Cytomolecular investigations using repetitive DNA probes contribute to the identification and characterization of Characidium sp. aff. C. vidali (Teleostei: Characiformes)

Maria Lígia Marques de Oliveira Fabilene Gomes Paim Érica Alves Serrano de Freitas Claudio Oliveira Fausto Foresti About the authors

Abstract

Characidium sp. aff. C. vidali is a species found in coastal streams in southeastern Brazil, which has karyotypic explanatory elements as the occurrence of microstructural variations, keeping the chromosomal macrostructure of the genus. The objective of this study was to apply cytomolecular tools in the chromosomes of Characidium sp. aff. C. vidali to identify characteristics in their karyotype contributing to cytogenetic definition of this species, adding information about the evolution of the chromosomal structure of the group. The species showed 2n = 50 chromosomes and from 1 to 4 additional B microchromosomes. FISH technique showed histone H3 and H4 genes in the short arm of pair 10, and microsatellites (CA)15, (CG)15, (GA)15 and (TTA)10 clustered in the subtelomeric portions of all A chromosomes, with total accumulation by supernumerary. The telomeric probe marked terminal regions of all chromosomes, in addition to the interstitial portion of four pairs, called ITS sites, with these markings being duplicated in two pairs, hence the double-ITS classification. C-banding revealed that supernumerary chromosomes are completely heterochromatic, that ITS sites are C-banding positive, but double-ITS sites are C-banding negative. So, throughout the evolution to Characidium, genomic events are occurring and restructuring chromosomes in populations.

Keywords:
Fish cytogenetics; Crenuchidae; Repetitive DNA; Molecular markers

Characidium sp. aff. C. vidali é uma espécie encontrada em riachos costeiros do sudeste do Brasil, que apresenta elementos cariotípicos elucidativos quanto à ocorrência de variações microestruturais, conservando a macroestrutura cromossômica do gênero. O objetivo deste estudo foi aplicar ferramentas citomoleculares para identificar características no cariótipo de Characidium sp. aff. C. vidali, que contribuam para a definição citogenética desta espécie, agregando informações quanto à evolução da estruturação cromossômica do grupo. A espécie apresentou 2n = 50 cromossomos, além de 1 a 4 microcromossomos B por célula. A FISH mostrou os genes de histona H3 e H4 sintênicos no braço curto do par 10, e os microssatélites (CA)15, (CG)15, (GA)15 e (TTA)10 clusterizados nas porções subteloméricas de todos os cromossomos do complemento A, com grande acúmulo nos supranumerários. A sonda telomérica identificou marcações terminais em todos os cromossomos, além de quatro pares marcados intersticialmente, chamados de sítios ITS, e dois pares com duas marcações intersticiais, chamados de double-ITS. O bandamento C revelou que os cromossomos supranumerários são completamente heterocromáticos, que os sítios ITS são banda C positivos, mas os sítios double-ITS são banda C negativos. Então, ao longo da evolução de Characidium, eventos genômicos estão ocorrendo e reestruturando cromossomos nas populações.

Palavras-chave:
Citogenética de peixes; Crenuchidae; DNA repetitivo; Marcadores moleculares


INTRODUCTION

Characidium Reinhardt, 1867, is considered the most species rich genus within the family Crenuchidae, with 65 valid species (Buckup, Van der Sleen, 2017Buckup PA, Van der Sleen P. Family Crenuchidae. In: Van der Sleen P, Albert JS, editors. Princeton: Princeton University Press; 2017. p.142–48.). These fishes are widely distributed in the Neotropical region, from eastern Panama to northeastern Argentina (Buckup, 2003Buckup PA. Family Crenuchidae (South American Darters). In: Reis RE, Kullander SO, Ferraris CJ Jr., editors. Check List of the Freshwater Fishes of South and Central America. Porto Alegre: Edipucrs; 2003. p.87–95.), with some ecological characteristics that restrict their habitat (Caramaschi, 1986Caramaschi EP. Distribuição da ictiofauna de riachos das Bacias do Tietê e do Paranapanema, junto ao divisor de águas (Botucatu, SP). [Dissertação]. São Paulo: Universidade Federal de São Carlos; 1986. PMid:3275190.). They generally are small fish, that lives in isolated populations at the head of streams (Maistro et al., 1998Maistro EL, Prieto-Mata E, Oliveira C, Foresti F. Unusual occurrence of a ZZ/ZW sex-chromosome system and supernumerary chromosomes in Characidium cf. fasciatum (Pisces, Characiformes, Characidiinae). Genetica. 1998; 104(1):1–7. https://doi.org/10.1023/A:1003242020259
https://doi.org/10.1023/A:1003242020259...
), which in most cases are environments of high altitudes and intense water flow, so cases of endemism and allopatric speciation are common for these fish (Buckup, 2003Buckup PA. Family Crenuchidae (South American Darters). In: Reis RE, Kullander SO, Ferraris CJ Jr., editors. Check List of the Freshwater Fishes of South and Central America. Porto Alegre: Edipucrs; 2003. p.87–95., 2011Buckup PA. The Eastern Brazilian Shield. In: Albert JS, Reis RE, editors. Berkeley and Los Angeles: University of California Press; 2011. p.203–10.). Therefore, it is probable that such factors directly impact the evolution of the karyotype structure of Characidium species, mostly in the microstructure of their chromosomes.

