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.

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.

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.

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.

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.

Genome assembly of 3 Amazonian Morpho butterfly species reveals Z-chromosome rearrangements between closely related species living in sympatry.

The genomic processes enabling speciation and species coexistence in sympatry are still largely unknown. Here we describe the whole-genome sequencing and assembly of 3 closely related species from the butterfly genus Morpho: Morpho achilles (Linnaeus, 1758), Morpho helenor (Cramer, 1776), and Morpho deidamia (Höbner, 1819). These large blue butterflies are emblematic species of the Amazonian rainforest. They live in sympatry in a wide range of their geographical distribution and display parallel diversification of dorsal wing color pattern, suggesting local mimicry. By sequencing, assembling, and annotating their genomes, we aim at uncovering prezygotic barriers preventing gene flow between these sympatric species. We found a genome size of  480 Mb for the 3 species and a chromosomal number ranging from 2n = 54 for M. deidamia to 2n = 56 for M. achilles and M. helenor. We also detected inversions on the sex chromosome Z that were differentially fixed between species, suggesting that chromosomal rearrangements may contribute to their reproductive isolation. The annotation of their genomes allowed us to recover in each species at least 12,000 protein-coding genes and to discover duplications of genes potentially involved in prezygotic isolation like genes controlling color discrimination (L-opsin). Altogether, the assembly and the annotation of these 3 new reference genomes open new research avenues into the genomic architecture of speciation and reinforcement in sympatry, establishing Morpho butterflies as a new eco-evolutionary model.

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.

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.

Parthenogenetic reproduction demonstrated in the diploid Spasalus puncticollis (Le Peletier & Serville 1825), n. stat., from the Antilles (Coleoptera, Scarabaeoidea, Passalidae).

Only females were observed in Spasalus crenatus (Mac Leay 1819) in the Antilles, from Puerto Rico to Saint-Vincent, whereas both sexes are in Trinidad and on the continent. No difference in endo- and ectodermic female genitalia could be noticed between the two populations. Chromosomes of specimens from Guadeloupe reveal a 26,XX karyotype, as in females of various sexual species of Passalini, which demonstrates its diploidy. Breedings were developed with isolated immature stages. After nine years, descendants from a single female are demonstrating their parthenogenetic reproduction. This is the first recorded parthenogenesis in Passalidae and a rare telytoky in diploid insects. Relationships between parthenogenesis, diploidy and insularity are discussed in the scheme of geographical parthenogenesis. No discriminant morphological character on adults could be found between the two populations, except the total length. The modes of reproduction distinguishing the two geographically separated populations suggest the presence of two taxa: S. crenatus on the continent and Trinidad; the parthenote S. puncticollis (Le Peletier & Serville 1825), n. stat., on the Arc of the Antilles.

Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas.

Amplification of the epidermal growth factor receptor gene on double minutes is recurrently observed in cells of advanced gliomas, but the structure of these extrachromosomal circular DNA molecules and the mechanisms responsible for their formation are still poorly understood. By using quantitative PCR and chromosome walking, we investigated the genetic content and the organization of the repeats in the double minutes of seven gliomas. It was established that all of the amplicons of a given tumor derive from a single founding extrachromosomal DNA molecule. In each of these gliomas, the founding molecule was generated by a simple event that circularizes a chromosome fragment overlapping the epidermal growth factor receptor gene. In all cases, the fusion of the two ends of this initial amplicon resulted from microhomology-based nonhomologous end-joining. Furthermore, the corresponding chromosomal loci were not rearranged, which strongly suggests that a postreplicative event was responsible for the formation of each of these initial amplicons.