Improved Basic Cytogenetics Challenges Holocentricity of Butterfly Chromosomes.

Mitotic chromosomes of butterflies, which look like dots or short filaments in most published data, are generally considered to lack localised centromeres and thus to be holokinetic. This particularity, observed in a number of other invertebrates, is associated with meiotic particularities known as "inverted meiosis," in which the first division is equational, i.e., centromere splitting-up and segregation of sister chromatids instead of homologous chromosomes. However, the accurate analysis of butterfly chromosomes is difficult because (1) their size is very small, equivalent to 2 bands of a mammalian metaphase chromosome, and (2) they lack satellite DNA/heterochromatin in putative centromere regions and therefore marked primary constrictions. Our improved conditions for basic chromosome preparations, here applied to 6 butterfly species belonging to families Nymphalidae and Pieridae challenges the holocentricity of their chromosomes: in spite of the absence of primary constrictions, sister chromatids are recurrently held together at definite positions during mitotic metaphase, which makes possible to establish karyotypes composed of acrocentric and submetacentric chromosomes. The total number of chromosomes per karyotype is roughly inversely proportional to that of non-acrocentric chromosomes, which suggests the occurrence of frequent robertsonian-like fusions or fissions during evolution. Furthermore, the behaviour and morphological changes of chromosomes along the various phases of meiosis do not seem to differ much from those of canonical meiosis. In particular, at metaphase II chromosomes clearly have 2 sister chromatids, which refutes that anaphase I was equational. Thus, we propose an alternative mechanism to holocentricity for explaining the large variations in chromosome numbers in butterflies: (1) in the ancestral karyotype, composed of about 62 mostly acrocentric chromosomes, the centromeres, devoid of centromeric heterochromatin/satellite DNA, were located at contact with telomeric heterochromatin; (2) the instability of telomeric heterochromatin largely contributed to drive the multiple rearrangements, principally chromosome fusions, which occurred during butterfly evolution.

Phylogenetic relationships between genera Dorcadion, Lamia, Morimus, Herophila and some other Lamiinae (Coleoptera: Cerambycidae) based on chromosome and CO1 gene sequence comparison.

A dual molecular and cytogenetic study was performed with the aim to improve the controversial systematic classification of some species of Lamiinae (Coleoptera: Cerambycidae). The karyotypes of species belonging to genera Morimus, Herophila, Dorcadion, Neodorcadion and Lamia share a number of characters, which differentiate them from other species, belonging to genera Phytoecia, Parmena and Monochamus. The karyotypes of the last three species comprise 20 chromosomes, mostly metacentric or sub-metacentric, as in the presumed Cerambycidae ancestors. The karyotypes of the former species share many characters derived from the Lamiinae ancestors by a number of chromosome fissions and inversions indicating their monophyly. Comparisons of the CO1 gene sequence also show the monophyly of Morimus, Lamia, Herophila and Dorcadion and their distant relationship with others. These convergent results allow us to propose a phylogenetic classification of these genera, which places the monospecific genus Lamia close to Dorcadion, clearly separates Dorcadion and Neodorcadion and places Herophila closer to Morimus than to Dorcadion/Lamia. The genus Morimus is the most derived. CO1 mutations loosely separate the forms M. asper and M. funereus, which have similar karyotypes and behaviour and copulate in captivity. The form M. ganglebaueri may have a funereus X asper hybrid origin.

High BrdU Sensitivity of Passeriformes Chromosomes: Conservation of BrdU-Sensitive Fragile Sites on Their Z Chromosomes during Evolution.

Amongst 15 bird species, representative of 7 orders, recurrent breakages evocating the presence of fragile sites were detected in the chromosomes of the 5 species belonging to Passeriformes. These breaks appeared when 5-bromodeoxyuridine (BrdU) was added to the cell culture medium at a dose inefficient for inducing chromosome structure alterations in other birds and mammals. They involved, similarly in male and female, 3 loci on the Z chromosome of 3 Turdus species (Turdidae). Labeling by BrdU antibody confirmed the correlation between BrdU incorporation into DNA and breakage, especially around and in the sites of breakage. Thus, 3 BrdU-sensitive fragile sites were present in the Z chromosomes of these birds. Three fragile sites were also detected at different locations in the Z chromosomes of the European robin (Erithacus rubecula, Muscicapidae), suggesting that a structural rearrangement occurred during the evolution of Turdidae and Muscicapidae. Chromosome banding confirmed this interpretation. Finally, in the more distantly related species Parus major (Paridae), the almost acrocentric Z chromosome displayed a single BrdU-sensitive fragile site in its short arm, and the W appeared to be pulverized by BrdU incorporation. Although it cannot be excluded that the BrdU-sensitive fragile sites may be involved in rearrangements, their conservation in many species, and possibly all Passeriformes, provides evidence that they do not constitute a pejorative character during evolution.

