High diversity of the chromosomal distribution of the 18S rDNA clusters and heterochromatin
Considering the major rRNA genes, two main patterns of distribution were detected (i) two rDNA sites (one chromosomal bivalent) harboring these genes, as observed in Dichotomius, Canthon staigi, Deltochilum elevatum and Ontherus apendiculatus; (ii) increased numbers of 18S rDNA clusters (ranging from 3 to 16 sites), as observed in Bubas bison, C. ensifer, C. cyanescens, D. mimas, Ontherus sulcator and three Deltochilum species. This suggests that the major rRNA genes are under a distinct evolutionary mechanism regarding cluster spreading.
The two main patterns for the major rDNA distribution were primarily observed for three tribes, Canthonini, Coprini and Phanaeini, which include several species analyzed. Among Coprini species, the clusters of the major rDNA clusters have not suffered intense chromosomal reorganization, as they are primarily associated with only one bivalent, as observed in Dichotomius species and Ontherus apendiiculatus. Phanaeini is characterized by an intense movement of the major rDNA clusters that resulted in the generation of different numbers of sites on several chromosomes, as observed in C. ensifer, which presents the highest number of rDNA clusters (16 sites) within the subfamily, and in Coleoptera . Intraspecific polymorphism with regard to the number of rDNA clusters was observed in Coprophaneus ensifer, C. cyanescens, and D. mimas. In the Canthonini tribe, variable patterns of rDNA clusters were observed, with species presenting with either no spreading of the major rDNA clusters, such as Canthon staigi and Deltochilum elevatum, or with scattered rDNA clusters, as observed for three of the Deltochilum representatives. These results indicate that the major rDNA clusters' ability to move is independent of taxonomic units and may be related to the heterochromatin dispersion (see discussion below).
The ancient condition in Scarabaeinae appears to be the occurrence of one autosomal bivalent as a nucleolar organizer. This theory is corroborated by the presence of the pattern in a large number of species within the group and the sister groups of the subfamily. That distribution pattern is also the most common pattern for Coleoptera as a whole, at least for representatives of Polyphaga . In addition to this common pattern that consists of only one chromosomal pair of major rDNA clusters, an intense repositioning of the major rDNA clusters in Scarabaeinae was involved in the increasing number of rDNA sites and the movement to different autosomes and sex chromosomes. The presence of major rDNA clusters associated with the sex chromosomes in different species could be related to either (i) the occurrence of large chromosomal rearrangements, such as fusions, as observed in Deltochilum calcaratum, D. morbillosum and E. caribaeus, species that have a derived neo-XY sex system, or (ii) the occurrence of transpositions, as observed in Coprophanaeus, D. mimas and Deltochilum verruciferum, which are species with the ancient Scarabaeinae diploid number (2n = 20). Although the occurrence of chromosomal fusions were proposed in some species with a reduced diploid number, the presence of rDNA clusters on the sex chromosomes could also be a result of transpositions if fusion involving only autosomes is considered, as in Dichotomius .
Although the variation in the number of major rDNA clusters can be attributed to chromosomal rearrangements in some species, there is no correlation between the variation in the rDNA sites and the diploid number. There are examples of species that have a reduction in the diploid number without a modification to the number of rDNA sites, while species with conservation of the ancestral diploid number and extensive repositioning and expansion of major rDNA clusters number have also been identified. There is evidence of the "movement" and "multiplication" of the major rDNA clusters without fusions or other chromosomal rearrangements . In Scarabaeinae, these modifications could be attributed to an ectopic recombination and transposition and to inversions and translocations within the genome. Similar mechanisms are responsible for intra- and interspecific variations in other insects, such as Acrididae grasshoppers  and in Lepidoptera . These results indicate distinct evolutionary trends that are related to the macro-chromosomal structure (diploid number, chromosome morphology and sex chromosomes) and the organization of the major rDNA genes in some insect genomes.
The analysis of heterochromatin and major rDNA dispersion revealed an interesting relationship pattern. Species with heterochromatin restricted to the centromeric/pericentromeric regions were primarily characterized as having a stable number of major rDNA that were restricted to one chromosomal bivalent. Only Ateuchus sp. had four clusters, while the presence of three clusters in D. semisquamosus was a polymorphic condition. However, extensive variability in the number of major rDNA sites was observed in the majority of representatives (except for Isocopris inhiata) in which heterochromatin was dispersed and occurred in large quantities within the karyotypes, e.g., large paracentromeric heterochromatic blocks and diphasic chromosomes. In species that showed a moderate dispersion of heterochromatin, the major rDNA clusters spread in two species and was restricted to one autosomal bivalent in another, Ontherus appendiculatus. Interestingly in species whose the relationship in position for heterochromatic blocks and major rDNA was possible to determine it was observed a general pattern for non co-localization in some representatives without dispersion for these two chromosomal markers, such as in Dichotomius . In species with spreading of these elements in general they were co-located, such as in Deltochilum and Coprophaneus [26, 27]. Our results indicate that the same evolutionary forces might be acting on these two components of the Scarabaeinae genome, resulting in the spreading of the major rDNA clusters along with heterochromatin. This hypothesized pattern of evolution might be favored by ectopic paring during chromocenter formation during the initial meiotic stage. Ectopic pairing is a common behavior in this insect group that appears to play an important role in nucleolar organization and chromosomal segregation [31, 32].
