siluriformes is an extremely large fish order with a wide distribution throughout tropical regions ( ferraris , 2007 ) . the number of known species in this region is about 3,100 , but may be considerably higher ( reis et al . , 2003 ; nelson , 2006 ; ferraris , 2007 ) . the largest family within the siluriformes is the loricariidae , with approximately 700 species distributed in eight subfamilies ( reis et al . , 2006 ; ferraris , 2007 ; chiachio et al . , 2008 ) loricariids occur in several habitats , from lagoons and swamps to rapids in sloping streams or rivers with rocky bottoms , at altitudes up to 3000 m. in large water channels , these fish are usually found on rocky bottoms facing into strong water currents ( garavello and garavello , 2004 ) or along the margins where the current is moderate ( burgess , 1989 ) . according to suzuki et al . ( 2000 ) , these fish exhibit a large diversity of adaptive strategies , with many species showing nest defense , parental care of eggs , brooder larvae behavior , and mouths adapted for feeding on algae and detritus . although the loricariidae is one of the largest fish families in the world , the number of cytogenetically studied species is still very low . there is marked inter - specific diversification in the diploid number , which ranges from 2n = 36 chromosomes in rineloricaria latirostris ( giuliano - caetano l , doctoral thesis , universidade federal de so carlos , 1998 ) to 2n = 84 in hypostomus sp . cytogenetically , the hypostominae is the best studied group within the loricariidae , but it is also the most complex , with the diploid number varying from 2n = 38 in ancistrus sp . a very interesting feature in the hypostominae ( particularly within hypostomini ) is the inverse relationship between the diploid number and the number of chromosomes with two arms , which suggests the occurrence of several events of centric fusion / fission ( robertsonian rearrangements ) during the evolution of this group ( artoni and bertollo , 2001 ) . the hypostomini consists of a single genus , hypostomus , whose representatives have a relatively small , stout body , without a depressed caudal peduncle and adipose fin ( armbruster , 2004 ) . , 2008a ; carvalho et al . , 2010 ) and is distributed from central america to southern south america ( ferraris , 2007 ) , has the greatest karyotypic diversity within the family ( artoni and bertollo , 1996 , 2001 ; artoni et al . , 1998 ) . according to artoni and bertollo ( 1996 ) , these fish exhibit non - conservative characteristics in diploid number , karyotypic macrostructure and chromosomal banding ( artoni and bertollo , 1996 ) . currently , most of the cytogenetic data on hypostomus relate to the diploid number , karyotypic formulas and location of the nor ( rubert et al . , 2008 ) . the diploid number ranges from 2n = 52 in hypostomus emarginatus ( artoni and bertollo , 2001 ) to 2n = 84 in hypostomus sp . some species have distinct karyotypic formulas and their chromosomal variation is accompanied by an increase in the number of subtelo / acrocentric chromosomes ( table 2 ) . according to artoni and bertollo ( 1996 ) , chromosomal rearrangements , such as centric fission and pericentric inversions , , with zz / zw ( artoni et al . , 1998 ) and ancistrus sp . 1 , with xx / x0 ( alves et al . , 2006 ) hypostomus has single or multiple nors in the terminal portion of the chromosomes , as observed for other species of this genus ( table 2 ) , with the number of silver - stained chromosomes varying from one ( artoni and bertollo , 1996 ; cereali et al . , 2008 ) to three ( artoni and bertollo , 1996 ; alves et al . , 2006 ) pairs . several hypostomus species are morphologically very similar ( schubart , 1964 ; schaefer , 1987 ; reis et al . , 1990 ; muller and weber , 1992 ; mazzoni et al . weber and montoya - burgos , 2002 ; oyakawa et al . , 2005 ; zawadzki et al . cytogenetic studies have been very useful taxonomically since several fish groups identified only on the basis of morphological studies have been further characterized as a cluster of two or more isolated genetic units . to improve our knowledge of the diversity and species relationships in hypostomus , in this study we undertook a cytogenetic analysis of five species in this genus . we provide information on the