From the cytogenetic point of view, Characidium is considered a genus with conserved karyotypes, since most species have diploid number of 2n = 50 chromosomes (metacentrics and submetacentrics). However, some studies identified the occurrence of structural variation in the karyotypes of some species and populations, such as occurrence of heteromorphic ZZ/ZW sex chromosomes (Maistro et al., 1998Maistro EL, Prieto-Mata E, Oliveira C, Foresti F. Unusual occurrence of a ZZ/ZW sex-chromosome system and supernumerary chromosomes in Characidium cf. fasciatum (Pisces, Characiformes, Characidiinae). Genetica. 1998; 104(1):1–7. https://doi.org/10.1023/A:1003242020259
https://doi.org/10.1023/A:1003242020259...
, 2004Maistro EL, de Jesus CM, Oliveira C, Moreira-Filho O, Foresti F. Cytogenetic analysis of A-, B-chromosomes and ZZ/ZW sex chromosomes of Characidium gomesi (Teleostei, Characiformes, Crenuchidae). Cytologia. 2004; 69(2):181–86. https://doi.org/10.1508/cytologia.69.181
https://doi.org/10.1508/cytologia.69.181...
; Centofante et al., 2001Centofante L, Bertollo LAC, Moreira-Filho O. Comparative cytogenetics among sympatric species of Characidium (Pisces, Characiformes). Diversity analysis with the description of a ZW sex chromosome system and natural triploidy. Caryologia. 2001; 54(3):253–60. https://doi.org/10.1080/00087114.2001.10589233
https://doi.org/10.1080/00087114.2001.10...
, 2003Centofante L, Bertollo LA, Buckup PA, Moreira-Filho O. Chromosomal divergence and maintenance of sympatric Characidium fish species (Crenuchidae, Characidiinae). Hereditas. 2003; 138(3):213–18. https://doi.org/10.1034/j.1601-5223.2003.01714.x
https://doi.org/10.1034/j.1601-5223.2003...
; Noleto et al., 2009Noleto RB, Amorim AP, Vicari M R, Artoni RF, Cestari MM. An unusual ZZ/ZW sex chromosome system in Characidium fishes (Crenuchidae, Characiformes) with the presence of rDNA sites. J Fish Biol. 2009; 75(2):448–53. https://doi.org/10.1111/j.1095-8649.2009.02342.x
https://doi.org/10.1111/j.1095-8649.2009...
; Pansonato-Alves et al., 2014Pansonato-Alves JC, Serrano ÉA, Utsunomia R, Camacho JP, Costa Silva GJ, Vicari MR et al. Single origin of sex chromosomes and multiple origins of B chromosomes in fish genus Characidium. PLoS ONE. 2014; 9(9):e107169. https://doi.org/10.1371/journal.pone.0107169
https://doi.org/10.1371/journal.pone.010...
; Serrano et al., 2017Serrano ÉA, Utsunomia R, Scudeller PS, Oliveira C, Foresti F. Origin of B chromosomes in Characidium alipioi (Characiformes, Crenuchidae) and its relationship with supernumerary chromosomes in other Characidium species. Comp Cytogenet. 2017; 11(1):81–95. https://doi.org/10.3897/CompCytogen.v11i1.10886
https://doi.org/10.3897/CompCytogen.v11i...
, 2019aSerrano-Freitas ÉA, Melo BF, Freitas-Souza D, Oliveira MLM, Utsunomia R, Oliveira C et al. Species delimitation in Neotropical fishes of the genus Characidium (Teleostei, Characiformes). Zool Scr. 2019a; 48(1):69–80. https://doi.org/10.1111/zsc.12318
https://doi.org/10.1111/zsc.12318...
,bSerrano-Freitas ÉA, Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Araya-Jaime C, Oliveira C et al. Satellite DNA content of B chromosomes in the characid fish Characidium gomesi supports their origin from sex chromosomes. Mol Genet Genomics. 2019b; 295:195–207. https://doi.org/10.1007/s00438-019-01615-2
https://doi.org/10.1007/s00438-019-01615...
), presence of supernumerary chromosomes (Maistro et al., 1998Maistro EL, Prieto-Mata E, Oliveira C, Foresti F. Unusual occurrence of a ZZ/ZW sex-chromosome system and supernumerary chromosomes in Characidium cf. fasciatum (Pisces, Characiformes, Characidiinae). Genetica. 1998; 104(1):1–7. https://doi.org/10.1023/A:1003242020259
https://doi.org/10.1023/A:1003242020259...
; Pansonato-Alves et al., 2010Pansonato-Alves JC, Paiva LRS, Oliveira C, Foresti F. Interspecific chromosomal divergences in the genus Characidium (Teleostei: Characiformes: Crenuchidae). Neotrop Ichthyol. 2010; 8(1):77–86. https://doi.org/10.1590/S1679-62252010000100010
https://doi.org/10.1590/S1679-6225201000...
, 2011aPansonato-Alves JC, Oliveira C, Foresti F. Karyotypic conservatism in samples of Characidium cf. zebra (Teleostei, Characiformes, Crenuchidae): Physical mapping of ribosomal genes and natural triploidy. Genet Mol Biol. 2011a; 34(2):208–13. https://doi.org/10.1590/S1415-47572011005000005
https://doi.org/10.1590/S1415-4757201100...
,bPansonato-Alves JC, Vicari MR, Oliveira C, Foresti F. Chromosomal diversification in populations of Characidium cf. gomesi (Teleostei, Crenuchidae). J Fish Biol. 2011b; 78(1):183–94. https://doi.org/10.1111/j.1095-8649.2010.02847.x
https://doi.org/10.1111/j.1095-8649.2010...
, 2014Pansonato-Alves JC, Serrano ÉA, Utsunomia R, Camacho JP, Costa Silva GJ, Vicari MR et al. Single origin of sex chromosomes and multiple origins of B chromosomes in fish genus Characidium. PLoS ONE. 2014; 9(9):e107169. https://doi.org/10.1371/journal.pone.0107169
https://doi.org/10.1371/journal.pone.010...
; Serrano et al., 2017Serrano ÉA, Utsunomia R, Scudeller PS, Oliveira C, Foresti F. Origin of B chromosomes in Characidium alipioi (Characiformes, Crenuchidae) and its relationship with supernumerary chromosomes in other Characidium species. Comp Cytogenet. 2017; 11(1):81–95. https://doi.org/10.3897/CompCytogen.v11i1.10886
https://doi.org/10.3897/CompCytogen.v11i...
, 2019aSerrano-Freitas ÉA, Melo BF, Freitas-Souza D, Oliveira MLM, Utsunomia R, Oliveira C et al. Species delimitation in Neotropical fishes of the genus Characidium (Teleostei, Characiformes). Zool Scr. 2019a; 48(1):69–80. https://doi.org/10.1111/zsc.12318
https://doi.org/10.1111/zsc.12318...
,bSerrano-Freitas ÉA, Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Araya-Jaime C, Oliveira C et al. Satellite DNA content of B chromosomes in the characid fish Characidium gomesi supports their origin from sex chromosomes. Mol Genet Genomics. 2019b; 295:195–207. https://doi.org/10.1007/s00438-019-01615-2
https://doi.org/10.1007/s00438-019-01615...
), distribution of repetitive DNA sequences (Vicari et al., 2008Vicari MR, Artoni RF, Moreira-Filho O, Bertollo LAC. Diversification of a ZZ/ZW sex chromosome system in Characidium fish (Crenuchidae, Characiformes). Genetica. 2008; 134:311. https://doi.org/10.1007/s10709-007-9238-2
https://doi.org/10.1007/s10709-007-9238-...
; Machado et al., 2011Machado TC, Pansonato-Alves JC, Pucci MB, Nogaroto V, Almeida MC, Oliveira C et al. Chromosomal painting and ZW sex chromosomes differentiation in Characidium (Characiformes, Crenuchidae). BMC Genet. 2011; 12(65). https://doi.org/10.1186/1471-2156-12-65
https://doi.org/10.1186/1471-2156-12-65...
; Pansonato-Alves et al., 2011aPansonato-Alves JC, Oliveira C, Foresti F. Karyotypic conservatism in samples of Characidium cf. zebra (Teleostei, Characiformes, Crenuchidae): Physical mapping of ribosomal genes and natural triploidy. Genet Mol Biol. 2011a; 34(2):208–13. https://doi.org/10.1590/S1415-47572011005000005
https://doi.org/10.1590/S1415-4757201100...
, 2014Pansonato-Alves JC, Serrano ÉA, Utsunomia R, Camacho JP, Costa Silva GJ, Vicari MR et al. Single origin of sex chromosomes and multiple origins of B chromosomes in fish genus Characidium. PLoS ONE. 2014; 9(9):e107169. https://doi.org/10.1371/journal.pone.0107169
https://doi.org/10.1371/journal.pone.010...
; Scacchetti et al., 2015aScacchetti PC, Utsunomia R, Pansonato-Alves JC, Costa-Silva GJ, Oliveira C, Foresti F. Extensive spreading of interstitial telomeric sites on the chromosomes of Characidium (Teleostei, Characiformes). Genetica. 2015a; 143:263–70. https://doi.org/10.1007/s10709-014-9812-3
https://doi.org/10.1007/s10709-014-9812-...
,bScacchetti PC, Utsunomia R, Pansonato JC, Vicari MR, Artoni RF, Oliveira C et al. Chromosomal mapping of repetitive DNAs in Characidium (Teleostei, Characiformes): genomic organization and the diversification of ZW sex chromosomes. Cytogenet Genome Res. 2015b; 146(2):163–46. https://doi.org/10.1159/000437165
https://doi.org/10.1159/000437165...
; Serrano et al., 2017Serrano ÉA, Utsunomia R, Scudeller PS, Oliveira C, Foresti F. Origin of B chromosomes in Characidium alipioi (Characiformes, Crenuchidae) and its relationship with supernumerary chromosomes in other Characidium species. Comp Cytogenet. 2017; 11(1):81–95. https://doi.org/10.3897/CompCytogen.v11i1.10886
https://doi.org/10.3897/CompCytogen.v11i...
, 2019aSerrano-Freitas ÉA, Melo BF, Freitas-Souza D, Oliveira MLM, Utsunomia R, Oliveira C et al. Species delimitation in Neotropical fishes of the genus Characidium (Teleostei, Characiformes). Zool Scr. 2019a; 48(1):69–80. https://doi.org/10.1111/zsc.12318
https://doi.org/10.1111/zsc.12318...
,bSerrano-Freitas ÉA, Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Araya-Jaime C, Oliveira C et al. Satellite DNA content of B chromosomes in the characid fish Characidium gomesi supports their origin from sex chromosomes. Mol Genet Genomics. 2019b; 295:195–207. https://doi.org/10.1007/s00438-019-01615-2
https://doi.org/10.1007/s00438-019-01615...
; Pucci et al., 2018Pucci MB, Nogaroto V, Moreira-Filho O, Vicari MR. Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes. Genet Mol Biol. 2018; 41(3):585–92. http://dx.doi.org/10.1590/1678-4685-GMB-2017-0121
http://dx.doi.org/10.1590/1678-4685-GMB-...
) and occurrence of natural triploidy (Centofante et al., 2001Centofante L, Bertollo LAC, Moreira-Filho O. Comparative cytogenetics among sympatric species of Characidium (Pisces, Characiformes). Diversity analysis with the description of a ZW sex chromosome system and natural triploidy. Caryologia. 2001; 54(3):253–60. https://doi.org/10.1080/00087114.2001.10589233
https://doi.org/10.1080/00087114.2001.10...
; Pansonato-Alves et al., 2011bPansonato-Alves JC, Vicari MR, Oliveira C, Foresti F. Chromosomal diversification in populations of Characidium cf. gomesi (Teleostei, Crenuchidae). J Fish Biol. 2011b; 78(1):183–94. https://doi.org/10.1111/j.1095-8649.2010.02847.x
https://doi.org/10.1111/j.1095-8649.2010...
). These particularities make the genus Characidium an interesting group for cytogenetic studies because rearrangements that occur in chromosome microstructure certainly can set the biological characteristics of the species and populations.

The eukaryote genome is composed of a large number of repetitive sequences, and the physical mapping as well as the knowledge of the molecular organization of these sequences has contributed significantly to a better understanding of the structural differences that occur in the karyotypes of many fish species (Cioffi, Bertollo, 2012Cioffi MB, Bertollo LAC. Chromosomal Distribution and Evolution of Repetitive DNAs in Fish. In: Garrido R, editor. Repetitive DNA. Genome Dynamics. Basel: Karger; 2012. p.197–221. https://doi.org/10.1159/000337950
https://doi.org/10.1159/000337950...
; López-Flores, Garrido-Ramos, 2012López-Flores I, Garrido-Ramos MA. The repetitive DNA content in eukaryotic genomes. In: Garrido-Ramos MA, editors. Repetitive DNA. Genome dynamics. Karger, Basel; 2012. p.1–28.). The accumulation of these repetitive elements in specific regions of the genome can generate breaks, inversions, deletions and amplifications in the chromosomes (Lim, Simmons, 1994Lim JK, Simmons MJ. Gross chromosome rearrangements mediated by transposable elements in Drosophila melanogaster. Bioessays. 1994; 16(4):269–75. https://doi.org/10.1002/bies.950160410
https://doi.org/10.1002/bies.950160410...
; Dimitri et al., 1997Dimitri P, Arcà B, Berghella L, Mei E. High genetic instability of heterochromatin after transposition of the LINE-like I factor in Drosophila melanogaster. Proc Natl Acad Sci USA. 1997; 94(15):8052–57. https://doi.org/10.1073/pnas.94.15.8052
https://doi.org/10.1073/pnas.94.15.8052...
; Raskina et al., 2008Raskina O, Barber JC, Nevo E, Belyayev A. Repetitive DNA and chromosomal rearrangements: speciation-related events in plant genomes. Cytogenet Genome Res. 2008; 120:351–57. https://doi.org/10.1159/000121084
https://doi.org/10.1159/000121084...
), which could reflect directly on the micro and/or macro structure of karyotype of a species or population.

There is a group of Characidium known to inhabit coastal drainages in Southeastern region of Brazil, and the species Characidium sp. aff. C. vidali, although not yet formerly described, has already been found in the Macaé River, São João River and Paraíba do Sul River (Leitão, Buckup, 2014Leitão RP, Buckup PA. A new Species of Characidium (Characiformes: Crenuchidae) from Coastal Basins of Serra do Mar, Southeastern Brazil. Copeia. 2014; 2014(1):14–22. http://doi.org/10.1643/CI-12-137
http://doi.org/10.1643/CI-12-137...
). Using cytomolecular tools, Scacchetti et al. (2015c)Scacchetti PC, Utsunomia R, Pansonato‐Alves JC, Costa‐Silva GJ, Vicari MR, Artoni RF et al. Repetitive DNA sequences and evolution of ZZ/ZW sex chromosomes in Characidium (Teleostei: Characiformes). PloS ONE. 2015c; 10(9):e0137231. https://doi.org/10.1371/journal.pone.0137231
https://doi.org/10.1371/journal.pone.013...
initiated investigations on the karyotype of a population of this species, verifying its importance in studies on the evolution of sex chromosomes within the genus Characidium, and in the species differentiation process, since Characidium sp. aff. C. vidali was found in sympatry with Characidium vidali Travassos, 1967. An interesting feature observed was the occurrence of supernumerary chromosomes present only in Characidium sp. aff. C. vidali, making the specie more interesting for cytogenetic studies related to the origin of the extra genomic elements.

