the subfamily scarabaeinae ( coleoptera : scarabaeidae ) comprises a group of insects with widespread geographic distribution and high diversity , consisting of approximately 6,000 species distributed in 11 tribes ( hanski and cambefort , 1991 ) . the tribe phanaeini encompasses 12 genera and 150 species restricted to neotropical and nearctic regions . phanaeus is considered one of the most derived genera in this tribe and comprises two subgenera : notiophanaeus , the more primitive , with 19 species divided into five groups , and phanaeus s.str . , with 35 species divided into eight groups . among the notiophanaeus groups , chalcomelas is considered an annectant taxon between the two subgenera ( edmonds , 1994 ; philips et al . , 2004 ; edmonds and zdek , 2012 ) . even though the number of cytogenetically analyzed phanaeini species is small , the results have shown a great variability , defining it as one of the most diverse tribes in terms of chromosomal characteristics within the subfamily scarabaeinae . among the 20 species studied , three different diploid numbers ( 2n = 12 , 19 and 20 ) and six sex chromosome mechanisms ( xyp , xyp , xy , xy , x0 and neo - xy ) were observed , contrasting with the majority of scarabaeinae species that display the karyotype 2n = 20 , xyp , considered modal and primitive for the group ( cabral - de - mello et al . , 2008 ; so far , six representatives of genus phanaeus ( corresponding to 14.29% of all described species ) were cytogenetically studied , four belonging to the subgenus phanaeus s.str . , and two to the subgenus notiophanaeus . the karyotypes observed in these species differed greatly from 2n = 20 , xyp , showing diploid number reduction to 2n = 12 and the occurrence of two distinct sex chromosome mechanisms in the genus , xy and neo - xy ( cabral - de - mello et al . differential cytogenetic techniques , such as c - banding , base - specific fluorochrome staining , silver nitrate ( agno3 ) staining and fluorescent in situ hybridization ( fish ) using major ribosomal dna ( 45s rdna ) as probe , were applied only in nine phanaeini representatives ( bione et al . . the use of these techniques has enabled a more detailed chromosomal analysis of different scarabaeinae species , helping to identify major events involved in the karyotype evolution of the group ( bione et al . , 2005a , b ; silva et al . , 2009 ; cabral - de - mello et al . , in order to contribute to a better understanding of the trends in the chromosomal diversification of phanaeus representatives , the species phanaeus ( notiophanaeus ) chalcomelas ( perty , 1830 ) ( chalcomelas group ) and p. ( n. ) splendidulus ( fabricius , 1781 ) ( splendidulus group ) were analyzed here by conventional and differential staining , and mapping of rdna genes by fish . the data obtained point to pericentric inversions and centric fusions as the main mechanisms responsible for the chromosomal differentiation in these species . furthermore , the amplification and dispersion of constitutive heterochromatin and rdna sequences may also be involved in their karyotype diversification . mitotic and meiotic chromosomes from six adult males of the species phanaeus ( notiophanaeus ) chalcomelas and 50 of p. ( n. ) splendidulus were analyzed . the six p. ( n. ) chalcomelas specimens were collected in the botanical garden ( jardn botnico ) of santa cruz de la sierra ( 174721 s : 631148 w ) , bolivia , and the 50 p. ( n. ) splendidulus specimens in the ecologic park ( parque ecolgico ) joo vasconcelos sobrinho of caruaru ( 081830 s : 360010 w ) , pernambuco , brazil . the individuals analyzed were deposited in the scientific collection of scarabaeidae , at the instituto de cincias biolgicas , universidade de pernambuco , brazil . subsequently , the materials were fixed in 3:1 carnoy ( ethanol : acetic acid ) and stored at 20 c . the cytological preparations were obtained by the classical method of testicular follicles squashing , followed by conventional staining with 2% lactoacetic orcein . c - banding was performed according to sumner ( 1972 ) , with modifications related to the exposure time of chromosomes to the different solutions , and the triple staining with cma3/da / dapi followed the method established by schweizer ( 1976 ) . silver nitrate ( agno3 ) staining was done according to howell and black ( 1980 ) , and fish according to cabral - de - mello et al . the probes