karyotypic organization of these species and discuss some aspects of karyotypic evolution in this group of fish . specimens of five species of hypostomus were collected in streams and rivers from the upper paran river basin and atlantic coastal rivers ( figure 1 , table 1 ) . the specimens were collected under a license from instituto brasileiro do meio ambiente e dos recursos naturais renovveis ( ibama ) . after the cytogenetic procedures , the fish were fixed in 10% formaldehyde and preserved in 70% ethanol for future taxonomic studies . voucher specimens were deposited in the ichthyological collection of the laboratrio de biologia e gentica de peixes ( lbp ) of the departamento de morfologia do instituto de biocincias , universidade estadual paulista 1981 ) and nucleolar organizer regions ( nors ) were detected by the silver impregnation technique of howell and black ( 1980 ) . chromosomal morphology was established based on the proportions of the arms , as proposed by levan et al . ( 1964 ) , and the chromosomal nomenclature commonly applied to fish ( a - acrocentric , m - metacentric , sm - submetacentric and st - subtelocentric ) was used . the five species analyzed ( table 2 ) showed diploid numbers ranging from 2n = 68 chromosomes in hypostomus sp . to 2n = 80 in hypostomus cf . all of the species , except for hypostomus regani , were analyzed karyotypically for the first time . heraldoi , from the mogi - guau river , had a diploid number of 2n = 72 chromosomes composed of 6 m , 6 sm , 26 st and 34 a ( figure 2a , table 2 ) . this diploid number was the same as in h. goyazensis ( alves et al . , 2006 ) , h. regani ( artoni and bertollo , 1996 ; alves et al . , 2006 ) and hypostomus sp . d2 ( artoni and bertollo , 1996 ) ( table 2 ) , although all of these species can be differentiated by their karyotypic organization . hypostomus regani , from the mogi - guau river , had 2n = 72 chromosomes , with a karyotypic formula of 6 m , 6 sm , 32 st and 28 a ( figure 2b , table 2 ) , which partially confirmed the results of artoni and bertollo ( 1996 ) and alves et al . ( 2006 ) , who observed the same diploid number as found here but different karyotypic formulas ( table 2 ) . hypstomus regani is one of the most widely - distributed species throughout the paran - paraguay river basin . ( 2008b ) identified genetically - structured populations of h. regani from the manso reservoir ( paraguay river basin ) , itaipu reservoir ( lower portion of the upper paran river basin ) and corumb reservoir ( upper portion of the upper paran river basin ) . these findings indicate that differences in the karyotypic formulas of h. regani populations are not uncommon . , from the mogi - guau river , had 2n = 68 chromosomes that consisted of 6 m , 6 sm , 32 st and 24 a ( figure 2c , table 2 ) . this diploid number was also found in h. ancistroides ( michele et al . , 1977 ; artoni and bertollo , 1996 ; alves et al . , 2006 ) . however , hypostomus sp . differs from h. ancistroides in its karyotypic structure ( table 2 ) . agna , from cavalo stream , had 2n = 74 chromosomes , with 8 m , 10 sm , 32 st and 24 a ( figure 3a , table 2 ) . this diploid number was also observed in h. paulinus , h. strigaticeps ( michele et al . , 1977 ) and h. albopunctatus ( artoni and bertollo , 1996 ) ( table 2 ) , but all of these species can also be differentiated by their karyotypic structure . topavae , from carrapato stream , had 2n = 80 chromosomes , consisting of 6 m , 8 sm , 42 st and 24 a ( figure 3b , table 2 ) . e ( artoni and bertollo , 1996 ) ( table 2 ) , although h. cf . based on cytogenetic studies , artoni and bertollo ( 2001 ) found that in the hypostominae higher chromosomal numbers are associated with a greater number of uniarmed chromosomes , whereas low diploid numbers are associated with a higher number of biarmed chromosomes . similarly , the high diploid numbers in hypostomus are associated with a high number of uniarmed chromosomes ( table 2 ) . ( 2003 , 2005 , 2006 ) suggested that the diploid number 2n = 54 and the presence of many biarmed chromosomes are primitive characteristics of the loricariidae . their conclusion was based mainly on the wide occurrence of this diploid number and karyotypic formulas in basal loricariid taxa , such as members of the subfamilies