Cytomolecular markers were used in the present study to investigate the genome of Characidium sp. aff. C. vidali aiming at the cytogenetic characterization of the species, contributing to a better understanding of the karyotype diversification process in this fish group. The mapping of repetitive DNA sequences permitted to identify characteristics of the karyotype structure throughout the evolutionary history of the species. It is considered that the information obtained can help to the construction of a cytogenetic map for the species of the genus Characidium, establishing better knowledge about the genomic structuring of the species, as well as the mechanisms involved in the chromosomal diversification process and speciation.

MATERIAL AND METHODS

Twenty-three specimens of Characidium sp. aff. C. vidali (10 females and 13 males) from the Bananeiras Stream, São João River basin, municipality of Silva Jardim, state of Rio de Janeiro (22º28’51.8”S 42º23’39”W) were analyzed. The individuals were deposited in fish collection of Laboratório de Biologia e Genética de Peixes, Universidade Estadual Paulista “Julio de Mesquita Filho” (UNESP), Botucatu, São Paulo, Brazil, LBP 28520.

Cell suspensions with mitotic chromosomes were obtained from anterior portion of the kidney, according to Foresti et al., (1981)Foresti F, Almeida-Toledo LF, Toledo- ASF. Polymorphic nature of nucleolus organizer regions in fishes. Cytogenet Cell Genet. 1981; 31(3):137–44. https://doi.org/10.1159/000131639
https://doi.org/10.1159/000131639...
. The heterochromatic regions were identified by C-banding (Sumner, 2003Sumner AT. Chromosomes: Organization and Function. London: Blackwell Publishing Company, 2003.), and the chromosomes were classified following Levan et al. (1964)Levan A, Fredga K, Sandberg AA. Nomenclature for centromeric position on chromosomes. Hereditas. 1964; 52(2):201–20. https://doi.org/10.1111/j.1601-5223.1964.tb01953.x
https://doi.org/10.1111/j.1601-5223.1964...
.

Genomic DNA from Characidium sp. aff. C. vidali was extracted using the Kit Wizard Genomic DNA Purification (PROMEGA), from liver fragments preserved in ethanol. Telomeric probes (TTAGGG), histone H3 and histone H4, were obtained by PCR (Polymerase Chain Reaction) and the primers used are in accordance with Ijdo et al. (1991)Ijdo JW, Wells RA, Baldini A, Reeders ST. Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR. Nucleic Acids Res. 1991; 19(17):4780. https://doi.org/10.1093/nar/19.17.4780
https://doi.org/10.1093/nar/19.17.4780...
, Colgan et al., (1998)Colgan DJ, McLauchlan A, Wilson GDF, Livingston SP, Edgecombe GD, Macaranas J et al. Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Aust J Zool. 1998; 46(5):419–37. https://doi.org/10.1071/ZO98048
https://doi.org/10.1071/ZO98048...
, and Pineau et al. (2005)Pineau P, Henry M, Suspène R, Marchio A, Dettai A, Debruyne R et al. A universal primer set for PCR amplification of nuclear histone H4 genes from all animal species. Mol Biol Evol. 2005; 22(3):582–88. https://doi.org/10.1093/molbev/msi053
https://doi.org/10.1093/molbev/msi053...
, respectively. The marking of the three probes were by PCR, with histone H3 and telomeric being marked with Biotin-16-dUTP (Roche), while histone H4 was marked with Digoxigenin-11-dUTP (Roche). In the present study, oligonucleotide probes with microsatellite sequences were also used (CA)15, (GA)15, (CG)15 and (TTA)10, all already marked directly with Carboxy-tetramethylrhodamine N-succinimidyl ester (TAMRA), by Sigma (Kubat et al., 2008Kubat Z, Hobza R, Vyskot B, Kejnovsky E. Microsatellite accumulation on the Y chromosome in Silene latifolia. Genome. 2008; 51(5):350–56. https://doi.org/10.1139/G08-024
https://doi.org/10.1139/G08-024...
).

The physical mapping of the probes was performed under high stringency conditions, on slides of males and females of Characidium sp. aff. C. vidali, according to Pinkel et al., (1986)Pinkel D, Straume T, Gray JW. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA. 1986; 83(9):2934–38. https://doi.org/10.1073/pnas.83.9.2934
https://doi.org/10.1073/pnas.83.9.2934...
. Signals were detected using anti-digoxigenin–Rhodamine for digoxigenin-11-dUTP and avidin–FITC (Sigma Aldrich, St Louis, MO, USA) for biotin-16-dUTP. Chromosomes were counterstained with DAPI (Vector Laboratories, Burlingame, Calif, USA), and the images analyzed with an epifluorescence microscope (Olympus BX61) were captured using the software Image Pro Plus 6.0 (Media Cybernetics, Rockville, Md. USA).

RESULTS

All specimens of Characidium sp. aff. C. vidali analyzed showed a basic diploid number of 2n = 50 chromosomes, composed of 32m + 18sm (Figs. 1A–D). All individuals analyzed presented micro B chromosomes mitotically unstable and eventually some difference in size, varying from one to four per cell in the individuals. Differences between the karyotypes of males and females were found only with the chromosomes of the second pair identified as sex-linked, being represented by two metacentrics of the same size (ZZ) in males (Figs. 1A, C) and by one metacentric (Z) and one submetacentric (W) in females, both medium-sized (Figs. 1B, D). Fig. 1 also shows an example of the species under study.

C-banding revealed heterochromatic blocks accumulated in a centromeric portion of all the chromosomes of the analyzed karyotypes, in addition some marked regions were observed in the terminal region in some pairs. In addition, a conspicuous block of constitutive heterochromatin was observed in the pericentromeric region of the long arm of the Z chromosome, while the W chromosome was completely heterochromatic. All supernumeraries present in the species were also considered to be completely heterochromatic (Figs. 1C, D, boxes).

FIGURE 1 |
Characidium sp. aff. C. vidali karyotypes arranged from mitotic metaphases after to conventional Giemsa staining and C-banding. A. and C. male karyotypes. B. and D. female karyotypes. B chromosomes are in the boxes. In evidence a preserved specimen under study. Photo of Characidium sp. aff. C. vidali by Bruno F. Melo.

The sequences for histone H3 (green) and H4 (red) genes, as revealed by the FISH technique were found clustered in synteny in the pericentromeric region of the short arm in a median sized chromosome pair (Fig. 2). The physical mapping of the four microsatellites motifs (CA)15, (CG)15, (GA)15 and (TTA)15 showed a preferential accumulation of these sequences in the subtelomeric regions of the A chromosomes, without differences between the karyotype of males and females (Fig. 3). In addition, the B chromosomes presented all tested microsatellites (Fig. 3).

FIGURE 2 |
Metaphase of Characidium sp. aff. C. vidali after FISH with histone H3 (green) and H4 (red) probe. Synteny marked in par 10. Scale bar = 10 µm.

FIGURE 3 |
Metaphase plates of Characidium sp. aff. C. vidali after fluorescent in situ hybridization (FISH) with four microsatellite motifs. B chromosomes present in the species are indicated. Scale bar = 10 µm.

Regarding the markings with telomeric probe TTAGGG, the chromosomes of Characidium sp. aff. C. vidali showed signals of hybridization in the terminal portions of all chromosomes, including the supernumeraries (Fig. 4A). Additionally, ITS (Interstitial Telomeric Sites) sites in pericentromeric regions of four chromosomal pairs and double-ITS sites, in interstitial portions of the short arm of two chromosome pairs were also observed (Fig. 4A). The sequential use of FISH and C-banding showed that ITS regions were coincident with constitutive heterochromatic blocks, while the double-ITS marks were C-band negative (Fig. 4B). B chromosomes showed telomeric marks only in the terminal portion and they were fully heterochromatic as revealed by C banding (Figs. 4A–B).

FIGURE 4 |
Metaphase of Characidium sp. aff. C. vidali after fluorescent in situ hybridization (FISH); A. With a telomeric probe (TTAGGG)n. B. After sequential C-banding. The arrows indicate Interstitial Telomeric Sites (ITS), and asterisks highlight double-ITS marks; B = B-chromosomes; Z, W = sex chromosomes. Scale bars = 10 µm.

DISCUSSION

Characidum are an ideal group for karyotype studies, due to the structural variation in karyotype in populations. In this study, it is the first time that the markers H3, H4, telomeric sequences and motifs CA, CG, TTA, GA are described for Characidum sp. aff. C. vidali that will reinforce the classic cytogenetic data already described and increase the knowledge about the composition of these sequences in the karyotypes of this species.

The results observed in the karyotype analysis of Characidium sp. aff. C. vidali, corroborate the findings of Scacchetti et al., (2015c)Scacchetti PC, Utsunomia R, Pansonato‐Alves JC, Costa‐Silva GJ, Vicari MR, Artoni RF et al. Repetitive DNA sequences and evolution of ZZ/ZW sex chromosomes in Characidium (Teleostei: Characiformes). PloS ONE. 2015c; 10(9):e0137231. https://doi.org/10.1371/journal.pone.0137231
https://doi.org/10.1371/journal.pone.013...
concerning the diploid number, presence of heteromorphic chromosomes linked to sex and distribution pattern of constitutive heterochromatin. The identification of B microchromosomes in all the cells analyzed, varying only in quantity, was also reported by the authors, drawing attention to the chromosomal structure of the species, which in both works maintained the diploid number of 50 chromosomes. In addition, the present work suggests the occurrence of evident characteristics in the genomic elements that are acting and reorganizing the karyotype microstructure during differentiation of the species and the evolutionary processes of this group of fish.

The physical mapping with H3 and H4 histone probes showed that in Characidium sp. aff. C. vidali these genes are colocalized in the short arm of the pair 10. This particularity was also observed by Pucci et al., (2018)Pucci MB, Nogaroto V, Moreira-Filho O, Vicari MR. Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes. Genet Mol Biol. 2018; 41(3):585–92. http://dx.doi.org/10.1590/1678-4685-GMB-2017-0121
http://dx.doi.org/10.1590/1678-4685-GMB-...
in Characidium zebra Eigenmann, 1909, and Characidium gomesi Travassos, 1956, both species collected in the Paiol Grande stream, while in C. gomesi from the São João River the two clusters were syntenic but located in the short arms of the chromosomes in pair 5. Additionally, Serrano et al. (2017)Serrano ÉA, Utsunomia R, Scudeller PS, Oliveira C, Foresti F. Origin of B chromosomes in Characidium alipioi (Characiformes, Crenuchidae) and its relationship with supernumerary chromosomes in other Characidium species. Comp Cytogenet. 2017; 11(1):81–95. https://doi.org/10.3897/CompCytogen.v11i1.10886
https://doi.org/10.3897/CompCytogen.v11i...
mapped the H3 histone gene in Characidium alipioi Travassos, 1955, verifying that the gene was located in the long arm of the chromosomes also in the par 10.

