50 88 6 22 10 12 0–1 1–4 11Fojo Stream A 50 88 6 22 10 12 0–2 1–6 11Carpas Lagoon A 50 88 6 22 10 12 0–1 2–7 11Piracuama River D 50 70 4 10 6 30 0 5–11 23Piracuama River D 50 86 6 24 6 14 0–1 1–3 23Barreiro Grande Stream B 50 94 6 30 8 6 0 1–4 8Cascatinha Stream A 50 86 6 16 14 14 0–2 4 26Santo Antônio River F 50 86 8 18 10 14 0 1–4 2Açungui River E 50 86 8 18 10 14 0 2–10 2Cascatinha Stream* A 75 129 9 24 21 21 2 4 26Pedras Stream* A 75 132 9 33 15 18 2 2–3 17Araquá River* A 75 132 6 45 6 18 1 1–3 17Tatupeba Stream A 48 88 8 26 6 8 3 4 26São Domingos Stream A 48 86 10 20 8 10 0 3 24Tamboara Stream A 48 84 10 24 6 8 0 7 24Canta Galo Stream A 48 82 6 22 6 14 0 – 20Canta Galo Stream A 48 84 6 22 8 12 0 1–3 20Tamanduá Stream A 48 86 6 28 4 10 0 1–2 15Marrecas Stream A 48 86 6 20 12 10 0 1–5 6, 7Ligeiro River A 48 82 10 22 2 14 0 1–4 4, 5Jaguariaíva River F 48 84 10 16 10 12 0 2–4 2Santo Antônio River F 48 84 10 16 10 12 0 2–5 2Tatupeba Stream A 46 82 8 22 6 10 2 2 25Curral das Éguas Stream B 46 6 22 8 14 0 1–5 6

Abbreviations. A: Paraná Basin; B: São Francisco Basin; C: Jucu Basin; D: Paraíba do Sul Basin; E: Ribeira de Iguape Basin; F: Jaguariaíva Basin;FN: fundamental number; m: metacentric; sm: submetacentric; st: subtelocentric; a: acrocentric; Bs: presence of extra chromosomes; NORs: nucleolarorganizer regions.*Triploid individuals.–Articles without description of NOR.a1: Present study; 2: Vicari et al. (2008); 3: Mizoguchi and Martins-Santos (1997); 4: Mizoguchi and Martins-Santos (1998a); 5: Mizoguchi andMartins-Santos (1998b); 6: Moreira-Filho and Bertollo (1991); 7: Mantovani et al. (2000); 8: Maistro et al. (1998); 9: Maistro, Foresti, et al. (1994);10: Porto-Foresti et al. (1997); 11: Ferro et al. (2001); 12: Néo, Moreira-Filho, et al. (2000); 13: Néo, Bertollo, et al. (2000); 14: Rocon-Stange andAlmeida-Toledo (1993); 15: Maistro et al. (2000); 16: Kavalco and Moreira-Filho (2003); 17: Maistro, Dias, et al. (1994); 18: Maistro et al. (1992);19: Morelli et al. (1983); 20: Souza et al. (1996); 21: Vicente et al. (1996); 22: Salvador and Moreira-Filho (1992); 23: Souza et al. (1995); 24: Alvesand Martins-Santos (2002); 25: Fernandes and Martins-Santos (2005); 26: Machado et al. (2012).

162 E. Abelini et al.

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In the population from Tagaçaba Stream, the 50 chro-mosomes were arranged into 10 metacentric (m), 22 sub-metacentric (sm), six subtelocentric (st) and 12acrocentric (a) chromosomes, with FN equal to 88, withno morphological chromosome difference between thesexes (Figure 1a). In the population from Tauá Stream,the diploid number of 2n = 50 chromosomes wasarranged into 10m + 18sm + 8st +14a, with FN equal to86, with no morphological chromosome differencebetween the sexes (Figure 1b).

Table 1 shows the results of 52 karyotype analysesobtained for the A. scabripinnis complex by differentauthors for different locations. Of these, 76.92% showeda diploid number of 2n = 50 chromosomes with differentkaryotype formulae, whereas in 19.23% the diploid num-ber was 2n = 48 chromosomes and only 3.84% had 2n= 46 chromosomes. So, at least the populations with dif-ferent diploid number must consist of taxonomic unitsdistinct from A. scabripinnis. The existence of threesympatric and syntopic cytotypes with different diploidnumbers (2n = 46, 2n = 48 and 2n = 50 chromosomes)observed in the same population from Tatupeba Stream,with no intermediate hybrids, corroborate the existenceof at least three different taxonomic units, identified as

belonging to the A. scabripinnis complex (Fernandes andMartins-Santos 2005). Triploid individuals were alsoobserved, with 3n = 75 chromosomes in the populationfrom the Araquá River and Pedras Stream (Maistro,Dias, et al. 1994) and Cascatinha Stream (Machado et al.2012).