used for fish consisted of cloned partial sequences of 5s and 18s rdnas , obtained by pcr from the genomic dna of the scarab beetle dichotomius semisquamosus ( genbank accession numbers gq443313 for the 18s rrna gene , and gq443312 for the 5s rrna gene ) . the probes were labeled with biotin-14-datp ( invitrogen , san diego , ca , usa ) and detected with an avidin - fitc conjugate ( sigma , st louis , mo , usa ) . the preparations were counterstained with propidium iodide ( 50 g / ml ) and mounted with vectashield mounting medium ( vector laboratories ltd , cambridgeshire , uk ) . for each species , approximately 50 metaphase cells were analyzed using conventional staining and about 30 metaphases were processed by differential techniques . the images were captured in an olympus bx61 microscope connected to an olympus dp71 digital camera , and organized using corel photo - paint x3 software. the morphological classification of chromosomes followed the criteria established by levan et al . the species phanaeus ( n. ) chalcomelas displayed 2n = 12 and neo - xy sex chromosomes ( figure 1a ) . the morphology of its chromosomes could not be precisely determined , but they may be meta - submetacentric . phanaeus ( n. ) splendidulus exhibited the modal karyotype for the family scarabaeidae , 2n = 20 , xyp ( figure 1c ) , with submetacentric pairs 1 and 9 and x chromosome , a metacentric pair 7 , acrocentric pairs 26 and 8 , and a punctiform y chromosome ( figure 1d ) . the c - banding technique showed chromosomes rich in constitutive heterochromatin in both species . in phanaeus ( n. ) chalcomelas there were large c - banded blocks in all chromosomes , with the pericentromeric heterochromatin extending along the chromosome arms ( figure 1b ) . in p. ( n. ) splendidulus moreover , pairs 36 and 8 showed additional large c - banded blocks in the terminal region of the long arms . pair 9 was diphasic , with an entirely heterochromatic short arm and an euchromatic long arm . c - banded blocks were observed in the pericentromeric region of the x chromosome and in part of its short arm . a small c - banded block was detected on the y element , but its small size and the low amount of hetero - chromatin did not allow a precise determination of the location or extent of the heterochromatic regions ( figure 1d ) . triple fluorochrome staining with cma3/da / dapi allowed the identification of cma3-positive ( rich in g+c base pairs ) heterochromatic blocks in all chromosomes of p. ( n. ) chalcomelas and p. ( n. ) splendidulus ( figures 1e and g ) . these blocks were dapi - negative ( figures 1f and h ) . in p. ( n. ) chalcomelas , three bivalents showed cma3 blocks restricted to the pericentromeric region , while the others displayed the same pattern revealed by c - banding . in p. ( n. ) splendidulus , the cma3 blocks corresponded to the c - banded blocks . phanaeus ( n. ) chalcomelas presented one autosomal pair carrying 18s rdna sequences ( figure 2a ) , while in p. ( n. ) splendidulus five pairs showed these sequences ( figure 2c ) . fish with a 5s rdna probe revealed the presence of one autosomal pair carrying these genes in both species analyzed ( figures 2b and d ) . in p. ( n. ) chalcomelas , the 18s and 5s rdna sites were located in different chromosomes : the 18s rdna was located in a pair relatively larger than the bivalent carrying the 5s rdna sequences ( figures 2a and b ) . in p. ( n. ) splendidulus , it was not possible to carry out this analysis , due to the similar size of the chromosomes . silver nitrate ( agno3 ) staining showed that three to five major rdna sites could be active as nucleolus organizer regions ( nors ) in p. ( n. ) splendidulus ( figures 2e  g ) . in p. ( n. ) chalcomelas , it was not possible to obtain cells in appropriate stages to identify active nors by this technique . the species phanaeus ( n. ) chalcomelas displayed 2n = 12 and neo - xy sex chromosomes ( figure 1a ) . the morphology of its chromosomes could not be precisely determined , but they may be meta - submetacentric . phanaeus ( n. ) splendidulus exhibited the modal karyotype for the family scarabaeidae , 2n = 20 , xyp ( figure 1c ) , with submetacentric pairs 1 and 9 and x chromosome , a metacentric pair 7 , acrocentric pairs 26 and 8 , and a punctiform y chromosome ( figure 1d ) . the c - banding technique showed chromosomes rich in constitutive heterochromatin in both species . in phanaeus ( n. ) chalcomelas there were large c - banded blocks in all chromosomes , with the pericentromeric heterochromatin extending along the chromosome arms ( figure 1b ) . in p. ( n. ) splendidulus , pericentromeric c - banded blocks were observed in all autosomes . moreover , pairs 36 and 8 showed additional large c - banded blocks in the terminal region of the long arms . pair 9 was diphasic , with an entirely heterochromatic short arm and an euchromatic long arm . c - banded blocks were observed in the pericentromeric region of the x chromosome and in part of its short arm . a small c - banded block was detected on the y element , but its small size and the low amount of hetero - chromatin did not allow a precise determination of the location or extent of the heterochromatic regions ( figure 1d ) . triple fluorochrome staining with cma3/da / dapi allowed the identification of cma3-positive ( rich in g+c base pairs ) heterochromatic blocks in all chromosomes of p. ( n. ) chalcomelas and p. ( n. ) splendidulus ( figures 1e and g ) . these blocks were dapi - negative ( figures 1f and h ) . in p. ( n. ) chalcomelas , three bivalents showed cma3 blocks restricted to the pericentromeric region , while the others displayed the same pattern revealed by c - banding . in p. ( n. ) splendidulus , the cma3 blocks corresponded to the c - banded blocks . phanaeus ( n. ) chalcomelas presented one autosomal pair carrying 18s rdna sequences ( figure 2a ) , while in p. ( n. ) splendidulus five pairs showed these sequences ( figure 2c ) . fish with a 5s rdna probe revealed the presence of one autosomal pair carrying these genes in both species analyzed ( figures 2b and d ) . in p. ( n. ) chalcomelas , the 18s and 5s rdna sites were located in different chromosomes : the 18s rdna was located in a pair relatively larger than the bivalent carrying the 5s rdna sequences ( figures 2a and b ) . in p. ( n. ) splendidulus , it was not possible to carry out this analysis , due to the similar size of the chromosomes . silver nitrate ( agno3 ) staining showed that three to five major rdna sites could be active as nucleolus organizer regions ( nors ) in p. ( n. ) splendidulus ( figures 2e  g ) . in p. ( n. ) chalcomelas , it was not possible to obtain cells in appropriate stages to identify active nors by this technique . the two species analyzed in this study , although belonging to the same subgenus , showed remarkable differences in their macro - chromosomal structure , and exhibited divergences with regard to the karyotype considered primitive and modal for the subfamily scarabaeinae ( 2n = 20 , xyp with meta - submetacentric chromosomes ) , reinforcing the high karyotype variability described in phanaeini ( bione et al . , 2005a ; cabral - de - mello et al . , 2008 the karyotype 2n = 20 , xyp observed in phanaeus ( n. ) splendidulus is similar to that considered ancestral for scarabaeinae , however , in this species a prevalence of acrocentric chromosomes ( six pairs ) was observed . this modification in chromosome morphology can be attributed to pericentric inversions in ancestral meta - submetacentric chromosomes . pericentric inversions have been described in other species of this subfamily , e. g. , bubas bison , paracopris ramosiceps and diabroctis mimas ( bione et al . comprise , along with autosomal fusions and fissions , x - autosome fusions and y chromosome loss , the main rearrangements responsible for the karyotype variability observed in scarabaeinae ( cabral - de - mello et al . , 2008 ) . the reduced diploid number ( 2n = 12 ) and the derivative sex chromosome mechanism ( neo - xy ) found in p. ( n. ) chalcomelas were also observed in four other species of the subgenus phanaeus s.str . edmonds ( 1994 ) reported other similarities between the species belonging to the chalcomelas group and phanaeus s.str . diploid number reductions have been reported in some beetles , and probably result from fusions involving only autosomes , or autosomes and sex chromosomes , as observed in scarabaeinae species such as eurysternus caribaeus , deltochilum calcaratum and deltochilum aff . morbillosum ( smith and virkki , 1978 ; arcanjo et al . , 2009 ; cabral - de - mello et al . , 2010b ) . apparently , fusions represent the main mechanism of chromosomal diversification in phanaeus , the only genus of phanaeini in which this event was described so far . the results presented