High Levels of Chromosomal Differentiation in Euchroma gigantea L . 1735 ( Coleoptera , Buprestidae )

Euchroma gigantea was karyotypically studied using conventional staining, C-banding, silver nitrate staining and ribosomal fluorescent in situ hybridization (rDNA FISH). Broad wide autosomal polymorphism and a complex sex determination system were found in this beetle. Karyotype complements ranging from 2n = 32, X1X2X3Y1Y2Y3 to 2n = 36,X1X2X3Y1Y2Y3 were detected in the sample analyzed. Punctiform supernumerary chromosomes were present in the different karyotypes. The karyotypic evolution of Brazilian E. gigantea may have taken two directions, reduction in the diploid number of 2n = 36 to 24 through centric fusions or 2n = 24 to 36 due to chromosomal fissions. In addition, pericentric inversions were also involved. The complex multiple sex mechanism of this species seems to be old and well established since it is found in specimens from different populations. Small pericentromeric blocks of constitutive heterochromatin were located on the autosomes and terminal blocks were also found on some small pairs. The sex chromosomes showed larger constitutive heterochromatin blocks. Silver nitrate staining during prophase I of meiosis showed labeling of the sex chromosome chain. However, the rDNA sites could only be precisely determined by FISH, which permitted the identification of these ribosomal sites on chromosomes X1 and X2 of this species.


Introduction
The monotypic beetle genus Euchroma (Buprestidae), also known as the jewel scarab beetle, is widely distributed in the Neotropics and some subspecies and varieties of this genus have been described in South America, such as Euchroma gigantea mrazi found in southeastern Brazil (Obenberger, 1928).
Chromosome studies involving representatives of the family Buprestidae are scarce and only about 83 species having so far been analyzed by conventional methods, corresponding to 0.55% of all species described.The Buprestidae is characterized by a wide chromosome variability, with the diploid number ranging from 2n = 12 (Melanophila acuminata) to 2n = 46 (Sphenoptera scovitzi), and sex chromosome mechanisms of the chiasmatic (neo XY, Xy r , X 1 Y 1 X 2 Y 2 X 3 Y 3 ) and achiasmatic type (Xy p , XO, Xy,) (Smith and Virkki, 1978;Mesa and Fontanetti, 1984;Karagyan and Kuznetsova, 2000;Karagyan, 2001;Karagyan et al., 2004).Various karyotypes have been described in E. gigantea, such as 2n = 26, Xy in specimens from Panama (Nichols, 1910), 2n = 24, X 1 X 2 X 3 Y 1 Y 2 Y 3 and 2n = 26, X 1 X 2 X 3 Y 1 Y 2 in specimens from the Brazilian state of São Paulo.In addition to chromosome polymorphism, this species presents the largest number of supernumerary chromosomes (16, 20 and 32) reported for the Coleoptera (Mesa and Fontanetti, 1984).
Most coleopteran species possess a simple sex mechanism, with only 3.4% of the species analyzed presenting a multiple sex mechanism.According to Virkki (1984), the Xy p sex system may not be the ancestral mechanism in this order, although it is apparently the most successful, being present in most species analyzed.Various families representative of the suborders Adephaga and Polyphaga present derived mechanisms such as XO or multiple sex systems.These mechanisms may have resulted from translocations between the X p and y p chromosomes and autosomes (Virkki, 1984).Examples of multiple systems have been described in the Tenebrionidae, Cincindelidae, Curculionidae and Chrysomelidae (Wahrman et al., 1973;Panzera et al., 1983;Postiglioni et al., 1987;Galián et al., 1995;Vitturi et al., 1996).
The aim of the research described in this paper was to analyze the meiotic and mitotic chromosomes of E. gigantea using conventional staining, C-banding, silver nitrate staining and fluorescent in situ hybridization (FISH) with a rDNA probe.Comparison of the results with data in the literature permitted a better chromosome characterization and the elaboration of a hypothesis on the karyotype evolution of this species.