Within these units – with different diploid numbers –differences in FN and number of chromosomes bearingthe NOR can be observed as well. Although many differ-ences may be due to the measurement criteria adopter byeach researcher, it is possible to establish effective differ-ences from the analysis of acrocentric chromosomes orfrom the FN.

In the present study, both analyzed populationsshowed diploid number corresponding to the majority ofindividuals analyzed for the A. scabripinnis complex andshowed different karyotype formulae among them andwhen compared to the other analyzed populations(Table 1). However, for the population from Tagaçaba,50% of females (six females) featured a metacentric Bchromosome in 100% of analyzed metaphases, similar tothe largest chromosome pair in complement A. That chro-mosome was fully heterochromatic and restricted tofemales (Figure 2d), as detected for other A. scabripinnis

Figure 2. (a) Location of NORs using Ag-NOR; (b) Giemsa-stained sequential somatic metaphase; (c) FISH with 18S probe;(d) constitutive heterochromatin pattern of the population of Astyanax paranae from Tagaçaba Stream. Arrows indicate NORs. Whitearrowhead indicates bitelomeric marker. Black arrowhead indicates heterochromatic B chromosome.


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complex populations with 2n = 50 chromosomes (Maistroet al. 1992; Mizoguchi and Martins-Santos 1997;Fernandes and Martins-Santos 2005). On the other hand,the metacentric B chromosome is not restricted to femalesand is commonly found among populations with 50, 48and 46 chromosomes (Fernandes and Martins-Santos2005; Machado et al. 2012). One hypothesis on the likelyorigin of this chromosome in the A. scabripinnis complexsuggests the occurrence of isochromosome formation(Vicente et al. 1996; Mestriner et al. 2000).

According to Miller et al. (1976), the analysis ofthe NOR using the silver nitrate technique (Ag-NOR)makes it possible to detect only those regions ofrDNA that were active in the previous interphase.Through that technique, the occurrence of NORs isdetected both in a single chromosome pair (singleNOR) and in those distributed over several pairs (mul-tiple NORs).

In this work, the study of that region using the Ag-NOR technique showed that, while the population fromTagaçaba Stream featured markings on the short arm ofsubmetacentric chromosome pair 11, coincident with thesecondary constriction visualized in one of the homo-

logues (Figure 2a, b), the population from Tauá Streamshowed the multiple-NOR system, with markings in twopairs of chromosomes: one submetacentric pair on theshort arm and another acrocentric pair on the terminalsection, in most analyzed metaphases (Figure 3a, b).Although this method is often used, it reveals only activegenes; on the other hand, the fluorescence in situ hybrid-ization (FISH) method detects rDNA genes regardless ofactivity. Therefore, the use of the FISH technique with18S rDNA probe, for the Tagaçaba population, revealedbright spots throughout the telomeric regions in 14 chro-mosomes, including pair 11. Moreover, it featured onechromosome with bitelomeric-type marking (Figure 2c),similar to that of other A. scabripinnis complex popula-tions with 2n = 50 chromosomes (Malacrida andGiuliano-Caetano 2003; Mantovani et al. 2005;Fernandes and Martins-Santos 2006a). AgNOR analysisshowed a single-NOR system for the Tagaçaba popula-tion, indicating that only one NOR-bearing chromosomepair is expressed in that population, whereas the FISHmethod more effectively determined the number andlocation of rDNA genes, evidencing the presence of 15chromosomes bearing that region. In addition, for the

Figure 3. (a) Location of NORs using Ag-NOR; (b) Giemsa-stained sequential somatic metaphase; (c) constitutive heterochromatinpattern for the population of Astyanax paranae from Tauá Stream.


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majority of metaphases analyzed in that population, het-eromorphy of the NOR was observed after Giemsa andAgNOR (Figure 2a, b).

The constitutive heterochromatin pattern, evidencedthorough the C-banding technique, showed differencesbetween the two populations, with pericentromeric mark-ings in almost all chromosomes in the population fromTagaçaba Stream (Figure 2d). For the Tauá Stream popu-lation, large telomeric blocks were found in at least sixpairs of acrocentric chromosomes and smaller centro-meric blocks in most chromosomes (Figure 3c). The pat-tern of this latter population has been the mostfrequently observed in the A. scabripinnis complex.However, C-banding distribution has varied from popula-tion to population. As a result, Souza et al. (1996),Mantovani et al. (2000) and Fernandes and Martins-San-tos (2003) propose that the model by Schweizer andLoidl (1987) is used to explain the dispersion of consti-tutive heterochromatin in telomeric regions. According tothose authors, chromosomes with similar arms tend toremain together during interphase, facilitating the transferof these regions to non-homologous chromosomes.