here are in agreement with the phylogenetic analysis made by price ( 2009 ) , based on combined molecular and morphological data . according to this phylogeny , p. ( n. ) splendidulus is located in a more basal group that is a sister of a clade including all other phanaeus species displaying the chromosomal reduction . the cytogenetic data for this genus indicate that more derived species have reduced diploid numbers . the great amount of constitutive heterochromatin ( ch ) observed in the karyotypes of the species analyzed here was also described in other scarabaeinae representatives , such as deltochilum calcaratum , d. aff . morbillosum ( deltochilini ) and some phanaeini species , such as coprophanaeus ( coprophanaeus ) cyanescens , c. ( megaphanaeus ) ensifer and diabroctis mimas ( bione et al . , 2005a ; cabral - de - mello et al . , 2010b ; oliveira et al . , 2010 however , the subfamily scarabaeinae shows different patterns of ch quantity and distribution , including species that conserve the pattern that is probably the ancestral condition of the family scarabaeidae , with a predominance of centromeric / pericentromeric heterochromatin blocks ( moura et al . , 2003 ; wilson and angus , 2004 , 2005 ; bione et al . , 2005b ) . although belonging to distinct genera , the karyotypes of all the phanaeini representatives studied display high amounts of ch , indicating that this condition may be characteristic to the tribe . this high amount of heterochromatin found both in phanaeus representatives and in other scarabaeinae suggests amplification / dispersion of this dna fraction in the genomes of these species . different mechanisms have been appointed to play major roles in the expansion and spreading of dna sequences in the genomes , such as unequal crossing over , occurrence of extrachromosomal circular dnas , slippage replication , rolling circle replication , and conversion - like mechanisms ( charlesworth et al . , 1994 ; ugarkovic and plohl , 2002 ; palomeque and lorite , 2008 ) . the heterochromatic blocks of the phanaeus species analyzed in this study presented a similar composition with regard to base pairs . however , in p. ( n. ) chalcomelas , cma3 staining revealed a pattern different from that observed by c - banding , indicating that part of the heterochromatin of this species has a distinct base pair content , which could be related to the presence of different repetitive dna families in the heterochromatic regions . these differences in the base pair content of c - banded blocks were already reported for some scarabaeinae species , e. g. , coprophanaeus ensifer , deltochilum aff . morbillosum , dichotomius bos , d. laevicollis , among others ( cabral - de - mello et al . , 2010b , 2011b ; oliveira et al . , 2010 chalcomelas and in p. ( n. ) splendidulus , the heterochromatin diversification involved an increase in the amount and variability of its base pair content . ugarkovic and plohl ( 2002 ) suggested that these variation mechanisms could occur concomitantly , promoting an increase in the number of satellite dna copies ( frequently associated to heterochromatin ) and modification in nucleotide sequences . according to the  library model  , proposed by fry and salser ( 1977 ) , closely related species share a set of repetitive sequences which can originate new variants by the accumulation of mutations . these sequences undergo dispersion by different mechanisms , such as unequal crossing - over and gene conversion . these processes could also explain the variability in the quantity and base pair composition observed in the hetero - chromatin of scarabaeinae species . the neo - xy sex bivalent of p. ( n. ) chalcomelas contains a higher amount of heterochromatin than the ancestral xyp mechanism present in species p. ( n. ) splendidulus . ( 2007 ) that the accumulation of heterochromatin contributes to the maintenance of the coleopteran neo - xy mechanism , promoting the isolation between ancestral gonosomal and autosomal segments . in some groups , such as rodents , bats and drosophila , the accumulation of heterochromatin seems to allow the permanence of x - autosome translocations in the karyotype , probably avoiding the deregulation of autosomal genes associated with x components ( parish et al . in contrast , neo - xy sex chromosome systems with heterochromatin restricted to the centromere were observed in eurysternus caribaeus , scarabaeinae ( arcanjo et al . , 2009 ) , indicating that the origin and differentiation of the neo - xy may occur by distinct mechanisms of accumulation / non - accumulation of heterochromatin , even in related species . the occurrence of major rdna sites on autosomes in both species studied was similar to the pattern that is most frequent in the order coleoptera , in which the majority of analyses were made using silver nitrate staining ( reviewed by schneider et al . , 2007 ) . in p. ( n. ) splendidulus , the presence of these sites was observed in some autosomal pairs , indicating dispersion of this gene cluster , including a polymorphic condition compared to the pattern described by cabral - de - mello et al . the presence of multiple rdna sites seems to be common in phanaeini and has been described in diabroctis mimas , coprophanaeus ( c. ) cyanescens and c. ( m. ) ensifer , which presents the highest number of 18s rdna sites in coleoptera ( bione et al . the dispersion of rdna sites in phanaeini could have occurred from only one pair ( one autosomal bivalent ) , which is apparently the ancestral characteristic of coleoptera ( revealed mostly by silver nitrate staining ) , present in about 60% of the species analyzed ( schneider et al . , 2007 ) . this hypothesis is reinforced by the occurrence of two species in the tribe with 18s rdna sites in one bivalent , phanaeus ( n. ) chalcomelas , analyzed here , and coprophanaeus ( metallophanaeus ) pertyi ( oliveira et al . , 2012 ) . as observed for instance in the coprophanaeus species ( oliveira et al . , 2010 , 2012 ) and p. ( n. ) splendidulus , the differences in number and location of major rdna sites appear to be related to processes such as amplification / dispersion rather than to macro - chromosomal rearrangements like translocations . in contrast to the variability in number of major rdna sites observed in phanaeini , both phanaeus species showed only one autosomal pair carrying 5s rrna genes , which was also observed in most of the scarabaeinae studied until now ( cabral - de - mello et al . the data obtained suggest that , in the genus phanaeus , the distinct rdna multigene families are controlled by distinct evolutionary forces for spreading / non - spreading , as described for other scarabaeinae groups ( cabral - de - mello et al . , 2011a ) . among the main chromosomal evolutionary mechanisms which might explain the great karyotype divergence between the species studied in this work , the relevant ones are apparently : ( 1 ) fusions between autosomes and at least one fusion between autosomes and sex chromosomes , generating the reduced diploid number and neo - xy mechanism in p. ( n. ) chalcomelas ; ( 2 ) pericentric inversions in chromosomes of p. ( n. ) splendidulus ; ( 3 ) amplification and dispersion of constitutive heterochromatin and 18s rdna sites . in addition , the data obtained demonstrate the intense dynamic of heterochromatin and major rdna sites in the tribe phanaeini .
the genus phanaeus is included in the tribe phanaeini , one of the most diverse tribes within the subfamily scarabaeinae in terms of chromosomal characteristics . 
 however , so far the species of this genus were not studied with differential cytogenetic techniques , limiting any inference of the probable mechanisms responsible for this diversity . in this work , 
 several techniques were applied with the aim of cytogenetically characterizing two phanaeus species . 
 the karyotype found for phanaeus ( notiophanaeus ) chalcomelas was 2n = 12 , neo - xy , and that of p. ( n. ) splendidulus was 2n = 20 , xyp , considered primitive for the family scarabaeidae . 
 the chromosomes of both species showed a high amount of constitutive heterochromatin ( ch ) , with blocks rich in base pairs gc ( cma3 + ) . 
 moreover , in p. ( n. ) chalcomelas the marks revealed by c - banding and fluorochrome staining were different in size , showing ch variability . 
 sites of 18s ribosomal dna ( rdna ) were identified in one autosomal pair of p. ( n. ) chalcomelas and in five autosomal pairs of p. ( n. ) splendidulus . on the other hand , 
 only one autosomal pair exhibited 5s rdna sequences in these species . 
 the results suggest that the karyotype differentiation of the phanaeus species studied here involved pericentric inversions and centric fusions , as well as mechanisms related to amplification and dispersion of ch and rdna sequences .