Evolution of the Sex Chromosomes in Beetles. I. The Loss of the Y Chromosome.

In the males of Coleoptera, the most frequent sex chromosome constitution is XY. At metaphase I of meiosis, the X and Y are linked by nucleolar proteins, forming the so-called parachute bivalent (Xyp), which is assumed to allow the non-synapsed X and Y to segregate correctly at anaphase I. However, X0 males are not exceptional, and we explored the relationships between the X and nucleolar proteins in the absence of the Y chromosome in 6 species belonging to different families/subfamilies. Using C-banding and silver staining, we show that nucleolar proteins always remain in contact with the X until anaphase I. These proteins are generally more abundant than in the Xyp bivalent, may remain associated with the NOR during diakinesis, and frequently link the X to 1 or 2 autosomal bivalents, which seem to play the same role as the Y. This role may also be played by B chromosomes, which appear to be more frequent in X0 than in XY males. In conclusion, following Y chromosome loss, various strategies using nucleolar proteins have been developed to facilitate the migration of the unique X at meiotic anaphase I.

The Relationship between the (In-)Stability of NORs and Their Chromosomal Location: The Example of Cercopithecidae and a Short Review of Other Primates.

Amongst Cercopithecidae, the species of the Cercopithecini tribe underwent a very active chromosome evolution, principally by fissions, which increased their chromosome number up to 72. In contrast, all the species of Papionini have fairly similar karyotypes with 42 chromosomes. In animals, nucleolus organizer regions (NORs) are generally considered as instable structures, which frequently vary in size, number, and location at both infra- and interspecific levels. Although in Cercopithecinae the NORs, involved in breaks, exchanges, and translocations, behave like fragile sites in somatic cells, their number and location appear to be very stable between species. Fluorescence in situ hybridization of a 28S rDNA probe on metaphase chromosomes displayed a unique interstitial location in either an acrocentric pair (in 12 species of Cercopithecini) or a metacentric pair (in 6 species of Papionini). A non-exhaustive survey of literature data on NOR location in other primates shows that numerical variations of the NORs principally depend on their location: most multiple NORs are in terminal positions, while almost all unique NORs are in interstitial positions. We propose that this correlation is the consequence of the selection against gametic imbalances involving the chromosomal material distal to the NORs, which is effective when they are interstitially, but not terminally, located. Thus, the consequences of the interstitial NOR instability for reproduction are essentially limited to their size variations, as observed in Cercopithecidae.

Heterozygosity and Chain Multivalents during Meiosis Illustrate Ongoing Evolution as a Result of Multiple Holokinetic Chromosome Fusions in the Genus Melinaea (Lepidoptera, Nymphalidae).

Mitotic and meiotic chromosomes from 2 taxa of the genus Melinaea, M. satevis cydon and M. "satevis" tarapotensis (Lepidoptera: Nymphalidae), and from hybrids produced in captivity were obtained using an improved spreading technique and were subsequently analyzed. In one of the taxa, the presence of trivalents and tetravalents at diakinesis/metaphase I is indicative of heterozygosity for multiple chromosome fusions or fissions, which might explain the highly variable number of chromosomes previously reported in this genus. Two large and complex multivalents were observed in the meiotic cells of the hybrid males (32 chromosomes) obtained from a cross between M. "s." tarapotensis (28 chromosomes) and M. s. cydon (40-43 chromosomes). The contribution of the 2 different haploid karyotypes to these complex figures during meiosis is discussed, and a taxonomic revision is proposed. We conclude that chromosome evolution is active and ongoing, that the karyotype of the common ancestor consisted of at least 48 chromosomes, and that evolution by chromosome fusion rather than fission is responsible for this pattern. Complex chromosome evolution in this genus may drive reproductive isolation and speciation, and highlights the difficulties inherent to the systematics of this group. We also show that Melinaea chromosomes, classically considered as holocentric, are attached to unique, rather than multiple, spindle fibers.

Karyotype diversity suggests that Laonastes aenigmamus (Laotian rock rat) (Rodentia, Diatomyidae) is a multi-specific genus.