Astyanax altiparanae

Both populations featured the same diploid number of50 chromosomes, but differed with regard to karyotypestructure, fundamental number and NORs.

In the population from the Iguaçu River, the karyo-type was arranged into 10m + 26sm + 6st + 8a and theFN was equal to 92, with no morphological chromosomedifference between the sexes (Figure 4a). In the popula-tion from Maringá Stream, the karyotype was 10m +22sm + 6st + 12a and to the FN was 88, with no mor-phological chromosome difference between the sexes(Figure 4b).

Table 2 shows the results of the 24 karyotype analy-ses of A. altiparanae by different authors for differentlocations. Of these, 100% showed a diploid number of2n = 50 chromosomes, but with different karyotype for-mulae, thus evidencing conservatism with regard to dip-loid number. The FN of the analyzed populations rangedbetween 86 for the Claro river and 100 for populationfrom Três Bocas Stream. For this latter population, thekaryotype formula differed from the others due to theabsence of acrocentric and submetacentric chromosomes

Figure 4. Conventional Giemsa karyotypes of Astyanax altiparanae: (a) population from the Iguaçu River; (b) population fromMaringá Stream.


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(Table 2). Although some differences may be related tothe different measurement methodologies adopted byeach researcher, the divergence observed, mainly in thenumber of acrocentric chromosomes, between the popu-lations of A. altiparanae described to date must representactual differences.

The Iguaçu River Basin features ichthyofauna with ahigh number of endemic species (Garavello et al. 1997).Prioli et al. (2002), using mitochondrial DNA and RAPDmarkers, analyzed populations of A. altiparanae from the

Upper Paraná River Basin and Iguaçu River Basin, geo-graphically separated from one another by Iguaçu Falls.These authors did not observe differences between thepopulations, indicating that A. altiparanae from theIguaçu River is not endemic and was likely introducedrecently into that basin. Likewise, the cytogenetic datapresented in this study are unable to characterize theIguaçu River population as a distinct species fromA. altiparanae; this also applies to populations ofA. altiparanae described for Maringá Stream in the

Table 2. Cytogenetic data on Astyanax altiparanae.


Mogi Guaçu A 50 88 10 24 4 12 0 — 3Tibagi River A 50 92 6 28 8 8 0 4 6Iguaçu River A 50 94 6 30 8 6 0 2 6Campo Novo River A 50 92 12 18 12 8 1 — 7Três Bocas Stream A 50 100 40 0 10 0 0 — 4Três Bocas Stream A 50 100 28 0 22 0 0 — 4Três Bocas Stream A 50 100 34 0 16 0 0 — 4Paranapanema River A 50 88 10 22 6 12 0 — 5Mogui-Guaçu River A 50 88 6 12 20 12 0 2 8Tietê River A 50 88 6 12 20 12 0 2 8Batalha River A 50 92 10 16 16 8 0 — 9Keçaba Stream A 50 88 6 26 6 12 0 7 10Paraná River A 50 88 6 26 6 12 0 4 10Índios River A 50 90 6 30 4 10 0 10 2Paraná River A 50 88 6 26 6 12 0 2 2Tatupeba Stream A 50 88 6 26 6 12 0 4 10Maringá Stream A 50 88 6 26 6 12 0 4 10Claro River A 50 90 10 26 4 10 0 4 11Claro River A 50 88 10 24 4 12 0 4 11Claro River A 50 86 10 22 4 14 0 4 11Paraná River A 50 88 10 22 6 12 0 — 12Monjolinho Stream A 50 94 12 18 20 6 0 4 13Maringá Stream A 50 88 10 22 6 12 0 2–3 1Iguaçu River A 50 92 10 26 6 8 0 2–5 1

Abbreviations. A: Paraná Basin; FN: fundamental number; m: metacentric; sm: submetacentric; st: subtelocentric; a: acrocentric; Bs: presence of extrachromosomes; NORs: nucleolar organizer regions.a1: Present study; 2: Fernandes and Martins-Santos (2004); 3: Morelli et al. (1983); 4: Takahashi et al. (1995); 5: Daniel-Silva and Almeida-Toledo(2001); 6: Domingues et al. (2007); 7: Hashimoto et al. (2008); 8: Martinez et al. (2012); 9: Hashimoto et al. (2011); 10: Fernandes and Martins-San-tos (2006b); 11: Pacheco et al. (2001); 12: Daniel-Silva and Almeida-Toledo (2005); Tenório et al. (2013).

Figure 5. Location of NORs using Ag-NOR. (a) Iguaçu River population; (b) Maringá Stream population. The arrows indicate thelocation of NORs.


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Figure 6. Constitutive heterochromatin pattern in Astyanax altiparanae: (a) Iguaçu River population; (b) Maringá Stream popula-tion.

Figure 7. Conventional Giemsa karyotype of Astyanax fasciatus from Maringá Stream.