Material and Methods
We analyzed 19 Euchroma gigantea L. 1735 specimens, the sample being made up of 15 males collected at the Igarassú Charles Darwin Ecological Refuge in the northeastern Brazilian state of Pernambuco (7°50'3" S, 34°54'23" W) and two males and two females collected from the Atlantic Rainforest in the Recife Zoo-Botanical Park in Pernambuco (8°3'14" S, 34°52'52" W).The beetles were and the testes and ovaries dissected out and fixed in Carnoy's solution (ethanol:acetic acid 3:1 v/v).Cytological preparations were obtained using the classical testicular and ovarian follicles squashing method and the chromosomes were stained with 2% (w) lacto-acetic orcein.The method of Sumner (1972) was used for C-banding and silver nitrate staining was carried out as described by Rufas et al. (1987).The FISH was performed according to the method of Moscone et al. (1996) using a probe containing Arabidopsis thaliana 45S ribosomal genes (18S, 5.8S, 25S) (Unfried et al., 1989;Unfried and Gruendler, 1990).The probes were labeled with biotin11-dUTP and detected with rat antibiotin antibody (Dakopatts M0743, Dako) and antiantibiotin antibody conjugated with tetramethyl-rhodamine isothiocyanate (TRITC).The preparations were counterstained with 2 μg mL -1 4'-6-diamidino-2-phenylindole (DAPI) AT-specific fluorochrome and mounted with Vectashield H-1000 (Vector).The slides submitted to the different techniques were analyzed using a Leica photomicroscope and Kodak Imagelink 25, TMAX 400 and Fugi film 400 for FISH.Photographic copies were obtained using Kodak Kodabrome F3 paper.