The histone genes H1, H2A, H2B, H3 and H4 form a complex multigenic family, encoding basic and essential proteins (Eirín-López et al., 2009Eirín-López JM, González-Romero R, Dryhurst D, Méndez J, Ausió J. Long-term evolution of histone families: old notions and new insights into their mechanisms of diversification across eukaryotes. In: Pontarotti P, editor. Evolutionary Biology.Berlin, Heidelberg: Springer; 2009. p.139-62. https://doi.org/10.1007/978-3-642-00952-5_8
https://doi.org/10.1007/978-3-642-00952-...
). The organization of these sequences has been the subject of many studies in fish. In some fish groups, the location of these genes are considered conserved, as the histone H1 in Astyanax Baird & Girard, 1854 (Hashimoto et al., 2011Hashimoto DT, Ferguson-Smith MA, Rens W, Foresti F, Porto-Foresti F. Chromosome mapping of H1 histone and 5S rRNA gene clusters in three species of Astyanax (Teleostei, Characiformes). Cytogenet Genome Res. 2011; 134(1):64–71. https://doi.org/10.1159/000323512
https://doi.org/10.1159/000323512...
) and Pseudoplatystoma Bleeker, 1862 (Hashimoto et al., 2013Hashimoto DT, Ferguson-Smith MA, Rens W, Prado FD, Foresti F, Porto-Foresti F. Cytogenetic mapping of H1 histone and ribosomal RNA genes in hybrids between catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum. Cytogenet Genome Res. 2013; 139(2):102–06. https://doi.org/10.1159/000345299
https://doi.org/10.1159/000345299...
), histone H3 in Hypostomus Lacepède, 1803 (Pansonato-Alves et al., 2013Pansonato-Alves JC, Serrano EA, Utsunomia R, Scacchetti PC, Oliveira C, Foresti F. Mapping five repetitive DNA classes in sympatric species of Hypostomus (Teleostei: Siluriformes: Loricariidae): analysis of chromosomal variability. Rev Fish Biol Fisher. 2013; 23:477–89. https://doi.org/10.1007/s11160-013-9303-0
https://doi.org/10.1007/s11160-013-9303-...
) and histone H1, H3, and H4 in Psalidodon bockmanni (Vari & Castro, 2007) (Silva et al., 2013Silva DMZA, Pansonato-Alves JC, Utsunomia R, Daniel SN, Hashimoto DT, Oliveira C et al. Chromosomal organization of repetitive DNA sequences in Astyanax bockmanni (Teleostei, Characiformes): Dispersive location, association and co-localization in the genome. Genetica. 2013; 141:329–36. https://doi.org/10.1007/s10709-013-9732-7
https://doi.org/10.1007/s10709-013-9732-...
). In Characidium, the H3 and H4 histone genes appear to be frequently colocalized and so far, in most cases, clustered in the chromosomes of par 10; therefore, the variations observed by Serrano et al., (2017)Serrano ÉA, Utsunomia R, Scudeller PS, Oliveira C, Foresti F. Origin of B chromosomes in Characidium alipioi (Characiformes, Crenuchidae) and its relationship with supernumerary chromosomes in other Characidium species. Comp Cytogenet. 2017; 11(1):81–95. https://doi.org/10.3897/CompCytogen.v11i1.10886
https://doi.org/10.3897/CompCytogen.v11i...
and Pucci et al., (2018)Pucci MB, Nogaroto V, Moreira-Filho O, Vicari MR. Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes. Genet Mol Biol. 2018; 41(3):585–92. http://dx.doi.org/10.1590/1678-4685-GMB-2017-0121
http://dx.doi.org/10.1590/1678-4685-GMB-...
would be consequence of structural rearrangements occurred in the karyotypes of the species. These findings increase the evidence that Characidium is a group of fish that frequently undergoes significant microstructural changes in its karyotype, although conserving the chromosomal macrostructure in most species and populations. The mapping of repetitive sequences particularly contributes to reveal this particularity.

The four microsatellite sequences mapped in this study showed an interesting similar pattern of distribution on the karyotype of Characidium sp. aff. C. vidali, with preferential accumulation of the sequences in the subtelomeric portions of the chromosomes. This distribution model observed in several species of the genus Characidium (Scacchetti et al., 2015bScacchetti PC, Utsunomia R, Pansonato JC, Vicari MR, Artoni RF, Oliveira C et al. Chromosomal mapping of repetitive DNAs in Characidium (Teleostei, Characiformes): genomic organization and the diversification of ZW sex chromosomes. Cytogenet Genome Res. 2015b; 146(2):163–46. https://doi.org/10.1159/000437165
https://doi.org/10.1159/000437165...
; Serrano et al., 2017Serrano ÉA, Utsunomia R, Scudeller PS, Oliveira C, Foresti F. Origin of B chromosomes in Characidium alipioi (Characiformes, Crenuchidae) and its relationship with supernumerary chromosomes in other Characidium species. Comp Cytogenet. 2017; 11(1):81–95. https://doi.org/10.3897/CompCytogen.v11i1.10886
https://doi.org/10.3897/CompCytogen.v11i...
) is also common among other Characiformes (Cioffi et al., 2010Cioffi MB, Kejnovsky E, 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. 2010; 132(4):289–96. https://doi.org/10.1159/000322058
https://doi.org/10.1159/000322058...
, 2012Cioffi MB, Bertollo LAC. Chromosomal Distribution and Evolution of Repetitive DNAs in Fish. In: Garrido R, editor. Repetitive DNA. Genome Dynamics. Basel: Karger; 2012. p.197–221. https://doi.org/10.1159/000337950
https://doi.org/10.1159/000337950...
; Poltronieri et al., 2014Poltronieri J, Marquioni V, Bertollo LAC, Kejnovsky E, Molina WF, Liehr T, Cioffi MB. Comparative chromosomal mapping of microsatellites in Leporinus species (Characiformes, Anostomidae): unequal accumulation on the W chromosomes. Cytogenet Genome Res. 2014; 142:40–45. https://doi.org/10.1159/000355908
https://doi.org/10.1159/000355908...
; Terencio et al., 2013Terencio ML, Schneider CH, Gross MC, Vicari MR, Farias IP, Passos KB et al. Evolutionary dynamics of repetitive DNA in Semaprochilodus (Characiformes, Prochilodontidae): a fish model for sex chromosome differentiation. Sex Dev. 2013; 7:325–33. https://doi.org/10.1159/000356691
https://doi.org/10.1159/000356691...
), representing a usual pattern among the components of this order. In most eukaryotic organisms, the microsatellites distribution does not occur randomly. As being highly repetitive sequences, they are usually found accumulated in the heterochromatic regions of the genome where the exchange rate is reduced (Lohe et al., 1993Lohe AR, Hilliker AJ, Roberts PA. Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics. 1993; 134(4): 1149–74. ; Cuadrado, Jouve, 2011Cuadrado Á, Jouve N. Novel simple sequence repeats (SSRs) detected by ND-FISH in heterochromatin of Drosophila melanogaster. BMC Genomics. 2011; 12:205. https://doi.org/10.1186/1471-2164-12-205
https://doi.org/10.1186/1471-2164-12-205...
; Pathak, Ali, 2012Pathak D, Ali S. Repetitive DNA: A tool to explore animal genomes/ transcriptomes. In: Germana M, Petrera F editors. Functional genomics. InTech, Published; 2012. p.155–80. https://doi.org/10.5772/48259
https://doi.org/10.5772/48259...
). The four microsatellites motifs analyzed in the present work showed a preferential accumulation near the subtelomeric regions in almost all chromosomes of the A complement, without differences between the sex of individuals. However, B chromosomes were almost completely positive for C-banding and conspicuous marks characterized the cumulative presence of the four motifs along this element.