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present study and by Fernandes and Martins-Santos(2006b). Even though variations in karyotype formulaare present, these differences must have occurred due tonon-Robertsonian translocations, particularly pericentro-meric inversions.

The analysis of the NOR showed a multiple-NORsystem for both populations. In the Iguaçu River popula-tion, five chromosomes showed markings distributed on

the short arms of submetacentric-type chromosomes(Figure 5a); in the population from Maringá Stream, themarkings were seen on the short arm of a submetacentricchromosome pair and on the long arm of a submeta-centric chromosome (Figure 5b). The multiple-NOR sys-tem is often seen within the genus Astyanax, includingA. altiparanae (Fernandes and Martins-Santos 2006b;Domingues et al. 2007), A. scabripinnis (Moreira-Filho

Table 3. Cytogenetic data for Astyanax fasciatus.


Araguari River A 46 86 14 16 10 6 0 4 3Campo Novo River A 46 90 10 14 20 2 1 — 7Meia Ponte River A 46 74 0 28 0 18 0 — 6Mogi-Guaçu River A 46 90 14 20 10 2 0 — 5Maringá Stream A 46 82 14 10 12 10 0 2 1Rosas Stream A 46 88 12 22 8 4 0 2 2Mogi-Guaçu River A 46 88 12 20 10 4 0 2 – 8 9Águas da Madalena Stream A 46 84 10 16 12 8 1 10 10Furnas 2 A 48 72 6 18 14 10 0 — 11Tibagi River A 48 72 6 18 14 10 0 — 11Lagoa Dourada A 48 72 6 18 14 10 0 — 11Cará-Cará River A 48 72 6 18 14 10 0 — 11Sapucaí River A 48 88 8 18 14 8 0 2 4Contas River C 48 92 8 24 12 4 0 5 8Preto do Costa River C 48 90 8 24 10 6 0 2 8Mineiro Stream C 48 90 8 18 16 6 0 2 8Juquiá River B 48 94 10 24 12 2 0 — 5Mogi-Guaçu River A 48 90 8 22 12 6 0 2 – 8 9Águas da Madalena Stream A 48 86 10 16 12 10 0 10 10Águas da Madalena Stream A 50 84 10 16 8 16 0 10 10Tibagi River A 50 76 8 18 14 10 0 — 11Lagoa Dourada A 50 76 8 18 14 10 0 — 11Cará-Cará River A 50 76 8 18 14 10 0 — 11Cará-Cará River A 49 74 7 18 14 10 0 — 11

Abbreviations. A: Paraná Basin; B: Ribeira do Iguape; C: Contas Basin; FN: fundamental number; m: metacentric; sm: submetacentric; st: subtelocen-tric; a: acrocentric; Bs: presence of extra chromosomes; NORs: nucleolar organizer regions.a1: Present study; 2: Fernandes et al. (2009); 3: Torres-Mariano and Morelli (2004); 4: Swerts et al. 1998); 5: Morelli et al. (1983); 6: Jim and Toledo(1975); 7: Hashimoto et al. (2011); 8: Medrado et al. (2008); 9: Pazza et al. (2006); 10: Ferreira-Neto et al. (2012); 11: Artoni et al. (2006).

Figure 8. Metaphases of Astyanax fasciatus from Maringá Stream. (a) Ag-NOR; (b) C-banded. The arrows indicate the sites ofAg-NOR staining.


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and Bertollo 1991; Rocon-Stange and Almeida-Toledo1993; Fernandes and Martins-Santos 2006a) andA. fasciatus (Fernandes et al. 2009), which indicates thatthis is a shared trait in the group.

The heterochromatin pattern of both populationswas quite similar, with markings in most chromosomes,distributed in pericentromeric and telomeric regions, aswell as interstitial blocks proximal to the centromere inpairs 8, 13, 14, 15 and 16 for the Iguaçu River popula-tion and pairs 8, 9, 12, 13, and 14 of submetacentricchromosomes for the population from Maringá Stream,corresponding to homologous chromosomes, especiallypairs 13 and 12, respectively, for both populations,which featured markings almost throughout the lengthof the short arm of these chromosomes (Figure 6a, b),in addition to the proximal heterochromatin block.Daniel-Silva and Almeida-Toledo (2001) and Fernandesand Martins-Santos (2004) analyzed Astyanaxaltiparanae populations in different rivers of the upperParaná river basin and also observed marked interstitialblocks of constitutive heterochromatin in most chromo-somes. Therefore, this marking pattern is a characteris-tic of A. altiparanae.

Astyanax fasciatus

All analyzed specimens showed the same diploid numberof 2n = 46 chromosomes, arranged into 14m + 10sm+12st + 10a and fundamental number equal to 82, with nomorphological chromosome difference between the sexes(Figure 7).