Results
Chromosome analysis of E. gigantea showed the presence of a multiple sex determination mechanism of the X 1 X 2 X 3 Y 1 Y 2 Y 3 type in males and wide autosomal poly-morphism.Five or six punctiform supernumerary chromosomes were also observed in the different karyotypes (Figure 1b,d).Two of the 15 specimens collected in Igarassú presented a 2n = 36, X 1 X 2 X 3 Y 1 Y 2 Y 3 karyotype in which all the autosomes were acrocentric (Figure 1a), 10 specimens showed a 2n = 34, X 1 X 2 X 3 Y 1 Y 2 Y 3 karyotype with one submetacentric and 13 acrocentric autosomal pairs (Figure 1b) while the other three specimens had a 2n = 32, X 1 X 2 X 3 Y 1 Y 2 Y 3 karyotype with two submetacentric and 11 acrocentric autosomal pairs (Figure 1 d).In addition, a heterozygous karyotype complement with 2n = 33 was observed in one specimen from the Recife population (Figure 1c), while the other specimens from this locality presented a 2n = 34,X 1 X 2 X 3 Y 1 Y 2 Y 3 karyotype.The X chromosomes were submetacentric, the Y 1 and Y 2 chromosomes were metacentric and the Y 3 chromosome was acrocentric (Figure 2b, c).The sex mechanism was complex and chiasmatic.During metaphase I, the sex chromosomes were connected by distal chiasmata and formed an alternate chain with a sequence of X 1 -Y 1 -X 2 -Y 2 -X 3 -Y 3 (Figure 2a).
In E. gigantea, constitutive heterochromatin (CH) was located in the pericentromeric region of all autosomes.In addition, some small pairs contained terminal CH blocks (Figure 3a).Chromosomes X 1 and X 3 possessed centromeric CH, and the long arm of chromosome X 1 was almost completely heterochromatic.In the case of the X 2 chromosome, CH was only present in the terminal region of the short arm.Chromosome Y 1 showed an almost completely heterochromatic short arm, in addition to an interstitial block on the long arm.The Y 2 presented pericentromeric CH extending along the two chromosome arms, and Y 3 possessed pericentromeric CH on the short arm (Figures 3a  and 3b).Silver nitrate staining of cells at the beginning of prophase I revealed labeling of the sex chromosome chain (Figure 3c).Analysis of spermatogonial metaphases by FISH using a ribosomal probe demonstrated the presence of two rDNA sites on different chromosomes.During metaphase I the sites were found to be associated with the sex chain, more precisely with chromosomes X 1 and X 2 (Fig- ures 3d and 3e).
In Coleoptera, examples of chromosome polymorphisms have been observed for different species of Ipis 2n = 16 to 2n = 32, Pissodes 2n = 25 to 2n = 34, Mulsantina 2n = 12 to 2n = 20, and Chilocorus 2n = 19 to 2n = 25.This variability has been attributed to the occurrence of centric fusions, fissions and pericentric inversions (Smith and Virkki, 1978;Boyce et al., 1994).The genus Chilocorus has a basic chromosome number of 2n = 22 and is found in North America, India and Europe.For example, Chilocorus stigma populations from North America present a multiple sex determination mechanism (X 1 X 2 Y), wide chromosomal polymorphism (2n = 19 to 2n = 25) and supernumerary chromosomes.Centric fusion and fission have been proposed to explain the karyotype variation observed in this species (Smith and Virkki, 1978).
Comparative karyotype analysis between our sample of E. gigantea from Pernambuco and specimens collected in the Brazilian state of São Paulo (Mesa and Fontanetti, 1984) throws some light on the chromosome evolution of this species.The known variation in chromosome constitution suggests that the most primitive karyotype would be 2n = 36 from which successive centric fusions resulted in derived karyotypes, starting with a single homozygous fu-  sion to produce 2n = 34, followed by two fusions to produce 2n = 32, five fusions to produce 2n = 26 and six fusions to produce 2n = 24.
On the other hand, cannot affirm that the fusions seen by us are the main rearrangement involved in the chromosomal evolution of E. gigantea, because molecular and biogeographic studies that establish ancestral groups do not exist.Another possible alternative is the rearrangement commenced with the 2n = 24, X 1 X 2 X 3 Y 1 Y 2 Y 3 karyotype, in which autosome-sexual chromosome translocations, autosome-autosome and pericentric inversions were initially involved generating the karyotypes described by Mesa and Fontanetti (1984) with 2n = 24, X 1 X 2 X 3 Y 1 Y 2 Y 3 and a fission producing the 2n = 26, X 1 X 2 X 3 Y 1 Y 2 Karyotype.Based on the karyotypes described in the present paper it seems that four fissions had probably occurred to produce 2n = 32, X 1 X 2 X 3 Y 1 Y 2 Y 3 , five fissions to produce 2n = 34, X 1 X 2 X 3 Y 1 Y 2 Y 3 and six fissions to produce 2n = 36, X 1 X 2 X 3 Y 1 Y 2 Y 3 .In addition to the rearrangements described above, we detected the occurrence of successive pericentric inversions responsible for the alteration in the proportion of chromosomal arms in the different karyotypes presented by E. gigantea.Karagyan et al. (2004) have pointed out that it is not yet possible to infer the ancestral karyotype for the Buprestidae, although a similarity has been proposed with the ancestral karyotype (2n = 20, Xyp) described for the Coleoptera in general and also observed in different species of the subfamilies Chrysochroinae, Buprestinae and Agrilinae but there is as yet no concrete evidence to define the ancestral karyotype for E. gigantea.The different karyotypes of E. gigantea and the probable rearrangements involving to produce the current karyotypes found in this species is shown in Figure 4. Mesa and Fontanetti (1984) have pointed out that successive translocations involving autosomes and sex chromosomes were responsible for the formation of the complex 3X + 3Y mechanism found in E. gigantea, which probably derived from an Xy or XO system.The presence of the X 1 X 2 X 3 Y 1 Y 2 Y 3 sex mechanism in all karyotype compositions found in E. gigantea suggests that this mechanism is old and well-established in this species.
Among coleopterans, the Buprestidae is the family with the largest number of supernumerary chromosomes (Smith and Virkki, 1978;Mesa and Fontanetti, 1984).We observed 16 to 32 supernumerary chromosomes in our southeastern Brazilian E. gigantea sample, while Mesa and Fontanetti (1984) have reported that E. gigantea from this region showed the lowest diploid number (2n = 24 and 26).However, in our study, the E. gigantea specimens from northeastern Brazil showed extensive autosomal polymorphism and also the largest diploid number (2n = 32 to 36), as well as a markedly smaller number (five) of supernumerary chromosomes.Derivation from A chromosomes is the most widely accepted of the various hypotheses on the origin of B chromosomes, which could have arisen either from centromeres remaining after chromosome fusions or from amplified pericentromeric fragments (Jones and Rees, 1982;Camacho et al., 2000).Events of these types might have been involved in the origin of B chromosomes in E. gigantea.Colombo and Remis (1997) have presented data on the grasshopper Sinipta dalmani showing that B chromosomes arise from centric fragments produced from the high frequency of karyotypic rearrangements such as inversions and centric fusions.
As in other coleopteran species, the pattern of CH distribution in E. gigantea is predominantly pericentromeric but we found that in this species the autosomal CH blocks were small and some autosomal pairs possessed minute terminal blocks, in contrast to the sex chromosomes which were characterized by large CH blocks.Atypical patterns of heterochromatin distribution have been described in other coleopterans.Some Carabidae species, such as Acapulcus Stenolophus teutonus, Acapulcus elegans and Bembidion minimum, not only possess large CH blocks on the autosomes and have completely heterochromatic X chromosome but also show C-band heteromorphism and terminal and subterminal CH blocks (Rozek, 1992;Rozek and Rudek, 1992).For example, Vitturi et al. (1996) observed pericentromeric CH blocks in two Blaps species, whereas Blaps gibba presented an additional proximal and interstitial block on chromosome pair 1 and terminal blocks on pairs 9 and 12.
Our silver nitrate staining and FISH results for E. gigantea agree with the pattern observed in other coleopterans that possess a multiple sex mechanism (Galián et al., 1995;Vitturi et al., 1996;Galián and Hudson, 1999) and for which the rDNA sites are located on the sex chromosomes.In general, the species analyzed so far have shown two hybridization signals, except for B. gibba which shows seven to eight signals scattered over the sex multivalent (Vitturi et al., 1996).In contrast, preferential staining of the autosomes is observed in representatives of the Carabidae (Galián et al., 1995;De La Rua et al., 1996;Galián and Hudson, 1999;Sanchez-Gea et al., 2000), Geotrupidae (Vitturi et al., 1999) and Scarabaeidae (Colomba et al., 2000) and which possess a simple sex mechanism.
To better understand the chromosome evolution of E. gigantea, studies investigating specimens from the North and Center-West region of Brazil are fundamental, in addition to the analysis of other specimens from the Southeast mainly employing FISH with a telomeric probe.Furthermore, a taxonomic revision of the genus including subspecies and varieties from different geographic regions, together with the available cytogenetic analysis, will be of great importance for better understanding the status of the genus Euchroma.

