Chromosomal diversification of diploid number, heterochromatin and rDNAs in two species of Phanaeus beetles (Scarabaeidae, Scarabaeinae)

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.


Introduction
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 Zídek, 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 (Xy p , XY p , Xy, XY, X0 and neo-XY) were observed, contrasting with the majority of Scarabaeinae species that display the karyotype 2n = 20, Xy p , considered modal and primitive for the group (Cabralde-Mello et al., 2008;Oliveira et al., 2010Oliveira et al., , 2012. 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, Xy p , 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., 2008, 2011a.
Differential cytogenetic techniques, such as C-banding, base-specific fluorochrome staining, silver nitrate (AgNO 3 ) 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., 2005a;Oliveira et al., 2010Oliveira et al., , 2012Cabral-de-Mello et al., 2011a). 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., 2010a,b, 2011a. 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.
The insects were anesthetized with ethyl ether and then their testes were dissected. 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% lacto-acetic 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 stain-ing with CMA 3 /DA/DAPI followed the method established by Schweizer (1976). Silver nitrate (AgNO 3 ) staining was done according to Howell and Black (1980), and FISH according to Cabral-de-Mello et al. (2010c). 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 mg/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. (1964).

Karyotypes
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, Xy p ( Figure 1c), with submetacentric pairs 1 and 9 and X chromosome, a metacentric pair 7, acrocentric pairs 2-6 and 8, and a punctiform Y chromosome ( Figure 1d). The karyotypes of both species showed chromosomes with gradual reduction in size.

Constitutive heterochromatin
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 3-6 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 heterochromatin did not allow a precise determination of the location or extent of the heterochromatic regions (Figure 1d).
Triple fluorochrome staining with CMA 3 /DA/DAPI allowed the identification of CMA 3 -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 CMA 3 + blocks restricted to the pericentromeric region, while the others displayed the same pattern revealed by C-banding. In P. (N.) splendidulus, the CMA 3 + blocks corresponded to the C-banded blocks.

Nucleolus organizer regions and ribosomal DNA sites
The chromosomal mapping of major rDNA clusters revealed that they were located in autosomes. 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 (AgNO 3 ) 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.

Discussion
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, Xy p with meta-submetacentric chromosomes), reinforcing the high karyotype variability described in Phanaeini (Bione et al., 2005a;Cabral-de-Mello et al., 2008;Oliveira et al., 2010).
The karyotype 2n = 20, Xy p 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., 2005a;Angus et al., 2007), and 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. (Smith and Virkki, 1978). Edmonds (1994) reported other similarities between the species belonging to the chalcomelas group and Phanaeus s.str., considering this group as an annectant between the two subgenera. 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 Cytogenetic study in scarab beetles 343 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;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, CMA 3 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 (Cabralde-Mello et al., 2010b, 2011bOliveira et al., 2010).
As a general trend, both in P. (N.) 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 heterochromatin of Scarabaeinae species.
The neo-XY sex bivalent of P. (N.) chalcomelas contains a higher amount of heterochromatin than the ancestral Xy p mechanism present in species P. (N.) splendidulus. These data corroborate the hypothesis raised by Dutrillaux et al. (2007) that the accumulation of heterochromatin contributes to the maintenance of the coleopteran neo-XY 344 Arcanjo et al. 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., 2002;Dobigny et al., 2004;Dutrillaux et al., 2007). 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. (2011a) in which four autosomal pairs displayed 18S rDNA sites. 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., 2005a;Oliveira et al., 2010).
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(Oliveira et al., , 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., 2011a,c). 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.