The restriction or spreading of the number of rDNA clusters might be associated with the presence or absence of an appropriate molecular mechanism associated with heterochromatin and involved in the ectopic recombination possibly caused by repeated DNAs. The ability of rDNA clusters to move and vary in number was first observed by Schubert (1984)  in Allium. Since then, some additional evidence has accumulated concerning the ability of rDNA to move within the genome. Recent studies have proposed that transposable elements are a potential source for the movement of rDNA [34, 35] and other genes [36, 37] to different regions of the genome.
The conservation of the 5S rRNA and histone H3 genes in Scarabaeinae karyotypes
In contrast to the variability in the number of major rDNA clusters, a high conservation in the number of 5S rRNA and histone H3 gene clusters was observed. For invertebrates, the mapping of these sequences was previously restricted to few species of mollusks, insects, crustaceans, annelids and echinoderms [9, 10, 12, 28, 38–42]. In insects, these types of studies have been mainly focused on grasshoppers [9, 10, 12, 42], and only 14 species of beetles had been previously studied, all of which belong to the genus Dichotomius . The co-localized clusters (one bivalent) for these two genes in some Scarabaeinae species could indicate that this is the ancient organization for these sequences, and they have not extensively changed in number since the origin of Scarabaeinae , despite the diversification of the species. An intense conservation of the number of histone gene clusters, with only one or two chromosomes containing clusters, has also been described in grasshoppers [9, 10], mollusks [40, 41] and fish species [44, 45], although variability has also been reported in these groups. These results might indicate that a strong purifying selection acts on the histone clusters, preventing the spread of these genes through the genome, as was proposed for the grasshoppers .
The 5S rDNA gene is highly conserved in Scarabaeinae representatives, and all species examined showed an overlap between the 5S rDNA and the histone H3 genes' signal at the same chromosomal region. This overlap was corroborated by the observation of overlapped signals in cells that were in the initial stages of meiosis and that had interphasic nuclei containing less condensed chromosomes. This indicates that these two multigene families could have a linked organization in the Scarabaeinae genomes. The associated dispersion of the 5S rRNA/histone H3 genes in D. mimas and the restriction of these two sequences to the × chromosome of E. caribaeus, likely due to unequal cross-over events between the × and Y chromosome in this species , reinforces the hypothesis that the 5S rDNA/histone H3 gene clusters are associated in the genome. Additional molecular studies are necessary to fully confirm this hypothesis. An associated or co-localized organization has also been described in mollusks , crustaceans [47–49], Dichotomius coleopterans  and Proscopiidae grasshoppers . Our results reinforce the idea that the association of the 5S rDNA and histone H3 clusters is not sporadic in coleopterans and that it appears to be common. Besides the association of 5S rDNA and histone H3 genes, co-localization or linked organization of major rDNA and histone genes were also reported in insect as described recently for example in Diuraphis noxia (Hemiptera) , Anthonomus grandis and A. texanus (Coleoptera) .
Unlike the results observed among the representatives of Scarabaeinae, the 5S rDNA cluster is highly dynamic among chromosomes and the genomic dynamism in some animal groups, such as in fish and Acrididae grasshoppers [12, 52, 53]. This stability in Scarabaeidae beetles could be the result of its association with the histone genes, which may result in the same purifying selection that appears to act against the spread of histone clusters.
In contrast to the co-localization of the 5S rDNA/histone H3 clusters, the 18S rDNA is not co-localized in the genomes of the Scarabaeinae species studied. Only Diabroctis mimas and Digitonthophagus gazella showed a co-localization of these sequences. These results could be explained by a transposition of the 18S rDNA cluster due to its intense movement in the genome of some species. This physical separation could be result in a functional advantage for these ribosomal sequences. The disassociation of the two multigene families that encode for rRNAs is a common pattern for eukaryotic and vertebrate chromosomes, including those in fishes [54–56]. However, some invertebrate species have co-localized rRNA clusters, including representatives of the annelids, mollusks and crustaceans; however, a non co-localized organization has also been described [38, 48, 57, 58].
The association/co-localization of multigene families in animal genomes has been reported for some sequences, including rRNAs, the histone genes and small nuclear RNAs (snRNAs). These associations/co-localizations are poorly understood, and their biological effect is still unclear. According to studies by Dover (1986)  and Liu and Fredga (1999) , the linkage is important to maintain multiple, conserved arrays. Kaplan et al. (1993)  hypothesized that the association of the repetitive multigene families might play a functional role in the organization of the nucleus. In the case of the 18S rDNA, 5S rDNA and histone H3 histone, the separation of the 18S and 5S rDNA arrays might convey a functional advantage, since the 18S rRNA gene is transcribed by RNA polymerase I and the 5S rRNA gene is transcribed by RNA polymerase III. However, the association of the histone H3 and 5S rRNA genes cannot be explained by a transcriptional advantage because these two sequences are transcribed by different polymerases.