neoplecostominae and hypoptopomatinae . the available data ( table 2 ) therefore corroborate and reinforce the hypothesis of artoni and bertollo ( 1996 ) that centric fissions and pericentric inversions have had an important role in the evolution of this fish group . our results showed that hypostomus species have single or multiple nors in the terminal position of their chromosomes , as observed in other species of this genus ( table 2 ) . in h. cf . heraldoi , nors occurred on the long arm of an acrocentric chromosomal pair ( pair 20 ) ( figure 2a , table 2 ) ; in h. regani , nors occurred on the short arms of two subtelocentric chromosomal pairs ( pairs 15 and 16 ) ( figure 2b , table 2 ) ; in hypostomus sp . , from the mogi - guau river , nors occurred on the short arms of two subtelocentric chromosomal pairs ( pairs 7 and 14 ) ( figure 2c , table 2 ) ; in h. aff . agna , nors occurred on the long arms of two chromosomal pairs , one submetacentric ( pair 5 ) and one subtelocentric ( pair 13 ) ( figure 3a , table 2 ) ; finally , in h. cf . topavae , nors occurred on two chromosomal pairs : on the short arms of a subtelocentric ( pair 11 ) and on the long arms of an acrocentric pair ( pair 30 ) ( figure 3b , table 2 ) . these finding highlight the extensive diversity in nor phenotype among the loricariidae . oliveira and gosztonyi ( 2000 ) stated that in the siluriformes the basal nor condition was probably a single nor at a terminal position on the chromosome . artoni and bertollo ( 1996 ) proposed that nors located terminally on the long arm of a single metacentric chromosomal pair represented the primitive condition in the hypostominae . based on these hypotheses , species with multiple nors would be derived in a monophyletic group . since most hypostomus species studied here had multiple nors , we suggest that these nors either originated independently among hypostomus species or originated only once in a monophyletic hypostomus group . the hypothesis that the five hypostomus species analyzed here represent a derived cytogenetic condition is coherent with the available biogeographic data for hypostomus . based on geological and molecular data , montoya - burgos ( 2003 ) estimated that the origin of the main clade of hypostomus was on the former amazon river basin . according to this author , the vicariant and dispersal events from amazonian areas to paran - paraguayan areas occurred about 1012 million years ago . thus , if the cytogenetic hypotheses are congruent with the biological evolution of hypostomus , then amazon basin species should have chromosomal numbers close to 2n = 54 and a single nor in the terminal position . however , although hypostomus species have been described from the amazon river basin ( weber , 2003 ; zawadzki et al . , 2008a ) only h. emarginatus has been karyotyped . if we consider that some authors consider h. emarginatus as pertaining to the genus squaliforma ( weber 3003 ; ferraris 2007 ; eschmeyer , 2011 ) , then , to date , no nominal hypostomus species from the amazon river basin have been karyotyped . clearly , an adequate understanding of the karyotypic evolutionary history of hypostomus requires detailed cytogenetic studies of amazonian species .
in this work , we analyzed the karyotypes of five hypostomus species . 
 hypostomus cf . 
 heraldoi , from the mogi - guau river , had 2n = 72 chromosomes , with a nucleolar organizer region ( nor ) in one chromosomal pair . 
 hypostomus regani , from the mogi - guau river had 2n = 72 chromosomes with nors in two chromosomal pairs . 
 hypostomus sp . 
 , from the mogi - guau river basin , had 2n = 68 chromosomes , with nors in two chromosomal pairs . 
 hypostomus aff . 
 agna , from cavalo stream , had 2n = 74 chromosomes with nors in two chromosomal pairs . 
 hypostomus cf . 
 topavae , from carrapato stream , had 2n = 80 chromosomes , with nors in two chromosomal pairs . 
 hypostomus species showed marked diversity in the karyotypic formula , which suggested the occurrence of several robertsonian rearrangements and pericentric inversions during the evolutionary history of this genus . 
 this hypothesis was supported by the occurrence of a large number of uniarmed chromosomes and multiple nors in a terminal position in most species and may be a derived condition in the loricariidae .