Laonastes aenigmamus (Khanyou) is a recently described rodent species living in geographically separated limestone formations of the Khammuan Province in Lao PDR. Chromosomes of 21 specimens of L. aenigmamus were studied using chromosome banding as well as fluorescent in situ hybridization (FISH) techniques using human painting, telomere repeats, and 28S rDNA probes. Four different karyotypes were established. Study with human chromosome paints and FISH revealed that four large chromosomes were formed by multiple common tandem fusions, with persistence of some interstitial telomeres. The rearrangements separating the different karyotypes (I to IV) were also reconstructed. Various combinations of Robertsonian translocations or tandem fusions involving the same chromosomes differentiate these karyotypes. These rearrangements create a strong gametic barrier, which isolates specimens with karyotype II from the others. C-banding and FISH with telomere repeats also exhibit large and systematized differences between karyotype II and others. These data indicate an ancient reproductive separation and suggest that Laonastes is not a mono-specific genus.

Interstitial NORs, Fragile Sites, and Chromosome Evolution: A Not So Simple Relationship - The Example of Melolontha melolontha and Genus Protaetia (Coleoptera: Scarabaeidae).

In the present study, the origin of recurrent rearrangements involving chromosome 6 in 3.2% of cells of Melolontha melolontha (Coleoptera, Scarabaeidae) was investigated. Various chromosome staining techniques, including C-banding, Giemsa and silver staining, as well as fluorescence in situ hybridization with a human 28S rDNA probe, were applied to M. melolontha chromosome spreads. In addition, related species of the genera Melolontha and Protaetia were studied. On chromosome 6 of M. melolontha, there is a fragile site-like structure which corresponds to an interstitial nucleolus organizer region (NOR). Despite this instability, the NOR remains unique and interstitial in this species, as well as in the other species studied. It is proposed that the intercalary position of the NOR both facilitates the detection of its fragile site-like instability and correlates with its relative stability during evolution. We explain this apparent paradox by strong counter-selection for imbalances of the chromosome fragment distal to the interstitial NORs, which would recurrently occur in the progeny of translocation carriers. Thus, the frequent telomeric position of the NORs in most animal and plant taxa would have no functional rationale but would be the consequence of selection against the meiotic transmission of chromosome imbalances.

Common fragile sites: mechanisms of instability revisited.

Common fragile sites (CFSs) are large chromosomal regions prone to breakage upon replication stress that are considered a driving force of oncogenesis. CFSs were long believed to contain sequences blocking fork progression, thus impeding replication completion and leading to DNA breaks upon chromosome condensation. However, recent studies show that delayed completion of DNA replication instead depends on a regional paucity in initiation events. Because the distribution and the timing of these events are cell type dependent, different chromosomal regions can be committed to fragility in different cell types. These new data reveal the epigenetic nature of CFSs and open the way to a reevaluation of the role played by these sites in the formation of chromosome rearrangements found in tumors from different tissues.

The heterochromatic chromosome caps in great apes impact telomere metabolism.

In contrast with the limited sequence divergence accumulated after separation of higher primate lineages, marked cytogenetic variation has been associated with the genome evolution in these species. Studying the impact of such structural variations on defined molecular processes can provide valuable insights on how genome structural organization contributes to organismal evolution. Here, we show that telomeres on chromosome arms carrying subtelomeric heterochromatic caps in the chimpanzee, which are completely absent in humans, replicate later than telomeres on chromosome arms without caps. In gorilla, on the other hand, a proportion of the subtelomeric heterochromatic caps present in most chromosome arms are associated with large blocks of telomere-like sequences that follow a replication program different from that of bona fide telomeres. Strikingly, telomere-containing RNA accumulates extrachromosomally in gorilla mitotic cells, suggesting that at least some aspects of telomere-containing RNA biogenesis have diverged in gorilla, perhaps in concert with the evolution of heterochromatic caps in this species.

Why Are X Autosome Rearrangements so Frequent in Beetles? A Study of 50 Cases.

Amongst the 460 karyotypes of Polyphagan Coleoptera that we studied, 50 (10.8%) were carriers of an X autosome rearrangement. In addition to mitotic metaphase analysis, the correct diagnosis was performed on meiotic cells, principally at the pachytene stage. The percentages of these inter-chromosomal rearrangements, principally fusions, varied in relation to the total diploid number of chromosomes: high (51%) below 19, null at 19, low (2.7%) at 20 (the ancestral and modal number), and slightly increasing from 7.1% to 16.7% from 22 to above 30. The involvement of the X in chromosome fusions appears to be more than seven-fold higher than expected for the average of the autosomes. Examples of karyotypes with X autosome rearrangements are shown, including insertion of the whole X in the autosome (ins(A;X)), which has never been reported before in animals. End-to-end fusions (Robertsonian translocations, terminal rearrangements, and pseudo-dicentrics) are the most frequent types of X autosome rearrangements. As in the 34 species with a 19,X formula, there was no trace of the Y chromosome in the 50 karyotypes with an X autosome rearrangement, which demonstrates the dispensability of this chromosome. In most instances, C-banded heterochromatin was present at the X autosome junction, which suggests that it insulates the gonosome from the autosome portions, whose genes are subjected to different levels of expression. Finally, it is proposed that the very preferential involvement of the X in inter-chromosome rearrangements is explained by: (1) the frequent acrocentric morphology of the X, thus the terminal position of constitutive heterochromatin, which can insulate the attached gonosomal and autosomal components; (2) the dispensability of the Y chromosome, which considerably minimizes the deleterious consequences of the heterozygous status in male meiosis, (3) following the rapid loss of the useless Y chromosome, the correct segregation of the X autosome-autosome trivalent, which ipso facto is ensured by a chiasma in its autosomal portion.