Cytogenetic studies of A. fasciatus have shown a var-iation in diploid number of 2n = 46 to 2n = 50 chromo-somes, with variations in karyotype formula with FNbetween 72 and 94 (Table 3).The differences in diploidnumber likely characterize different taxonomic units, andkaryotype formula differences within each unit occur dueto structural chromosomal rearrangements. Therefore, A.fasciatus as A. scabripinnis form a species complex.

The analysis of the NOR through silver nitrateimpregnation showed only one active pair, indicating asingle-NOR system, with the presence of a terminalmarking on the long arm of a pair of submetacentric-typechromosomes (Figure 8a). Among the populations withthe same diploid number, similar markings wereobserved in the population from Rosas Stream(Fernandes et al. 2009) and a multiple-NOR system withup to four chromosomes marked were observed in theAraguari River population (Torres-Mariano and Morelli2004). Although featuring only one silver-marked pair,the Rosas Stream population evidenced four pairs ofmarkings with 18S probe through FISH analysis, indicat-ing that only one submetacentric pair was expressed inthat population, similar to the same active pair in thepopulation of the present study. The C-banding patternshowed evident constitutive heterochromatin blocks,present in the telomeric regions in several chromosomescharacteristic of the specie (Figure 8b).

The present data strengthen the accentuated cytoge-netical variability of the genus Astyanax, based on thekaryotype formula, fundamental number, presence/absence of B chromosome, NORs and C-banding pat-tern. Such biodiversity might be maximized by the non-migratory behavior of some species, such as A. paranae,which are able to form isolated populations and sufferspecific selection pressures.

AcknowledgmentsThe authors thank the Brazilian agency Coordenação de Aper-feiçoamento de Pessoal de Nível Superior (CAPES) for finan-cial support.

ReferencesAlves AL, Martins-Santos IC. 2002. Cytogenetics studies in two

populations of Astyanax scabripinnis with 2n=48 chromo-somes (Teleostei, Characidae). Cytologia. 67(2):117–122.

Artoni RF, Shibata OA, Gross MC, Schneider CH, AlmeidaMC, Vicari MR, Bertollo LAC. 2006. Astyanax aff fascia-tus Cuvier, 1819 (Teleostei; Characidae): evidences of aspecies complex in the upper River Tibagi basin (Parana,Brazil). Neotrop Ichthyol. 4(2):197–202.

Bertaco VA, Lucena CAS. 2006. Two new species of Astyanax(Ostariophysi: Characiformes: Characidae) from easternBrazil with a synopsis of the Astyanax scabripinnis speciescomplex. Neotrop Ichthyol. 4(1):53–60.

Bertollo LAC, Takahashi CS, Moreira-Filho O. 1978.Cytotaxonomic considerations on Hoplias lacerdae (Pisces,Erythrinidade). Brazil J Genet. 1(2):103–120.

Britski HA. 1972. Peixes de água doce do Estado de SãoPaulo: Sistemática. In: Branco SM, editor. Poluição ePsicultura. v. 1, São Paulo: Faculdade de Saúde Pública daUSP e Instituto de Pesca - CPRN-SA. p. 79–108.

Daniel-Silva MFZ, Toledo LF. 2001. Chromosome R-bandingpattern and conservation of a marker chromosome in fourspecies, genus Astyanax (Characidae, Tetragonopterinae).Caryologia. 54(3):209–215.

Daniel-Silva MFZ, Almeida-Toledo LF. 2005. Chromosomeevolution in fish: BrdU replication patterns demonstratechromosome homeologies in two species of the genus Asty-anax. Cytogenet Genome Res. 109(4):497–501.

Domingues MDS, Vicari MR, Abilhoa V, Wamser JP, CestariMM, Bertollo L a. C, Almeida MC DeArtoni RF. 2007.Cytogenetic and comparative morphology of two allopatricpopulations of Astyanax altiparanae Garutti & Britski,2000 (Teleostei: Characidae) from upper rio Paraná basin.Neotrop Ichthyol. 5(1):37–44.

Fernandes CA, Martins-Santos IC. 2003. Cytogenetic character-ization of two populations of Astyanax scabripinnis (Pisces,Characiformes) of the Ivaí basin PR Brazil. Cytologia. 68(3):289–293.

Fernandes CA, Martins-Santos IC. 2004. Cytogenetic studies intwo populations of Astyanax altiparanae (Pisces, Characi-formes). Hereditas. 141(3):328–332.

Fernandes CA, Martins-Santos IC. 2005. Sympatric occurrenceof three cytotypes and four morphological types of B chro-mosomes of Astyanax scabripinnis (Pisces, Characiformes)in the River Ivaí Basin, state of Paraná, Brazil. Genetica.124(2–3):301–306.

