fishes are the largest group of vertebrates , with a wide variety of adaptive responses to diversified aquatic habitats and ecological limitations . also with respect to sex determination , unlike the situation in most vertebrates , where the male and female sexes are represented by two different individuals ( gonochorism ) , hundreds of fish species are known to be hermaphroditic ( devlin and nagahama , 2002 ; schartl , 2004 ) . sex chromosomes are fundamental in many vertebrate species for the development of either a male or a female ( livernois et al . , 2012 ) . few species of fish are carriers of heteromorphic chromosomal systems when compared to the enormous taxa diversity ( oliveira et al . , 2007 ) . however , fishes have a remarkable variety of sex determination chromosome systems ( xy , x0 , x1x2y , xy1y2 , zw , z0 , zw1w2 , z1z1z2z2/z1z2 , w1w2 , wxz ) ( moreira - filho et al . , 1993 ; devlin and nagahama , 2002 ; oliveira et al . , 2008 ) , in which the zz / zw simple system with female heterogamety is the most frequent one among neotropical fishes ( artoni and bertollo , 2002 ; bellafronte et al . , 2011 ; machado et al . 2011 ) . the family parodontidae occurs throughout south america and its species are classified in three genera : parodon , apareiodon and saccodon ( pavanelli , 2003 ) . generally they are robust fish , with strong pectoral , ventral and caudal fins , a fusiform body , and a dorsal profile which is more arched than the ventral one ( travassos , 1957 ) . parodontidae taxonomy is controversial because the family members lack diagnostic morphological traits that are sufficiently reliable for accurate phylogenetic analysis ( pavanelli and britski , 2003 ; ingenito lfs , 2008 , ph.d thesis , universidade federal do rio de janeiro , brazil ) . ingenito argued that existing morphological phylogenetic evidence for the genus apareiodon is insufficient to support its maintenance , and that apareiodon should be regarded as a junior synonym of parodon . cytogenetic analyses of both parodon and apareiodon revealed a conserved diploid number of 54 chromosomes , but with remarkable heterogeneity in the distribution of heterochromatin , nor activity , number and location of 18s rdna , 5s rdna and satellite dna familiy sites , and morphologically differentiated sex chromosome systems ( for a review see bellafronte et al . , 2011 ) . from the parodontidae species that have been cytogenetically analyzed , six have sex chromosomes : zz / zw1w2 for apareiodon affinis steindachner , 1879 , and zz / zw for parodon hilarii reinhardt , 1867 , parodon moreirai ingenito & buckup , 2005 , apareiodon vladii pavanelli , 2006 , apareiodon sp . and apareiodon ibitiensis campos , 1944 ( moreira - filho et al . , 1980 , 1993 ; , 2002 ; vicente et al . , 2003 ; rosa et al . , 2006 ; vicari et al . 2009 ) . in parodontidae , satellite dna has great importance for the understanding of karyotype differentiation . so far , two satellite dnas have been described for this fish group : pph2004 ( vicente et al . , 2003 ) and wap ( schemberger et al . , 2011 ) . the physical mapping of the pph2004 sequence showed that this satellite dna is present on chromosomes z and w ( also on autosomal chromosomes ) ; however , it is not a part of the heterochromatic amplified region of the w chromosome heteromorphism ( bellafronte et al . , 2011 ) . the wap satellite dna probe is capable of detecting the amplification region and the chromosome w heteromorphism , corroborating the common origin of this system in parodontidae , apart from homologies to other chromosome sites ( schemberger et al . , 2011 ) . the integrated analysis of chromosomal markers has also allowed to deduce the chromosomal differentiation of the family , which is organized in : ( i ) species without morphologically differentiated sex chromosomes ( apareiodon piracicabae eigenmann , 1907 and apareiodon vittatus garavello , 1977 ) , ( ii ) species with differentiated sex chromosomes and without satellite dna pph2004 ( a. ibitiensis , apareiodon sp . , a. vladii ) , and ( iii ) species with proto sex chromosomes and/or heteromorphic sex chromosome systems and presence of satellite dna pph2004 ( a. affinis , p. hilarii , p. moreirai , parodon nasus kner , 1859 and parodon pongoensis allen , 1942 ) ( schemberger et al . , 2011 ) . based on these data , the present study aimed at characterizing the species apareiodon hasemani eigenmann , 1916 ( from the so francisco river basin , brazil ) with respect to chromosome number , morphology , banding , sex system , presence or absence of certain molecular cytogenetic markers , as well as to establish karyoevolutionary relationships