The accumulation of repetitive sequences in genome, can change the structures of chromosomes, and mapping these regions in DNA can provide information about origin, structure organization, and functions of specific chromosomes (Ruiz-Ruano et al., 2015Ruiz-Ruano FJ, Cuadrado Á, Montiel EE, Camacho JPM, Lopez-Leon MD. Next generation sequencing and FISH reveal uneven and nonrandom microsatellite distribution in two grasshopper genomes. Chromosoma. 2015; 124:221–34. https://doi.org/10.1007/s00412-014-0492-7
https://doi.org/10.1007/s00412-014-0492-...
). In this sense, many studies have attempted to investigate the location of microsatellite sequences on sex-linked chromosomes to understand about the participation of these repetitive regions in the chromosome differentiation process (Kubat et al., 2008Kubat Z, Hobza R, Vyskot B, Kejnovsky E. Microsatellite accumulation on the Y chromosome in Silene latifolia. Genome. 2008; 51(5):350–56. https://doi.org/10.1139/G08-024
https://doi.org/10.1139/G08-024...
; Poltronieri et al., 2014Poltronieri J, Marquioni V, Bertollo LAC, Kejnovsky E, Molina WF, Liehr T, Cioffi MB. Comparative chromosomal mapping of microsatellites in Leporinus species (Characiformes, Anostomidae): unequal accumulation on the W chromosomes. Cytogenet Genome Res. 2014; 142:40–45. https://doi.org/10.1159/000355908
https://doi.org/10.1159/000355908...
; Ziemniczak et al., 2014Ziemniczak K, Traldi JB, Nogaroto V, Almeida MC, Artoni RF, Moreira-Filho O et al. In situ localization of (GATA)n and (TTAGGG)n repeated DNAs and W sex chromosome differentiation in Parodontidae (Actinopterygii: Characiformes). Cytogenet Genome Res. 2014; 144:325–32. https://doi.org/10.1159/000370297
https://doi.org/10.1159/000370297...
; Scacchetti et al., 2015bScacchetti PC, Utsunomia R, Pansonato JC, Vicari MR, Artoni RF, Oliveira C et al. Chromosomal mapping of repetitive DNAs in Characidium (Teleostei, Characiformes): genomic organization and the diversification of ZW sex chromosomes. Cytogenet Genome Res. 2015b; 146(2):163–46. https://doi.org/10.1159/000437165
https://doi.org/10.1159/000437165...
, cScacchetti PC, Utsunomia R, Pansonato‐Alves JC, Costa‐Silva GJ, Vicari MR, Artoni RF et al. Repetitive DNA sequences and evolution of ZZ/ZW sex chromosomes in Characidium (Teleostei: Characiformes). PloS ONE. 2015c; 10(9):e0137231. https://doi.org/10.1371/journal.pone.0137231
https://doi.org/10.1371/journal.pone.013...
). In the case of Characidium sp. aff. C. vidali the accumulation of the four motifs highlighted the B chromosomes of the species. It can be supposed that the accumulation could occur due to the low rate of recombination of these chromosomes, and the fact that they are completely heterochromatic (Lohe et al., 1993Lohe AR, Hilliker AJ, Roberts PA. Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics. 1993; 134(4): 1149–74. ; Cuadrado, Jouve, 2011Cuadrado Á, Jouve N. Novel simple sequence repeats (SSRs) detected by ND-FISH in heterochromatin of Drosophila melanogaster. BMC Genomics. 2011; 12:205. https://doi.org/10.1186/1471-2164-12-205
https://doi.org/10.1186/1471-2164-12-205...
). It is interesting to note that the same four motifs were mapped in Characidium gomesi (Scacchetti et al., 2015bScacchetti PC, Utsunomia R, Pansonato JC, Vicari MR, Artoni RF, Oliveira C et al. Chromosomal mapping of repetitive DNAs in Characidium (Teleostei, Characiformes): genomic organization and the diversification of ZW sex chromosomes. Cytogenet Genome Res. 2015b; 146(2):163–46. https://doi.org/10.1159/000437165
https://doi.org/10.1159/000437165...
), as well as (CA)15 and (GA)15 sequences in C. alipioi (Serrano et al., 2017Serrano ÉA, Utsunomia R, Scudeller PS, Oliveira C, Foresti F. Origin of B chromosomes in Characidium alipioi (Characiformes, Crenuchidae) and its relationship with supernumerary chromosomes in other Characidium species. Comp Cytogenet. 2017; 11(1):81–95. https://doi.org/10.3897/CompCytogen.v11i1.10886
https://doi.org/10.3897/CompCytogen.v11i...
), and although the two species also carry heterochromatic B chromosomes, no microsatellite clusters were observed in their supernumerary element. This indicates that the B chromosomes of the three species differ in their structural composition as revealed by their specific degree of heterochromatic enrichment, and probably in Characidium sp. aff. C. vidali the four motifs have accumulated after the establishment of the supernumerary chromosome in the karyotype of the population.

The distribution of microsatellites in the genome of a population can be attributed to different events such as uneven crossing-over, ectopic recombination, disturbs in DNA repair or slippage, and action of transposable elements (Dover, 1993Dover GA. Evolution of genetic redundancy for advanced players. Curr Opin Genet Dev. 1993; 3:902–10. https://doi.org/10.1016/0959-437x(93)90012-e
https://doi.org/10.1016/0959-437x(93)900...
; McMurray, 1995McMurray CT. Mechanisms of DNA expansion. Chromosoma. 1995; 104:2–13. https://doi.org/10.1007/BF00352220
https://doi.org/10.1007/BF00352220...
; Hancock, 1996Hancock JM. Simple sequences and the expanding genome. Bioessays. 1996; 18(5):421–25. https://doi.org/10.1002/bies.950180512
https://doi.org/10.1002/bies.950180512...
; Ruiz-Ruano et al., 2015Ruiz-Ruano FJ, Cuadrado Á, Montiel EE, Camacho JPM, Lopez-Leon MD. Next generation sequencing and FISH reveal uneven and nonrandom microsatellite distribution in two grasshopper genomes. Chromosoma. 2015; 124:221–34. https://doi.org/10.1007/s00412-014-0492-7
https://doi.org/10.1007/s00412-014-0492-...
). In Characidium, we believe that the spread of microsatellites occurred by ectopic recombination, as pointed out by Pucci et al., (2018)Pucci MB, Nogaroto V, Moreira-Filho O, Vicari MR. Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes. Genet Mol Biol. 2018; 41(3):585–92. http://dx.doi.org/10.1590/1678-4685-GMB-2017-0121
http://dx.doi.org/10.1590/1678-4685-GMB-...
when mapped transposable elements in some species of the genus and observed a low accumulation of these sequences in the genomes. In addition, the similar pattern of microsatellite allocated in the subterminal regions of the chromosomes may be further evidence that reinforces the macrostructural homogeneity of the karyotypes in this group of fish.

In the vertebrate genome, the identification of telomeric sequences also increase knowledge about the chromosomal structure of the species. Such sequences are made up of six nucleotides TTAGGG that repeat in tandem (Meyne et al., 1989Meyne J, Ratliff RL, Moyzis RK. Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proc Natl Acad Sci USA. 1989; 86(18):7049–53. https://doi.org/10.1073/pnas.86.18.7049
https://doi.org/10.1073/pnas.86.18.7049...
; Guerra et al., 2004Guerra M. FISH: conceitos e aplicações na citogenética. Ribeirão Preto: Sociedade Brasileira de Genética; 2004.), and the present study showed the physical mapping of these sites in Characidium sp. aff. C. vidali. All chromosomes of the species showed the telomeric region marked, including the supernumerary elements. As telomeric sequences confer integrity and protect DNA from damage (Zakian, 1995Zakian VA. Telomeres: beginning to understand the end. Science. 1995; 270(5242):1601–1607. https://doi.org/10.1126/science.270.5242.1601
https://doi.org/10.1126/science.270.5242...
; de Lange 2005de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes & development. 2005; 19(18):2100–10. http://doi.org/10.1101/gad.1346005
http://doi.org/10.1101/gad.1346005...
; Palm, de Lange, 2008Palm W, de Lange T. How shelterin protects mammalian telomeres. Annu Rev Genet. 2008; 42:301–34. https://doi.org/10.1146/annurev.genet.41.110306.130350
https://doi.org/10.1146/annurev.genet.41...
; Luke, Lingner, 2009Luke B, Lingner J. TERRA: telomeric repeat‐containing RNA. The EMBO journal. 2009; 28(17):2503–10. https://doi.org/10.1038/emboj.2009.166
https://doi.org/10.1038/emboj.2009.166...
; Chan, Chang, 2010Chan SS, Chang S. Defending the end zone: studying the players involved in protecting chromosome ends. FEBS letters. 2010; 584(17):3773–78. https://doi.org/10.1016/j.febslet.2010.06.016
https://doi.org/10.1016/j.febslet.2010.0...
; Feuerhahn et al., 2010Feuerhahn S, Iglesias N, Panza A, Porro A, Lingner J. TERRA biogenesis, turnover and implications for function. FEBS letters. 2010; 584(17):3812-18. https://doi.org/10.1016/j.febslet.2010.07.032
https://doi.org/10.1016/j.febslet.2010.0...
; O’Sullivan, Karlseder, 2010O’Sullivan RJ, Karlseder J. Telomers: protecting chromosomes against genome instability. Nat Rev Mol Cell Biol. 2010; 11(3):171–81. https://doi.org/10.1038/nrm2848
https://doi.org/10.1038/nrm2848...
), these sequences play an important role also in the stability of the extra elements in the genome and could explain the presence of B chromosomes in the carrier individuals.

In Characidium sp. aff. C. vidali telomeric probes hybridized with the terminal portion of all chromosomes in the karyotype, including the supernumerary elements. Additionally, interstitial telomeric sequence (ITS) signals are highlighted in several regions of many chromosomes. These marks were previously found in other species of the genus varying only in number of labeled homologous chromosomes (Scacchetti et al., 2015aScacchetti PC, Utsunomia R, Pansonato-Alves JC, Costa-Silva GJ, Oliveira C, Foresti F. Extensive spreading of interstitial telomeric sites on the chromosomes of Characidium (Teleostei, Characiformes). Genetica. 2015a; 143:263–70. https://doi.org/10.1007/s10709-014-9812-3
https://doi.org/10.1007/s10709-014-9812-...
). C-banding revealed that interstitial sites accumulate constitutive heterochromatin, and this could mean that ITSs behave as satellite components, turning possible their spread to more internal chromosomal regions (Pagnozzi et al., 2000Pagnozzi JM, Silva MJJ, Yonenaga-Yassuda Y. Intraspecific variation in the distribution of the interstitial telomeric (TTAGGG)n sequences in Micoureus demerarae (Marsupialia: Didelphidae). Chromosome Res. 2000; 8:585–91. https://doi.org/10.1023/A:1009229806649
https://doi.org/10.1023/A:1009229806649...
, 2003Pagnozzi JM, Ditchfield AD, Yonenaga-Yassuda Y. Mapping the distribution of the interstitial telomeric (TTAGGG)n sequences in eight species of Brazilian marsupials (Didelphidae) by FISH and the correlation with constitutive heterochromatin. Do ITS represent evidence for fusion events in American marsupials? Cytogenet Genome Res. 2003; 98:278–84. https://doi.org/10.1159/000071049
https://doi.org/10.1159/000071049...
; Metcalfe et al., 2004Metcalfe CJ, Eldridge MDB, Johnston PG. Mapping the distribution of the telomeric sequence (T2AG3)n in the 2n = 14 ancestral marsupial complement and in the macropodines (Marsupialia: Macropodidae) by fluorescence in situ hybridization. Chromosome Research. 2004; 12(4):405–14. https://doi.org/10.1023/B:CHRO.0000034133.77878.88
https://doi.org/10.1023/B:CHRO.000003413...
; Scacchetti et al., 2015aScacchetti PC, Utsunomia R, Pansonato-Alves JC, Costa-Silva GJ, Oliveira C, Foresti F. Extensive spreading of interstitial telomeric sites on the chromosomes of Characidium (Teleostei, Characiformes). Genetica. 2015a; 143:263–70. https://doi.org/10.1007/s10709-014-9812-3
https://doi.org/10.1007/s10709-014-9812-...
). This could explain the variation in the number of interstitial sites in the species here studied and in other six species of Characidium analized by Scacchetti et al. (2015a)Scacchetti PC, Utsunomia R, Pansonato-Alves JC, Costa-Silva GJ, Oliveira C, Foresti F. Extensive spreading of interstitial telomeric sites on the chromosomes of Characidium (Teleostei, Characiformes). Genetica. 2015a; 143:263–70. https://doi.org/10.1007/s10709-014-9812-3
https://doi.org/10.1007/s10709-014-9812-...
. An interesting point observed was the occurrence of chromosomes bearing double-ITS marks, not evidenced by C-banding, in addition to single-ITS marks C-banding positive. One can consider the hypothesis of the possible occurrence of a position effect acting in the determination of the negative heterochromatic pattern of regions with double ITS that, when changing from the pericentromeric position to a region along the short arm of the chromosomes would promote changes in these segments previously heterochromatic, modifying their structural behavior. Alternatively, even due to the occurrence of changes in the composition and structure of the sequences in these segments, modifying and distending the heterochromatic components in these blocks, turning them euchromatic.