Fernandes CA, Martins-Santos IC. 2006a. Chromosomallocation of 5S and 18S rRNA genes in three sympatric cy-totypes of Astyanax scabripinnis (Characiformes, Characi-dae) from the Ivaí river basin, state of Paraná , Brazil.Caryologia. 59(3):253–259.


Dow

nloa

ded

by [

Gaz

i Uni

vers

ity]

at 0

4:59

21

Aug

ust 2

014

Fernandes CA, Martins-Santos IC. 2006b. Mapping of the 18Sand 5S ribosomal RNA genes in Astyanax altiparanae Ga-rutti & Britski, 2000 (Teleostei, Characidae) from the upperParaná river basin, Brazil. Genet Mol Biol. 29(3):464–468.

Fernandes CA, Martins-Santos IC, Bailly D. 2009. Karyotypeanalysis and mapping of the 18S and 5S ribosomal genesin Astyanax fasciatus (Teleostei, Characiformes) fromParanapanema River Basin. Cytologia. 74(3):295–300.

Fernandes CA, Piscor D, Bailly D, Silva VFB, Martins-SantosIC. 2010. Cytogenetic studies comparing three Characidaefish species from the Iguatemi River Basin, Brazil. Cytolo-gia. 75(4):329–333.

Ferreira-Neto M, Artoni RF, Vicari MR, Moreira-Filho O,Camacho JPM, Bakkali M, Oliveira C, Foresti F. 2012.Three sympatric karyomorphs in the fish Astyanax fasciatus(Teleostei, Characidae) do not seem to hybridize in naturalpopulations. Compar Cytogenet. 6(1):29–40.

Ferro DA, Néo DM, Moreira-Filho O, Bertollo LAC. 2001.Nucleolar organizing regions, 18S and 5S rDNA in Astya-nax scabripinnis (Pisces, Characidae): populations distribu-tion and functional diversity. Genetica. 110(1):55–62.

Froese R, Pauly D. 2013. FishBase [Word Wide Web Elec-tronic Publication]. www.fishbase.org.

Garavello JC, Pavanelli CS, Suzuki HI. 1997. Caracterizaçãoda ictiofauna do rio Iguaçu. In: Agostinho AA, Gomes LC,editors. Reservatório de Segredo: bases ecológicas para omanejo. Maringá: EDUEM; p. 61–84.

Garutti V, Britski HA. 2000. Descrição de uma espécie nova deAstyanax (Teleostei: Characidae) da bacia do alto rioParaná e considerações sobre as demais espécies do gênerona bacia. Comun. Mus. Ciênc. Tecnol. PUCRS. Sér. Zool.13(1):65–88.

Hashimoto DT, Ferguson-Smith MA, Rens W, Foresti F, Porto-Foresti F. 2011. Chromosome mapping of H1 histone and5S rRNA gene clusters in three species of Astyanax (Teleo-stei, Characiformes). Cytogenet Genome Res. 134(1):64–71.

Hashimoto DT, Gonçalves VR, Bortolozzi J, Foresti F, Porto-Foresti F. 2008. First report of a B chromosome in a natu-ral population of Astyanax altiparanae (Characiformes,Characidae). Genet Mol Biol. 31(1):275–278.

Howell WM, Black DA. 1980. Controlled silver-staining ofnucleolus organizer regions with the protective coloidaldeveloper: a 1-step method. Experientia. 36(8):1014–1015.

Jim SM, Toledo V. 1975. Citogenética de Astyanax bimaculatus(Characidae, Tetragonopterinae). Cienc Cult. 27(supl.):1122–1124.

Kavalco KF, Moreira-Filho O. 2003. Cytogenetical analyses infour species of the genus Astyanax (Pisces, Characidae)from Paraíba do Sul. Caryologia. 56(4):453–461.

Levan A, Fredga K, Sandberg AA. 1964. Nomenclature forcentromeric positions on chromosomes. Hereditas. 52(2):201–220.

Lima FCT, Malabarba LR, Buckup PA, Pezzi Da Silva JF, RpVari, Harold A, Benine R, Oyakawa OT, Pavanelli CS,Menezes NA, Lucena CAS, et al. 2003. Genera IncertaeSedis in Characidae. In: Reis RE, Kullander SO, FerrarisCJ Jr, editors. Check list of the freshwater fishes of Southand Central America. Porto Alegre: Edipucrs; p. 106–169.

Machado SN, Neto MF, Bakkali M, Vicari MR, Artoni RF,Oliveira C, Foresti F. 2012. Natural triploidy and Bchromosomes in Astyanax scabripinnis (Characiformes,Characidae): a new occurrence. Caryologia. 65(1):40–46.

Maistro EL, Oliveira C, Foresti F. 2000. Sympatric occurrenceof two cytotypes of Astyanax scabripinnis paranae (Char-aciformes, Characidae). Genet Mol Biol. 23(2):365–369.