based in other cytogenetic studies performed in the family parodontidae . chromosome studies were carried out in 18 specimens ( 7 males and 11 females ) of a. hasemani , collected in the main channel of the so francisco river , in pirapora city , state of minas gerais , brazil ( 172117.47 s and 445718.45 w ) . the analyzed samples were deposited in the ncleo de pesquisas em limnologia , ictiologia e aquicultura ( nupelia ) , universidade estadual de maring , brazil , with voucher number nup11512 . samples were collected in compliance with the ethics committee on animal experimentation ( process ceua 07/2011 ) of the universidade estadual de ponta grossa , brazil ) and with authorization to collect the biological material ( brazilian federal license : ministrio do meio ambiente / instituto brasileiro do meio ambiente e dos recursos renovveis mma / ibama / sisbio number 10538 - 1 ) and the instituto estadual de florestas de minas gerais . mitotic chromosomes were obtained from the anterior kidney , according to the methodology described by bertollo et al . the heterochromatin was detected using the methodology of c - banding ( sumner , 1972 ) . c - banded chromosomes were stained with propidium iodide ( 50 g ml ) according to lui et al . the nucleolar organizer regions were detected using the silver nitrate method ( ag - nor ) described by howell and black ( 1980 ) . fluorescence in situ hybridization ( fish ) experiments were performed in the parodontidae specimens using probes for 18s rdna ( hatanaka and galetti jr , 2004 ) , 5s rdna ( martins and galetti jr , 1999 ) , pph2004 satellite dna ( vicente et al . , 2003 ) and wap satellite dna ( schemberger et al . , 2011 ) . the 18s rdna , 5s rdna and pph2004 cloned probes were labeled with biotin or digoxigenin via pcr , using plasmid vector primers ( t7 promoter and m13 reverse ) . the pcr amplification was done with : 20 ng of template dna , 1x reaction buffer , 2 mm mgcl2 , 40 m datp , dgtp and dctp , 28 m dttp , 12 m biotin 16-dutp , or digoxigenin 11 dutp ( roche applied science ) , 0.3 m of each primer and 1 u taq dna polymerase ( invitrogen ) . the wap probe was labeled via degenerate oligonucleotide - primed polymerase chain reaction ( dop - pcr ) , using 11-dutp - digoxigenin ( roche applied science ) . this pcr amplification was done with : 100 ng of template dna , 1x reaction buffer , 2 mm mgcl2 , 40 m datp , dgtp and dctp , 28 m dttp , 12 m 11-dutp - digoxigenin , 2 m dop primer , and 1 u taq dna polymerase ( invitrogen ) . the fish procedure was performed under high stringency conditions ( 2.5 ng/l probe , 50% formamide , 2x ssc , 10% dextran sulfate ) following the methodology described by pinkel et al . the signal was detected using the anti - streptavidin antibody conjugated to alexa fluor 488 ( invitrogen ) and an anti - digoxigenin antibody conjugated to rhodamine ( roche applied science ) . the chromosomes were counterstained with dapi ( 0.2 g / ml ) , covered with vectashield mounting medium ( vector ) and analyzed with the aid of an olympus bx41 epifluorescence microscope equipped with a dp71 digital image capture system ( olympus ) . ( 1964 ) and classified as metacentric ( m ) , submetacentric ( sm ) , or subtelocentric ( st ) . the species a. hasemani showed 2n = 54 meta - submetacentric chromosomes , with a fundamental number equal to 108 for both sexes ( figure 1 ) . however , a heteromorphism in size and morphology was observed for chromosome pair 4 , restricted to females , which had a medium - sized submetacentric chromosome ( z ) and a large metacentric chromosome ( w ) ( figure 1 ) . the c - banding technique revealed centromeric heterochromatin regions in some chromosomes , as well as interstitial and terminal bands ( figure 1b and 1d ) . the zz pair showed centromeric and terminal c - bands on both chromosome arms . in all metaphases of females , the w chromosome had similar centromeric and terminal heterochromatin bands on the long arm , when compared to the z chromosome . the heterochromatin segments , however , occupied almost the entire short arm of the w chromosome , being twice as large as in the z chromosome ( figure 1b , d ) . the nucleolar organizer regions stained with silver ( ag - nors ) showed terminal signals on the long arm of one chromosome pair , pair 11 , which was subtelocentric ( figure 2 , box ) , while fish with 18s rdna probes showed up to five terminal signals on the long arm of pairs 11 ( larger and more evident marks ) , 7 and 22 ( smaller in size ) , and all these signals generally coinciding with gc - rich regions ( figure 2a ) . fish with 5s rdna probes showed only one marked pair , located in the interstitial