Multiple Chromosome Fissions, Including That of the X Chromosome, in Aulacocyclus tricuspis Kaup (Coleoptera, Passalidae) from New Caledonia: Characterization of a Rare but Recurrent Pathway of Chromosome Evolution in Animals.

The male karyotype of Aulacocyclus tricuspis Kaup 1868 (Coleoptera, Scarabaeoidea, Passalidae, Aulacocyclinae) from New Caledonia contains an exceptionally high number of chromosomes, almost all of which are acrocentric (53,X1X2Y). Unlike the karyotypes of other species of the pantropical family Passalidae, which are principally composed of metacentric chromosomes, this karyotype is derived by fissions involving almost all the autosomes after breakage in their centromere region. This presupposes the duplication of the centromeres. More surprising is the X chromosome fragmentation. The rarity of X chromosome fission during evolution may be explained by the deleterious effects of alterations to the mechanisms of gene dosage compensation (resulting from the over-expression of the unique X chromosome in male insects). Herein, we propose that its occurrence and persistence were facilitated by (1) the presence of amplified heterochromatin in the X chromosome of Passalidae ancestor, and (2) the capacity of heterochromatin to modulate the regulation of gene expression. In A. tricuspis, we suggest that the portion containing the X proper genes and either a gene-free heterochromatin fragment or a fragment containing a few genes insulated from the peculiar regulation of the X by surrounding heterochromatin were separated by fission. Finally, we show that similar karyotypes with multiple acrocentric autosomes and unusual sex chromosomes rarely occur in species of Coleoptera belonging to the families Vesperidae, Tenebrionidae, and Chrysomelidae. Unlike classical Robertsonian evolution by centric fusion, this pathway of chromosome evolution involving the centric fission of autosomes has rarely been documented in animals.

Hormone escape is associated with genomic instability in a human prostate cancer model.

Lack of hormone dependency in prostate cancers is an irreversible event that occurs through generation of genomic instability induced by androgen deprivation. Indeed, the cytogenetic profile of hormone-dependent (HD) prostate cancer remains stable as long as it received a hormone supply, whereas the profile of hormone-independent (HID) variants acquired new and various alterations. This is demonstrated here using a HD xenografted model of a human prostate cancer, PAC120, transplanted for 11 years into male nude mice and 4 HID variants obtained by surgical castration. Cytogenetic analysis, done by karyotype, FISH, CGH and array-CGH, shows that PAC120 at early passage presents numerous chromosomal alterations. Very few additional alterations were found between the 5th and 47th passages, indicating the stability of the parental tumor. HID variants largely maintained the core of chromosomal alterations of PAC120 - losses at 6q, 7p, 12q, 15q and 17q sites. However, each HID variant displayed a number of new alterations, almost all being specific to each variant and very few shared by all. None of the HID had androgen receptor mutations. Our study indicates that hormone castration is responsible for genomic instability generating new cytogenetic abnormalities susceptible to alter the properties of cancer cell associated with tumor progression, such as increased cell survival and ability to metastasize.

Establishment of human colon cancer cell lines from fresh tumors versus xenografts: comparison of success rate and cell line features.

Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease.

Different behaviour of C-banded peri-centromeric heterochromatin between sex chromosomes and autosomes in Polyphagan beetles.

Heterochromatin variation was studied after C-banding of male karyotypes with a XY sex formula from 224 species belonging to most of the main families of Coleoptera. The karyotypes were classified in relation with the ratio heterochromatin/euchromatin total amounts and the amounts of heterochromatin on autosomes and gonosomes were compared. The C-banded karyotypes of 19 species, representing characteristic profiles are presented. This analysis shows that there is a strong tendency for the homogenization of the size of the peri-centromeric C-banded heterochromatin on autosomes. The amount of heterochromatin on the X roughly follows the variations of autosomes. At contrast, the C-banded heterochromatin of the Y, most frequently absent or very small and rarely amplified, looks quite independent from that of other chromosomes. We conclude that the Xs and autosomes, but not the Y, possibly share some, but not all mechanisms of heterochromatin amplification/reduction. The theoretical models of heterochromatin expansion are discussed in the light of these data.

Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide.

Common fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription-replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription-replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.

Preferential binding of a G-quadruplex ligand to human chromosome ends.

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, 3H-360A (2,6-N,N'-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-3H]. We verified the in vitro selectivity of 3H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that 3H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3'-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with 3H-360A. We found that 3H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.

Genetic alterations associated with acquired temozolomide resistance in SNB-19, a human glioma cell line.

Gliomas are highly lethal neoplasms that cannot be cured by currently available therapies. Temozolomide is a recently introduced alkylating agent that has yielded a significant benefit in the treatment of high-grade gliomas. However, either de novo or acquired chemoresistance occurs frequently and has been attributed to increased levels of O6-methylguanine-DNA methyltransferase or to the loss of mismatch repair capacity. However, very few gliomas overexpress O6-methylguanine-DNA methyltransferase or are mismatch repair-deficient, suggesting that other mechanisms may be involved in the resistance to temozolomide. The purpose of the present study was to generate temozolomide-resistant variants from a human glioma cell line (SNB-19) and to use large-scale genomic and transcriptional analyses to study the molecular basis of acquired temozolomide resistance. Two independently obtained temozolomide-resistant variants exhibited no cross-resistance to other alkylating agents [1,3-bis(2-chloroethyl)-1-nitrosourea and carboplatin] and shared genetic alterations, such as loss of a 2p region and loss of amplification of chromosome 4 and 16q regions. The karyotypic alterations were compatible with clonal selection of preexistent resistant cells in the parental SNB-19 cell line. Microarray analysis showed that 78 out of 17,000 genes were differentially expressed between parental cells and both temozolomide-resistant variants. None are implicated in known resistance mechanisms, such as DNA repair, whereas interestingly, several genes involved in differentiation were down-regulated. The data suggest that the acquisition of resistance to temozolomide in this model resulted from the selection of less differentiated preexistent resistant cells in the parental tumor.

Long-term controlled immortalization of a primate hepatic progenitor cell line after Simian virus 40 T-Antigen gene transfer.

Hepatoblasts are bipotent progenitors of both hepatocytes and cholangiocytes. The lack of stable in vitro culture systems for such cells makes it necessary to generate liver progenitor cell lines by means of immortalization. In this study, we describe the long-term behaviour of a clone of simian foetal hepatic progenitor cells immortalized by Simian virus 40 (SV40) large T-antigen (T-Ag) flanked by loxP sites. Immortalization was associated with the re-expression of telomerase activity, which decreased at late passages (population doubling 120) after more than a year in culture. This decrease was concomitant to telomere shortening and karyotypic instability. However, the chromosomes carrying the p53 gene remained intact and long-term immortalized progenitor cells maintained contact inhibition and proliferative properties. They also displayed the features of a normal bipotent phenotype. We constructed a retroviral vector expressing an inducible Cre recombinase and transferred it into the immortalized progenitors. Activation of the Cre recombinase by 4-hydroxy-tamoxifen induced SV40 T-Ag excision, leading to the death of cells expressing Cre recombinase. Immortalized progenitors at late passages stopped growing and eventually disappeared after transplantation into the livers of immunocompromised mice. These cells provide a novel model to study hepatic differentiation and carcinogenesis.

Chromosome comparison of 17 species / sub-species of African Goliathini (Coleoptera, Scarabaeidae, Cetoniinae).

The mitotic karyotypes of 17 species of African Goliathini (Cetoniinae) are described using various chromosome banding techniques. All but one are composed of 20 chromosomes, mostly metacentric, forming a karyotype assumed to be close to that of the Polyphaga ancestor. The most derived karyotypes are those of Goliathus goliatus Drury, 1770, with eight pairs of acrocentrics and Chlorocana africana Drury, 1773, with only14 chromosomes. In species of the genera Cyprolais Burmeister, 1842, Megalorhina Westwood, 1847, Stephanocrates Kolbe, 1894 and Stephanorrhina Burmeister, 1842, large additions of variable heterochromatin are observed on both some particular autosomes and the X chromosome. Species of the genera Eudicella White, 1839 and Dicronorrhina Burmeister, 1842 share the same sub-metacentric X. Although each species possesses its own karyotype, it remains impossible to propose robust phylogenetic relationships on the basis of chromosome data only.