Many studies try to identify and understand the dispersion process of telomeric sequences in the internal portion of the chromosomes of several organisms (Meyne et al., 1989Meyne J, Ratliff RL, Moyzis RK. Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proc Natl Acad Sci USA. 1989; 86(18):7049–53. https://doi.org/10.1073/pnas.86.18.7049
https://doi.org/10.1073/pnas.86.18.7049...
; Pagnozzi et al., 2000Pagnozzi JM, Silva MJJ, Yonenaga-Yassuda Y. Intraspecific variation in the distribution of the interstitial telomeric (TTAGGG)n sequences in Micoureus demerarae (Marsupialia: Didelphidae). Chromosome Res. 2000; 8:585–91. https://doi.org/10.1023/A:1009229806649
https://doi.org/10.1023/A:1009229806649...
, 2003Pagnozzi JM, Ditchfield AD, Yonenaga-Yassuda Y. Mapping the distribution of the interstitial telomeric (TTAGGG)n sequences in eight species of Brazilian marsupials (Didelphidae) by FISH and the correlation with constitutive heterochromatin. Do ITS represent evidence for fusion events in American marsupials? Cytogenet Genome Res. 2003; 98:278–84. https://doi.org/10.1159/000071049
https://doi.org/10.1159/000071049...
; Bolzán, Bianchi, 2006Bolzán AD, Bianchi MS. Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations. Mutat Res. 2006; 612(3):189–214. https://doi.org/10.1016/j.mrrev.2005.12.003
https://doi.org/10.1016/j.mrrev.2005.12....
; Lin, Yan, 2008Lin WK, Yan J. Endings in the middle: current knowledge of interstitial telomeric sequences. Mutat Res. 2008; 658(1–2 ):95–110. https://doi.org/10.1016/j.mrrev.2007.08.006
https://doi.org/10.1016/j.mrrev.2007.08....
; Ruiz-Herrera et al., 2008Ruiz-Herrera A, Nergadze SG, Santagostino M, Giulotto E. Telomeric repeats far from the ends: mechanisms of origin and role in evolution. Cytogenet Genome Res. 2008; 122(3–4):219–28. https://doi.org/10.1159/000167807
https://doi.org/10.1159/000167807...
; Cioffi et al., 2010Cioffi MB, Kejnovsky E, 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. 2010; 132(4):289–96. https://doi.org/10.1159/000322058
https://doi.org/10.1159/000322058...
; Scacchetti et al., 2015aScacchetti PC, Utsunomia R, Pansonato-Alves JC, Costa-Silva GJ, Oliveira C, Foresti F. Extensive spreading of interstitial telomeric sites on the chromosomes of Characidium (Teleostei, Characiformes). Genetica. 2015a; 143:263–70. https://doi.org/10.1007/s10709-014-9812-3
https://doi.org/10.1007/s10709-014-9812-...
). In general, these sites are interpreted as remnants of chromosomal rearrangements, which can modify the karyotype structure of the taxon (Holmquist, Dancis, 1979Holmquist GP, Dancis B. Telomere replication, kinetochore organizers, and satellite DNA evolution. Proc Natl Acad Sci USA. 1979; 76(9):4566–70. https://doi.org/10.1073/pnas.76.9.4566
https://doi.org/10.1073/pnas.76.9.4566...
; Hastie, Allshire, 1989Hastie ND, Allshire RC. Human telomeres: fusion and interstitial sites. Trends in Genetics. 1989; 5:326-31. https://doi.org/10.1016/0168-9525(89)90137-6
https://doi.org/10.1016/0168-9525(89)901...
; Meyne et al., 1989Meyne J, Ratliff RL, Moyzis RK. Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proc Natl Acad Sci USA. 1989; 86(18):7049–53. https://doi.org/10.1073/pnas.86.18.7049
https://doi.org/10.1073/pnas.86.18.7049...
; Pagnozzi et al., 2003Pagnozzi JM, Ditchfield AD, Yonenaga-Yassuda Y. Mapping the distribution of the interstitial telomeric (TTAGGG)n sequences in eight species of Brazilian marsupials (Didelphidae) by FISH and the correlation with constitutive heterochromatin. Do ITS represent evidence for fusion events in American marsupials? Cytogenet Genome Res. 2003; 98:278–84. https://doi.org/10.1159/000071049
https://doi.org/10.1159/000071049...
; Cioffi et al., 2010Cioffi MB, Kejnovsky E, 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. 2010; 132(4):289–96. https://doi.org/10.1159/000322058
https://doi.org/10.1159/000322058...
). However, there are cases where variation in number and structure of the chromosomes are evident but ITS are not observed, indicating that the sequences (TTAGGG)n may have been eliminated or suffered structural modification throughout the evolution of the genome (Ocalewicz et al., 2013Ocalewicz K, Furgala-Selezniow G, Szmyt M, Lisboa R, Kucinski M, Lejk AM et al. Pericentromeric location of the telomeric DNA sequences on the European grayling chromosomes. Genetica. 2013; 141:409–16. https://doi.org/10.1007/s10709-013-9740-7
https://doi.org/10.1007/s10709-013-9740-...
). In this context, the presence of interstitial telomeric marks in Characidium cannot be interpreted as absolute evidence of structural chromosomal rearrangements, since the genus is known by its conserved karyotype macrostructure. Scacchetti et al. (2015a)Scacchetti PC, Utsunomia R, Pansonato-Alves JC, Costa-Silva GJ, Oliveira C, Foresti F. Extensive spreading of interstitial telomeric sites on the chromosomes of Characidium (Teleostei, Characiformes). Genetica. 2015a; 143:263–70. https://doi.org/10.1007/s10709-014-9812-3
https://doi.org/10.1007/s10709-014-9812-...
proposed that species with ITS belong to a small group of phylogenetically related Characidium species and the spread of this mark within the genomes would be a consequence of ectopic transposition and/or events of recombination. The physical mapping of telomeric sequences here described were similar to those presented by Scacchetti et al. (2015a)Scacchetti PC, Utsunomia R, Pansonato-Alves JC, Costa-Silva GJ, Oliveira C, Foresti F. Extensive spreading of interstitial telomeric sites on the chromosomes of Characidium (Teleostei, Characiformes). Genetica. 2015a; 143:263–70. https://doi.org/10.1007/s10709-014-9812-3
https://doi.org/10.1007/s10709-014-9812-...
, evidencing a possible relationship between the species (MLMO, work in progress).

The results obtained revealed that Characidium sp. aff. C. vidali shares the common and conserved diploid number of the genus, and corroborates the hypothesis that the ecological characteristics of the species with restrict habits to headwaters of small streams favors the occurrence of endemism and speciation by allopatry, a particularly common situation for these fish. It is clear that such factors directly affect the evolution of the karyotype structure of Characidium populations, mostly in the composition of the microstructure of their chromosomes. The mapping of the sequences performed here provide an identification of the species by means of cytomolecular tools, in addition to aggregating information about the evolution of repetitive portions of the genome within Characidium. The findings also reveal that, despite presenting a conserved chromosomal macrostructure, different micro alterations can occur in the genome of the species and provide an important and significative mechanism of diversification and, consequently, for speciation in this group of fish.

ACKNOWLEDGEMENTS

The authors are thankful to the Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP) and Laboratório de Biologia e Genética de Peixes (LBGP) to support. Our gratitude to Bruno F. Melo by pictures taken of the fishes. This study was financed by CAPES (Coordenadoria de Aperfeiçoamento de Ensino Superior). The procedures were carried out in accordance with the National Council for Control of Animal Experimentation (CONCEA) and Ethics Committee on Use of Animals (CEUA) (protocol 971) of the BIOSCIENCE INSTITUTE/UNESP. CO received financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (grants 2018/20610–1, 2016/09204–6, 2014/26508–3) and Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq (proc. 306054/2006–0).