region of the long arm of chromosome pair 14 ( figure 2a ) . apareiodon hansemani did not show pph2004 satellite dna sites ( data not shown ) , but wap satellite dna sites were found in the terminal regions of some chromosomes of the karyotype ( figure 2b ) . the z chromosome showed the satellite dna wap in the terminal region of the short and long arms and in the proximal region of the short arm ( figure 2bb ) . in turn , the w chromosome also showed a wap amplification from the proximal region of the short arm ( figure 2b ) . karyotype analyses of apareiodon hasemani ( figure 1 ) show a diploid number of 54 chromosomes , similarly to other species of parodontidae , except for the population of a. affinis from the upper paran river basin ( moreira - filho et al . , 1980 ) , which has 2n = 54/55 chromosomes . the chromosome morphology of a. hasemani consists mainly of meta - submetacentric chromosomes , a characteristic shared with a. affinis ( moreira - filho et al . , 1980 ) , p. hilarii ( moreira - filho et al . , 1993 ) and p. moreirai ( centofante et al . , 2002 ) . in addition to meta - submetacentric chromosomes , other species also have subtelocentric chromosomes ( moreira - filho et al . , 1985 ; jesus and moreira - filho 2000a , b ; bellafronte et al . , 2005 ; vicari et al . , 2006 ; rosa et al . , populations of a. affinis from the lower paran and paraguay river basins showed a structural polymorphism with the presence of acrocentric chromosomes , probably caused by pericentric inversions ( jesus et al . the location of the major nors sites of a. hasemani in the terminal region of the long arm of a subtelocentric chromosome pair , revealed by silver nitrate staining and the 18s rdna probes ( figure 2a , box ) , is a character shared by other species of the genus apareiodon and by p. nasus ( bellafronte et al . , 2005 ; vicari et al . , 2006 ; rosa et al . , 2006 ; bellafronte et al . , 2009 , 2011 ) . in populations of a. affinis in the lower paran and paraguay basins , additional sites were also found in a. hasemani , in a characteristic shared with a. ibitiensis and a. vittatus .. dispersal events may have originated the additional 18s rdna sites in these species , since they are found in more than one chromosome pair ( jesus and moreira - filho , 2000a ; bellafronte et al . , 2009 , 2011 , this study ) . considering that nors are generally associated with terminal heterochromatin , chromosome rearrangements and transpositions become more likely , which may result in dispersion in the genome ( moreira filho et al . , 1984 ) . fish with 5s rdna probes in parodontidae revealed that these cistrons are often located on the short arm of one of the submetacentric chromosome pairs ( centofante et al . additional sites may , however , occur in different locations in a. vladii , p. hilarii , p. pongoensis and p. nasus , which show a case of synteny with the 18s rdna gene ( vicente et al . , 2001 ; apareiodon hasemani and most species of parodontidae show only one submetacentric pair carrying 5s rdna sites ; however , the cistron is located in the subterminal region of the long arm . it is likely that this pattern originated from a pericentric inversion , since in other parodontidae species and in the outgroup anostomidae the 5s rdna sites are located on the short arm ( bellafronte et al . , 2011 ) . 2004 ) , where a. intermedius shows all 5s rdna sites located on acrocentric chromosomes , while a. guiton has a marked subtelocentric pair besides the aforementioned acrocentric chromosomes , probably originated by a pericentric inversion . apareiodon hasemani shares a zz / zw heteromorphic sex chromosome system with p. hilarii , p. moreirai , a. vladii , a. ibitiensis and apareiodon sp . the distribution of constitutive heterochromatin in a. hasemani ( figure 1b , d ) is characterized by centromeric bands in some chromosome pairs , large interstitial and terminal blocks , and blocks adjacent to the nors , which have also been found in several other species of parodontidae ( moreira - filho et al . , 1984 ; jesus and moreira - filho 2000a , b ; vicente et al . , 2001 , 2003 ; centofante et al . , 2002 ; bellafronte et al . , 2005 ; rosa et al . , 2006 ; vicari et al . , moreover , the z sex chromosome exhibited a correspondence of proximal and terminal bands on short arms with those of other parodontids bearing sex chromosomes ( bellafronte et al . ( 2011 ) inferred that the z and w chromosomes of parodontidae were initially a homologous pair , and that heterocromatinization through the accumulation of wap sequences was a decisive step in the differentiation of the w chromosome . on the other hand , the terminal