Chromosomal differentiation in Euchroma gigantea 433 Figure 2 -
Figure 2 -Conventionally stained meiotic and mitotic chromosomes of Euchroma gigantea.(a) Metaphase I with 2n = 34.Observe the alternate chain formed by the (X 1 Y 1 X 2 Y 2 X 3 Y 3 ) sex chromosomes.(b) and (c) Sex chromosomes of a male and of a female, respectively.Bar = 10 μm.

Figure 3
Figure 3 -C-banding pattern, AgNO 3 staining and fluorescent in situ hybridization with a ribosomal probe of meiotic and mitotic chromosomes of Euchroma gigantea.(a) Mitotic metaphase of a male.(b) Schematic representation of the C-banding pattern of the sex chromosomes.The arrows indicate the sex chromosomes.(c) Zygotene.The arrowhead indicates the AgNO 3 -stained nucleolar organizer region.(d) Spermatogonial metaphase showing the two rDNA sites.(e) Metaphase I indicating the location of the sites on the X 1 and X 2 chromosomes (arrowheads).Bar = 10 μm.

Chromosomal differentiation in Euchroma gigantea 435 Figure 4 -
Figure4-Schematic representation of the different chromosome numbers observed in Euchroma gigantea and of the probable rearrangements that occurred during the chromosome evolution of this species.A -hypothetical karyotypes; B -karyotypes described byMesa and Fontanetti (1984) and C -karyotypes observed in this work.