REFERENCES

  • Bolzán AD, Bianchi MS Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations. Mutat Res. 2006; 612(3):189–214. https://doi.org/10.1016/j.mrrev.2005.12.003
    » https://doi.org/10.1016/j.mrrev.2005.12.003
  • Buckup PA Family Crenuchidae (South American Darters). In: Reis RE, Kullander SO, Ferraris CJ Jr., editors. Check List of the Freshwater Fishes of South and Central America. Porto Alegre: Edipucrs; 2003. p.87–95.
  • Buckup PA The Eastern Brazilian Shield. In: Albert JS, Reis RE, editors. Berkeley and Los Angeles: University of California Press; 2011. p.203–10.
  • Buckup PA, Van der Sleen P Family Crenuchidae. In: Van der Sleen P, Albert JS, editors. Princeton: Princeton University Press; 2017. p.142–48.
  • Caramaschi EP Distribuição da ictiofauna de riachos das Bacias do Tietê e do Paranapanema, junto ao divisor de águas (Botucatu, SP). [Dissertação]. São Paulo: Universidade Federal de São Carlos; 1986. PMid:3275190.
  • Centofante L, Bertollo LAC, Moreira-Filho O Comparative cytogenetics among sympatric species of Characidium (Pisces, Characiformes). Diversity analysis with the description of a ZW sex chromosome system and natural triploidy. Caryologia. 2001; 54(3):253–60. https://doi.org/10.1080/00087114.2001.10589233
    » https://doi.org/10.1080/00087114.2001.10589233
  • Centofante L, Bertollo LA, Buckup PA, Moreira-Filho O Chromosomal divergence and maintenance of sympatric Characidium fish species (Crenuchidae, Characidiinae). Hereditas. 2003; 138(3):213–18. https://doi.org/10.1034/j.1601-5223.2003.01714.x
    » https://doi.org/10.1034/j.1601-5223.2003.01714.x
  • Chan SS, Chang S Defending the end zone: studying the players involved in protecting chromosome ends. FEBS letters. 2010; 584(17):3773–78. https://doi.org/10.1016/j.febslet.2010.06.016
    » https://doi.org/10.1016/j.febslet.2010.06.016
  • Cioffi MB, Kejnovsky E, 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. 2010; 132(4):289–96. https://doi.org/10.1159/000322058
    » https://doi.org/10.1159/000322058
  • Cioffi MB, Bertollo LAC Chromosomal Distribution and Evolution of Repetitive DNAs in Fish. In: Garrido R, editor. Repetitive DNA. Genome Dynamics. Basel: Karger; 2012. p.197–221. https://doi.org/10.1159/000337950
    » https://doi.org/10.1159/000337950
  • Colgan DJ, McLauchlan A, Wilson GDF, Livingston SP, Edgecombe GD, Macaranas J et al Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Aust J Zool. 1998; 46(5):419–37. https://doi.org/10.1071/ZO98048
    » https://doi.org/10.1071/ZO98048
  • Cuadrado Á, Jouve N Novel simple sequence repeats (SSRs) detected by ND-FISH in heterochromatin of Drosophila melanogaster BMC Genomics. 2011; 12:205. https://doi.org/10.1186/1471-2164-12-205
    » https://doi.org/10.1186/1471-2164-12-205
  • Dimitri P, Arcà B, Berghella L, Mei E High genetic instability of heterochromatin after transposition of the LINE-like I factor in Drosophila melanogaster Proc Natl Acad Sci USA. 1997; 94(15):8052–57. https://doi.org/10.1073/pnas.94.15.8052
    » https://doi.org/10.1073/pnas.94.15.8052
  • Dover GA Evolution of genetic redundancy for advanced players. Curr Opin Genet Dev. 1993; 3:902–10. https://doi.org/10.1016/0959-437x(93)90012-e
    » https://doi.org/10.1016/0959-437x(93)90012-e
  • Eirín-López JM, González-Romero R, Dryhurst D, Méndez J, Ausió J Long-term evolution of histone families: old notions and new insights into their mechanisms of diversification across eukaryotes. In: Pontarotti P, editor. Evolutionary Biology.Berlin, Heidelberg: Springer; 2009. p.139-62. https://doi.org/10.1007/978-3-642-00952-5_8
    » https://doi.org/10.1007/978-3-642-00952-5_8
  • Feuerhahn S, Iglesias N, Panza A, Porro A, Lingner J TERRA biogenesis, turnover and implications for function. FEBS letters. 2010; 584(17):3812-18. https://doi.org/10.1016/j.febslet.2010.07.032
    » https://doi.org/10.1016/j.febslet.2010.07.032
  • Foresti F, Almeida-Toledo LF, Toledo- ASF Polymorphic nature of nucleolus organizer regions in fishes. Cytogenet Cell Genet. 1981; 31(3):137–44. https://doi.org/10.1159/000131639
    » https://doi.org/10.1159/000131639
  • Guerra M FISH: conceitos e aplicações na citogenética. Ribeirão Preto: Sociedade Brasileira de Genética; 2004.
  • Hancock JM Simple sequences and the expanding genome. Bioessays. 1996; 18(5):421–25. https://doi.org/10.1002/bies.950180512
    » https://doi.org/10.1002/bies.950180512
  • Hashimoto DT, Ferguson-Smith MA, Rens W, Foresti F, Porto-Foresti F Chromosome mapping of H1 histone and 5S rRNA gene clusters in three species of Astyanax (Teleostei, Characiformes). Cytogenet Genome Res. 2011; 134(1):64–71. https://doi.org/10.1159/000323512
    » https://doi.org/10.1159/000323512
  • Hashimoto DT, Ferguson-Smith MA, Rens W, Prado FD, Foresti F, Porto-Foresti F Cytogenetic mapping of H1 histone and ribosomal RNA genes in hybrids between catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum Cytogenet Genome Res. 2013; 139(2):102–06. https://doi.org/10.1159/000345299
    » https://doi.org/10.1159/000345299
  • Hastie ND, Allshire RC Human telomeres: fusion and interstitial sites. Trends in Genetics. 1989; 5:326-31. https://doi.org/10.1016/0168-9525(89)90137-6
    » https://doi.org/10.1016/0168-9525(89)90137-6
  • Holmquist GP, Dancis B Telomere replication, kinetochore organizers, and satellite DNA evolution. Proc Natl Acad Sci USA. 1979; 76(9):4566–70. https://doi.org/10.1073/pnas.76.9.4566
    » https://doi.org/10.1073/pnas.76.9.4566
  • Ijdo JW, Wells RA, Baldini A, Reeders ST Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR. Nucleic Acids Res. 1991; 19(17):4780. https://doi.org/10.1093/nar/19.17.4780
    » https://doi.org/10.1093/nar/19.17.4780
  • Kubat Z, Hobza R, Vyskot B, Kejnovsky E Microsatellite accumulation on the Y chromosome in Silene latifolia Genome. 2008; 51(5):350–56. https://doi.org/10.1139/G08-024
    » https://doi.org/10.1139/G08-024
  • de Lange T Shelterin: the protein complex that shapes and safeguards human telomeres. Genes & development. 2005; 19(18):2100–10. http://doi.org/10.1101/gad.1346005
    » http://doi.org/10.1101/gad.1346005
  • Leitão RP, Buckup PA A new Species of Characidium (Characiformes: Crenuchidae) from Coastal Basins of Serra do Mar, Southeastern Brazil. Copeia. 2014; 2014(1):14–22. http://doi.org/10.1643/CI-12-137
    » http://doi.org/10.1643/CI-12-137
  • Levan A, Fredga K, Sandberg AA Nomenclature for centromeric position on chromosomes. Hereditas. 1964; 52(2):201–20. https://doi.org/10.1111/j.1601-5223.1964.tb01953.x
    » https://doi.org/10.1111/j.1601-5223.1964.tb01953.x
  • Lim JK, Simmons MJ Gross chromosome rearrangements mediated by transposable elements in Drosophila melanogaster Bioessays. 1994; 16(4):269–75. https://doi.org/10.1002/bies.950160410
    » https://doi.org/10.1002/bies.950160410
  • Lin WK, Yan J Endings in the middle: current knowledge of interstitial telomeric sequences. Mutat Res. 2008; 658(1–2 ):95–110. https://doi.org/10.1016/j.mrrev.2007.08.006
    » https://doi.org/10.1016/j.mrrev.2007.08.006
  • Lohe AR, Hilliker AJ, Roberts PA Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster Genetics. 1993; 134(4): 1149–74.
  • López-Flores I, Garrido-Ramos MA The repetitive DNA content in eukaryotic genomes. In: Garrido-Ramos MA, editors. Repetitive DNA. Genome dynamics. Karger, Basel; 2012. p.1–28.
  • Luke B, Lingner J TERRA: telomeric repeat‐containing RNA. The EMBO journal. 2009; 28(17):2503–10. https://doi.org/10.1038/emboj.2009.166
    » https://doi.org/10.1038/emboj.2009.166
  • Machado TC, Pansonato-Alves JC, Pucci MB, Nogaroto V, Almeida MC, Oliveira C et al Chromosomal painting and ZW sex chromosomes differentiation in Characidium (Characiformes, Crenuchidae). BMC Genet. 2011; 12(65). https://doi.org/10.1186/1471-2156-12-65
    » https://doi.org/10.1186/1471-2156-12-65
  • Maistro EL, Prieto-Mata E, Oliveira C, Foresti F Unusual occurrence of a ZZ/ZW sex-chromosome system and supernumerary chromosomes in Characidium cf. fasciatum (Pisces, Characiformes, Characidiinae). Genetica. 1998; 104(1):1–7. https://doi.org/10.1023/A:1003242020259
    » https://doi.org/10.1023/A:1003242020259
  • Maistro EL, de Jesus CM, Oliveira C, Moreira-Filho O, Foresti F Cytogenetic analysis of A-, B-chromosomes and ZZ/ZW sex chromosomes of Characidium gomesi (Teleostei, Characiformes, Crenuchidae). Cytologia. 2004; 69(2):181–86. https://doi.org/10.1508/cytologia.69.181
    » https://doi.org/10.1508/cytologia.69.181
  • McMurray CT Mechanisms of DNA expansion. Chromosoma. 1995; 104:2–13. https://doi.org/10.1007/BF00352220
    » https://doi.org/10.1007/BF00352220
  • Metcalfe CJ, Eldridge MDB, Johnston PG Mapping the distribution of the telomeric sequence (T2AG3)n in the 2n = 14 ancestral marsupial complement and in the macropodines (Marsupialia: Macropodidae) by fluorescence in situ hybridization. Chromosome Research. 2004; 12(4):405–14. https://doi.org/10.1023/B:CHRO.0000034133.77878.88
    » https://doi.org/10.1023/B:CHRO.0000034133.77878.88
  • Meyne J, Ratliff RL, Moyzis RK Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proc Natl Acad Sci USA. 1989; 86(18):7049–53. https://doi.org/10.1073/pnas.86.18.7049
    » https://doi.org/10.1073/pnas.86.18.7049
  • Noleto RB, Amorim AP, Vicari M R, Artoni RF, Cestari MM An unusual ZZ/ZW sex chromosome system in Characidium fishes (Crenuchidae, Characiformes) with the presence of rDNA sites. J Fish Biol. 2009; 75(2):448–53. https://doi.org/10.1111/j.1095-8649.2009.02342.x