Maistro EL, Foresti F, Oliveira C. 1994. New occurrence of amacro B-chromosome in Astyanax scabripinnis paranae

(Pisces, Characiformes, Characidae). Brazil J Genet.17(2):153–156.

Maistro EL, Foresti F, Oliveira C, Almeida-Toledo LF. 1992.Occurence of macro B chromosomes in Astyanax scabri-pinnis paranae (Pisces, Characiformes, Characidae).Genetica. 87(2):101–106.

Maistro EL, Dias AL, Foresti F, Oliveira C, Moreira-Filho O.1994. Natural triploidy in Astyanax scabripinnis (Pisces,Characidae) and simultaneous occurrence of macro B-chro-mosomes. Caryologia. 47(3–4):233–239.

Maistro EL, Oliveira C, Foresti F. 1998. Comparative cytoge-netic and morphological analysis of Astyanax scabripinnisparanae (Pisces, Characidae, Tetragonopterinae). GenetMol Biol. 21(2):201–206.

Malacrida ACCP, Giuliano-Caetano L. 2003. Natural triploidyin Astyanax aff. scabripinnis (Pisces, Characidae) of theTibagi river basin. PR. Cytologia. 68(3):267–270.

Mantovani M, Abel LD, Moreira-Filho O. 2005. Conserved 5Sand variable 45S rDNA chromosomal localisation revealedby FISH in Astyanax scabripinnis (Pisces, Characidae).Genetica. 123(3):211–216.

Mantovani M, Abel LD, Mestriner CA, Moreira-Filho O. 2000.Accentuated polymorphism of heterochromatin and nucleo-lar organizer regions in Astyanax scabripinnis (Pisces,Characidae): tools for understanding karyotypic evolution.Genetica. 109(3):161–168.

Martinez ERM, Alves AL, Silveira SM, Foresti F, Oliveira C.2012. Cytogenetic analysis in the incertae sedis speciesAstyanax altiparanae Garutti and Britzki, 2000 andHyphessobrycon eques Steindachner, 1882 (Characiformes,Characidae) from the upper Paraná river basin. ComparCytogenet. 6(1):41–51.

Medrado AS, Figueiredo AVA, Waldschmidt AM, AffonsoPRADM, Carneiro PLS. 2008. Cytogenetic and morpholog-ical diversity in populations of Astyanax fasciatus (Teleo-stei, Characidae) from Brazilian northeastern river basins.Genet Mol Biol. 31(1):208–214.

Melo FAG. 2001. Revisão taxonômica das espécies do gêneroAstyanax Baird e Girard, 1854 (Teleostei, Characiformes,Characidae) da região da Serra dos Órgãos. Arquivos doMuseu Nacional do Rio de Janeiro. 59:1–46.

Mestriner CA, Galetti Jr. PM, Valentini SR, Ruiz IRG, AbelLDS, Moreira-Filho O, Camacho JPM. 2000. Structuraland functional evidence that a B chromosome in the char-acid fish Astyanax scabripinnis is an isochromosome.Heredity. 85(1):1–9.

Miller DA, Dev VG, Tantravahi R, Miller OJ. 1976. Suppres-sion of human nucleolus organizer activity in mouse-humansomatic cells hybrid. Explor Cell Res. 101(2):235–243.

Mizoguchi SMHN, Martins-Santos IC. 1997. Macro and micro-chromosomes B in females of Astyanax scabripinnis.Hereditas. 127(3):249–253.

Mizoguchi SMHN, Martins-Santos IC. 1998a. Cytogenetic andmorphometric differences in populations of Astyanax scab-ripinnis (Pisces, Characidae) from Maringá region, Pr. Bra-zil. Genet Mol Biol. 21(1):55–61.

Mizoguchi SMHN, Martins-Santos IC. 1998b. Activation pat-terns of the nucleolar organizer region in Astyanax scabri-pinnis populations (Pisces, Characidae). Cytologia. 63(3):259–265.

Moreira-Filho O, Bertollo LAC. 1991. Astyanax scabripinnis(Pisces, Characidae): a species complex. Rev Brasil Genét.14(2):331–357.

Morelli S, Bertollo LAC, Foresti F. 1983. Cytogenetic consider-ations on the genus Astyanax (Pisces, Characidae). I Karyo-typic variability. Caryologia. 36(3):235–244.

Néo DM, Bertollo LAC, Moreira-Filho O. 2000. Morphologicaldifferentiation and possible origin of B chromosomes in


Dow

nloa

ded

by [

Gaz

i Uni

vers

ity]

at 0

4:59

21

Aug

ust 2

014

http://www.fishbase.org

natural Brazilian population of Astyanax scabripinnis(Pisces, Characidae). Genetica. 108(3):211–215.