heterochromatic block in the long arm of the z and w chromosomes of a. hasemani ( figure 2b ) led to a size increase of the long arm of these chromosomes , in relation to other species in the group . thus , with the increase in the long arm of the z chromosome due to this terminal heterochromatin , the heterochromatic differentiation of the w chromosome in the short arm region did not lead to a sufficient size increase to exceed the total length of the long arm ( figure 3 ) , a common state observed among the other parodontid carriers of heteromorphic sex chromosomes . in this proposed scenario , not only would the w chromosome change in shape and size , but the z chromosome would also have increased in size in relation to a possible ancestral condition . this mechanism is in agreement with the model for the evolution of heteromorphic sex chromosomes ( muller , 1964 ; charlesworth , 1978 ; green , 1990 ) . during the evolution of the heteromorphic sex chromosomes xy or zw , there is a suppression of meiotic recombination between a pair of homomorphic chromosomes ( homologues ) , which progressively accumulate sex determining genes , followed by structural chromosomal rearrangements such as deletions , insertions , accumulation of transposable elements , and expansion of repetitive sequences ( schartl , 2004 ; kasahara , 2009 ; livernois et al . , 2012 ) . in recent years , repetitive dna sequences have been widely studied in fishes , providing important information on the differentiation and evolution of sex chromosomes . these studies have shown that the differentiation of sex chromosomes is frequently associated with the accumulation of such dna sequences on chromosomes ( vicente et al . fish with the sequence pph2004 in a. hasemani showed that this satellite dna is not present in the karyotype or in species with heteromorphic zw sex chromosomes : a. ibitiensis , apareiodon sp . and a. vladii ( bellafronte et al . , 2011 ) . furthermore , the wap satellite dna probe ( schemberger et al . , 2011 ) detected the w chromosome amplification region of a. hasemani , suggesting that this system is common to other parodontid carriers of sex chromosomes . in the family parodontidae , all species studied so far have positive sites for this marker , suggesting a common origin of this satellite dna and therefore of the sex chromosome systems ( schemberger et al . , 2011 ) . the simple zz / zw sex chromosome systems may have originated from a paracentric inversion of a wap terminal site to the proximal region of the short arm of a metacentric chromosome pair , with the subsequent amplification of this sequence leading to differentiation of the w sex chromosome in most species . through the use of chromosome markers , schemberger et al . ( 2011 ) demonstrated the existence of closely related species groups : ( i ) those without heteromorphic sex chromosomes ( a. piracicabae and a. vittatus ) ; ( ii ) those with proto sex chromosomes and the presence of satellite dna pph2004 ( p. nasus and p. pongoensis ) ; ( iii ) those with a heteromorphic sex chromosome system ( a. ibitiensis and a. vladii ) ; ( iv ) those with a heteromorphic sex chromosome system and the presence of satellite dna pph2004 ; and ( v ) a species with a multiple sex chromosome system and satellite dna pph2004 ( a. affinis ) . apareiodon hasemani shows chromosomal markers similar to those found in a. ibitiensis , apareiodon sp . and a. vladii ( bellafronte et al . , 2011 ; schemberger et al . , 2011 ) ; however , the location of the 5s rdna can be considered derived in a. hasemani . the main brazilian rivers are separated by barriers that prevent the dispersal of species and populations , favoring the occurrence of events that cause the isolation of groups ( weitzman et al . , 1988 ) . by such conditions , unique karyotypes and distinct sex chromosomes systems in fish may have become fixed independently in different species of a genus , promoting diversification and contributing to the speciation process ( centofante et al . , 2001 ) . although allopatry may have contributed to the origin of sex chromosome systems , moreira - filho et al . ( 1985 ) reported the occurrence of four species of parodontidae in sympatry and syntopy , so the different sex chromosomes systems in some species would play an important role in their isolation . this study found that the repetitive dna in parodontidae seemed to play an important role in the chromosome diversification of the studied species , as in a. hasemani . furthermore , a. hasemani presented derived chromosomal regions with respect to 5s rdna , contributing to the genetic isolation of the population and differentiation of strains .