    » https://doi.org/10.1111/j.1095-8649.2009.02342.x
  • Ocalewicz K, Furgala-Selezniow G, Szmyt M, Lisboa R, Kucinski M, Lejk AM et al Pericentromeric location of the telomeric DNA sequences on the European grayling chromosomes. Genetica. 2013; 141:409–16. https://doi.org/10.1007/s10709-013-9740-7
    » https://doi.org/10.1007/s10709-013-9740-7
  • O’Sullivan RJ, Karlseder J Telomers: protecting chromosomes against genome instability. Nat Rev Mol Cell Biol. 2010; 11(3):171–81. https://doi.org/10.1038/nrm2848
    » https://doi.org/10.1038/nrm2848
  • Pagnozzi JM, Silva MJJ, Yonenaga-Yassuda Y Intraspecific variation in the distribution of the interstitial telomeric (TTAGGG)n sequences in Micoureus demerarae (Marsupialia: Didelphidae). Chromosome Res. 2000; 8:585–91. https://doi.org/10.1023/A:1009229806649
    » https://doi.org/10.1023/A:1009229806649
  • Pagnozzi JM, Ditchfield AD, Yonenaga-Yassuda Y Mapping the distribution of the interstitial telomeric (TTAGGG)n sequences in eight species of Brazilian marsupials (Didelphidae) by FISH and the correlation with constitutive heterochromatin. Do ITS represent evidence for fusion events in American marsupials? Cytogenet Genome Res. 2003; 98:278–84. https://doi.org/10.1159/000071049
    » https://doi.org/10.1159/000071049
  • Palm W, de Lange T How shelterin protects mammalian telomeres. Annu Rev Genet. 2008; 42:301–34. https://doi.org/10.1146/annurev.genet.41.110306.130350
    » https://doi.org/10.1146/annurev.genet.41.110306.130350
  • Pansonato-Alves JC, Paiva LRS, Oliveira C, Foresti F Interspecific chromosomal divergences in the genus Characidium (Teleostei: Characiformes: Crenuchidae). Neotrop Ichthyol. 2010; 8(1):77–86. https://doi.org/10.1590/S1679-62252010000100010
    » https://doi.org/10.1590/S1679-62252010000100010
  • Pansonato-Alves JC, Oliveira C, Foresti F Karyotypic conservatism in samples of Characidium cf. zebra (Teleostei, Characiformes, Crenuchidae): Physical mapping of ribosomal genes and natural triploidy. Genet Mol Biol. 2011a; 34(2):208–13. https://doi.org/10.1590/S1415-47572011005000005
    » https://doi.org/10.1590/S1415-47572011005000005
  • Pansonato-Alves JC, Vicari MR, Oliveira C, Foresti F Chromosomal diversification in populations of Characidium cf. gomesi (Teleostei, Crenuchidae). J Fish Biol. 2011b; 78(1):183–94. https://doi.org/10.1111/j.1095-8649.2010.02847.x
    » https://doi.org/10.1111/j.1095-8649.2010.02847.x
  • Pansonato-Alves JC, Serrano EA, Utsunomia R, Scacchetti PC, Oliveira C, Foresti F Mapping five repetitive DNA classes in sympatric species of Hypostomus (Teleostei: Siluriformes: Loricariidae): analysis of chromosomal variability. Rev Fish Biol Fisher. 2013; 23:477–89. https://doi.org/10.1007/s11160-013-9303-0
    » https://doi.org/10.1007/s11160-013-9303-0
  • Pansonato-Alves JC, Serrano ÉA, Utsunomia R, Camacho JP, Costa Silva GJ, Vicari MR et al Single origin of sex chromosomes and multiple origins of B chromosomes in fish genus Characidium PLoS ONE. 2014; 9(9):e107169. https://doi.org/10.1371/journal.pone.0107169
    » https://doi.org/10.1371/journal.pone.0107169
  • Pathak D, Ali S Repetitive DNA: A tool to explore animal genomes/ transcriptomes. In: Germana M, Petrera F editors. Functional genomics. InTech, Published; 2012. p.155–80. https://doi.org/10.5772/48259
    » https://doi.org/10.5772/48259
  • Pineau P, Henry M, Suspène R, Marchio A, Dettai A, Debruyne R et al A universal primer set for PCR amplification of nuclear histone H4 genes from all animal species. Mol Biol Evol. 2005; 22(3):582–88. https://doi.org/10.1093/molbev/msi053
    » https://doi.org/10.1093/molbev/msi053
  • Pinkel D, Straume T, Gray JW Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA. 1986; 83(9):2934–38. https://doi.org/10.1073/pnas.83.9.2934
    » https://doi.org/10.1073/pnas.83.9.2934
  • Poltronieri J, Marquioni V, Bertollo LAC, Kejnovsky E, Molina WF, Liehr T, Cioffi MB Comparative chromosomal mapping of microsatellites in Leporinus species (Characiformes, Anostomidae): unequal accumulation on the W chromosomes. Cytogenet Genome Res. 2014; 142:40–45. https://doi.org/10.1159/000355908
    » https://doi.org/10.1159/000355908
  • Pucci MB, Nogaroto V, Moreira-Filho O, Vicari MR Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes. Genet Mol Biol. 2018; 41(3):585–92. http://dx.doi.org/10.1590/1678-4685-GMB-2017-0121
    » http://dx.doi.org/10.1590/1678-4685-GMB-2017-0121
  • Raskina O, Barber JC, Nevo E, Belyayev A Repetitive DNA and chromosomal rearrangements: speciation-related events in plant genomes. Cytogenet Genome Res. 2008; 120:351–57. https://doi.org/10.1159/000121084
    » https://doi.org/10.1159/000121084
  • Ruiz-Herrera A, Nergadze SG, Santagostino M, Giulotto E Telomeric repeats far from the ends: mechanisms of origin and role in evolution. Cytogenet Genome Res. 2008; 122(3–4):219–28. https://doi.org/10.1159/000167807
    » https://doi.org/10.1159/000167807
  • Ruiz-Ruano FJ, Cuadrado Á, Montiel EE, Camacho JPM, Lopez-Leon MD Next generation sequencing and FISH reveal uneven and nonrandom microsatellite distribution in two grasshopper genomes. Chromosoma. 2015; 124:221–34. https://doi.org/10.1007/s00412-014-0492-7
    » https://doi.org/10.1007/s00412-014-0492-7
  • Scacchetti PC, Utsunomia R, Pansonato-Alves JC, Costa-Silva GJ, Oliveira C, Foresti F Extensive spreading of interstitial telomeric sites on the chromosomes of Characidium (Teleostei, Characiformes). Genetica. 2015a; 143:263–70. https://doi.org/10.1007/s10709-014-9812-3
    » https://doi.org/10.1007/s10709-014-9812-3
  • Scacchetti PC, Utsunomia R, Pansonato JC, Vicari MR, Artoni RF, Oliveira C et al Chromosomal mapping of repetitive DNAs in Characidium (Teleostei, Characiformes): genomic organization and the diversification of ZW sex chromosomes. Cytogenet Genome Res. 2015b; 146(2):163–46. https://doi.org/10.1159/000437165
    » https://doi.org/10.1159/000437165
  • Scacchetti PC, Utsunomia R, Pansonato‐Alves JC, Costa‐Silva GJ, Vicari MR, Artoni RF et al Repetitive DNA sequences and evolution of ZZ/ZW sex chromosomes in Characidium (Teleostei: Characiformes). PloS ONE. 2015c; 10(9):e0137231. https://doi.org/10.1371/journal.pone.0137231
    » https://doi.org/10.1371/journal.pone.0137231
  • Serrano ÉA, Utsunomia R, Scudeller PS, Oliveira C, Foresti F Origin of B chromosomes in Characidium alipioi (Characiformes, Crenuchidae) and its relationship with supernumerary chromosomes in other Characidium species. Comp Cytogenet. 2017; 11(1):81–95. https://doi.org/10.3897/CompCytogen.v11i1.10886
    » https://doi.org/10.3897/CompCytogen.v11i1.10886
  • Serrano-Freitas ÉA, Melo BF, Freitas-Souza D, Oliveira MLM, Utsunomia R, Oliveira C et al Species delimitation in Neotropical fishes of the genus Characidium (Teleostei, Characiformes). Zool Scr. 2019a; 48(1):69–80. https://doi.org/10.1111/zsc.12318
    » https://doi.org/10.1111/zsc.12318
  • Serrano-Freitas ÉA, Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Araya-Jaime C, Oliveira C et al Satellite DNA content of B chromosomes in the characid fish Characidium gomesi supports their origin from sex chromosomes. Mol Genet Genomics. 2019b; 295:195–207. https://doi.org/10.1007/s00438-019-01615-2
    » https://doi.org/10.1007/s00438-019-01615-2
  • Silva DMZA, Pansonato-Alves JC, Utsunomia R, Daniel SN, Hashimoto DT, Oliveira C et al Chromosomal organization of repetitive DNA sequences in Astyanax bockmanni (Teleostei, Characiformes): Dispersive location, association and co-localization in the genome. Genetica. 2013; 141:329–36. https://doi.org/10.1007/s10709-013-9732-7
    » https://doi.org/10.1007/s10709-013-9732-7
  • Sumner AT Chromosomes: Organization and Function. London: Blackwell Publishing Company, 2003.
  • Terencio ML, Schneider CH, Gross MC, Vicari MR, Farias IP, Passos KB et al Evolutionary dynamics of repetitive DNA in Semaprochilodus (Characiformes, Prochilodontidae): a fish model for sex chromosome differentiation. Sex Dev. 2013; 7:325–33. https://doi.org/10.1159/000356691
    » https://doi.org/10.1159/000356691
  • Vicari MR, Artoni RF, Moreira-Filho O, Bertollo LAC Diversification of a ZZ/ZW sex chromosome system in Characidium fish (Crenuchidae, Characiformes). Genetica. 2008; 134:311. https://doi.org/10.1007/s10709-007-9238-2
    » https://doi.org/10.1007/s10709-007-9238-2
  • Zakian VA Telomeres: beginning to understand the end. Science. 1995; 270(5242):1601–1607. https://doi.org/10.1126/science.270.5242.1601
    » https://doi.org/10.1126/science.270.5242.1601
  • Ziemniczak K, Traldi JB, Nogaroto V, Almeida MC, Artoni RF, Moreira-Filho O et al In situ localization of (GATA)n and (TTAGGG)n repeated DNAs and W sex chromosome differentiation in Parodontidae (Actinopterygii: Characiformes). Cytogenet Genome Res. 2014; 144:325–32. https://doi.org/10.1159/000370297
    » https://doi.org/10.1159/000370297

ADDITIONAL NOTES

  • HOW TO CITE THIS ARTICLE

    Oliveira MLM, Paim FG, Freitas EAS, Oliveira C, Foresti F. Cytomolecular investigations using repetitive DNA probes contribute to the identification and characterization of Characidium sp. aff. C. vidali (Teleostei: Characiformes). Neotrop Ichthyol. 2021; 19(2):e200045. https://doi.org/10.1590/1982-0224-2020-0045

Publication Dates

  • Publication in this collection
    11 June 2021
  • Date of issue
    2021

History

  • Received
    1 July 2020
  • Accepted
    3 Mar 2021
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E-mail: neoichth@nupelia.uem.br