Néo DM, Moreira-Filho O, Camacho JPM. 2000. Altitudinalvariation for B chromosome frequency in the characid fishAstyanax scabripinnis. Heredity. 85(2):136–141.

Pacheco RB, Giuliano-Caetano L, Dias AL. 2001. Cytotypesand Multiple NORs in an Astyanax altiparanae population(Pisces, Tetragonopterinae). Chromosome Sci. 5(2):109–114.

Pacheco RB, Giuliano-Caetano L, Julio Junior HFJ, Dias AL.2010. Cytogenetic data on Astyanax jacuhiensis (Characi-dae) in the lago Guaíba and tributaries, Brazil. Neotrop Ich-thyol. 8(3):667–671.

Pazza R, Kavalco KF, Bertollo LAC. 2006. Chromosome poly-morphism in Astyanax fasciatus (Teleostei, Characidae). 1.Karyotype analysis, Ag-NORs and mapping of the 18S and5S ribosomal genes in sympatric karyotypes and their pos-sible hybrid forms. Cytogenet Genome Res. 112(3–4):313–319.

Pinkel D, Straume T, Gray JW. 1986. Cytogenetic analysisusing quantitative, high-sensitivity, fluorescence hybridiza-tion. Proc Natl Acad Sci. 83(9):2934–2938.

Porto-Foresti F, Oliveira C, Maistro EL, Foresti F. 1997. Esti-mated frequency of B-chromosomes and population densityof Astyanax scabripinnis paranae in a small stream. BrazilJ Genet. 20(3):337–380.

Prioli SMA, Prioli AJ, Júlio Jr. HF, Pavanelli CS, Oliveira AV,Carrer H, Carraro DM, Prioli LM. 2002. Identification ofAstyanax altiparanae (Teleostei, Characidae) in the IguaçuRiver, Brazil, based on mitochondrial DNA and RAPDmarkers. Genet Mol Biol. 25(4):421–430.

Rocon-Stange EA, Almeida-Toledo LF. 1993. SupernumeraryB chromosomes restricted to males in Astyanax scabripin-nis (Pisces, Characidae). Rev Brasil Genét. 16(3):601–615.

Salvador LB, Moreira-Filho O. 1992. B chromosomes in Astya-nax scabripinnis (Pisces, Characidae). Heredity. 69(1):50–56.

Schweizer D, Loidl J. 1987. A model for heterochromatin dis-persal and the evolution of C-band patterns. Chromosom T.9:61–74.

Souza IL, Moreira-Filho O, Bertollo LAC. 1995. Cytogeneticdiversity in the Astyanax scabripinnis (Pisces, Characidae)complex II. Different cytotypes living in sympatry. Cytolo-gia. 60(2):273–281.

Souza IL, Moreira-Filho O, Galetti PM Jr. 1996. Heterochroma-tin differentiation in the characid fish Astyanax scabripinnis.Brazil J Genet. 19(3):405–410.

Sumner AT. 1972. A simple technique for demonstrating cen-tromeric heterochromatin. Explor Cell Res. 75(1):304–306.

Swerts CP, Castro GC, Maistro EL, Foresti F, Oliveira C. 1998.Análises citogenéticas preliminares na espécie Astyanaxfasciatus (Pisces, Characidae) provenientes do rio Sapucaí,represa de Furnas. MG. Genet Mol Biol. 21(supl. 3):66–66.

Takahashi EK, Giuliano-Caetano L, Dias AL. 1995. Estudocitogenético em Astyanax bimaculatus. Análise meiótica.In: 41o Congresso Nacional de Genética. Caxambu, Brasil.p. 450.

Tenorio RCCO, Vitorino CA, Souza IL, Oliveira C, Venere PC.2013. Comparative cytogenetics in Astyanax (Characifor-mes: Characidae) with focus on the cytotaxonomy of thegroup. Neotrop Ichthyol. 11(3):553–564.

Torres-Mariano AR, Morelli S. 2004. Chromosomal analysis ofAstyanax fasciatus (Pisces, Characidae) fron the Araguaririver, Uberlândia, MG. Brazil. Brazil J Biol. 66(1):161–165.

Vicari MR, Noleto RB, Artoni RF, Moreira-Filho O, BertolloLAC. 2008. Comparative cytogenetics among species ofthe Astyanax scabripinnis complex: evolutionary and bio-geographical inferences. Genet Mol Biol. 31(1):173–179.

Vicente VE, Moreira-Filho O, Camacho JPM. 1996. Sex-ratiodistortion associated with the presence of a B chromosomein Astyanax scabripinnis (Teleostei, Characidae). CytogenetGenome Res. 74(1–2):1–2.

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification anddirect sequencing of fungal ribosomal RNA genes forphylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ,White TJ, editors. PCR protocols: a guide to methodsand applications. San Diego (CA): Academic Press;p. 315–322.


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