B1 was the ancestor B chromosome variant in the western Mediterranean area in the grasshopper Eyprepocnemis plorans.

We analyzed the distribution of 2 repetitive DNAs, i.e. ribosomal DNA (rDNA) and a satellite DNA (satDNA), on the B chromosomes found in 17 natural populations of the grasshopper Eyprepocnemis ploransplorans sampled around the western Mediterranean region, including the Iberian Peninsula, Balearic Islands, Sicily, and Tunisia. Based on the amount of these repetitive DNAs, 4 types of B variants were found: B1, showing an equal or higher amount of rDNA than satDNA, and 3 other variants, B2, B24 and B5, bearing a higher amount of satDNA than rDNA. The variants B1 and B2 varied in size among populations: B1 was about half the size of the X chromosome in Balearic Islands, but two-thirds of the X in Iberian populations at Alicante, Murcia and Albacete provinces. Likewise, B2 was about one-third the size of the X chromosome in populations from the Granada province but half the size of the X in the populations collected at Málaga province. The widespread geographical distribution of the B1 variant makes it the best candidate for being the ancestor B chromosome in the whole western Mediterranean region.

Cytogenetic analysis of Lagria villosa (Coleoptera, Tenebrionidae): emphasis on the mechanism of association of the Xy(p) sex chromosomes.

The Xy(p) sex determination mechanism is the system most frequent and ancestral to Coleoptera. Moreover, the presence of argyrophilous material associated with the sex bivalent is described as being responsible for the maintenance and association of these chromosomes. There are no karyotype data available regarding the genus Lagria and no consensus in the literature regarding the argyrophilous material present in the lumen of sex bivalent. Therefore, the aim of this work was to investigate the mechanism of sex chromosome bivalent association in Lagria villosa by analyzing the argyrophilous nature of the material present in the Xy(p) lumen. It was also intended to characterize L. villosa cytogenetically. The analysis of meiotic cells showed 2n = 18 = 16+Xy(p) for males and 2n = 18 = 16+XX in females and the meiotic formula was 2n = 8(II)+Xy(p). The C-banding showed blocks of pericentromeric heterochromatin in all chromosomes except in the y(p) chromosome. In these regions, the use of fluorochromes revealed the presence of heterochromatin containing GC rich DNA sequences. The study of synaptonemal complex showed a gradual increase in the electron-density of the axial elements of the sex chromosomes and their association with strongly electron-dense material. The pepsin pretreatment revealed that the material impregnated by silver is protein.

A new contribution to the cytogenetic knowledge of Alticinae (Coleoptera, Chrysomelidae).

Seven species of Alticinae, mostly from Spain, have been chromosomally surveyed from male meiotic or mitotic cells. The two Spanish species of Oedionychus, O. cinctus and O. limbatus, have shown a karyotype of 2n=16+X+Y, with a lower number of autosomes than in their congeneric Neotropical species, but sharing with them the giant distance-paired sex-chromosomes. The Uruguayan Macrohaltica transversa has a 11+X+y meioformula, which agrees with that found in four congeneric species and in most ones of the allied genus Altica. The meioformula of Hermaeophaga ruficollis 7+Xy, differs only slightly from that reported in another congeneric species. Moreover those of Longitarsus aeruginosus 11+Xy, L. australis 13+Xy, and Psylliodes obscuroaeneus 15+Xy(p,) are similar to some of those previously published for other species in both genera.

C-bands on chromosomes of 32 beetle species (Coleoptera: Elateridae, Cantharidae, Oedemeridae, Cerambycidae, Anthicidae, Chrysomelidae, Attelabidae and Curculionidae).

C-banding patterns of 32 beetle species from the families Elateridae, Cantharidae, Oedemeridae, Cerambycidae, Anthicidae, Chrysomelidae, Attelabidae and Curculionidae were studied using the C-banding technique. Mitotic and meiotic chromosomes were previously described for 14 species. From among 18 species that had never been cytogenetically studied, we determined the diploid and haploid chromosome numbers and the sex determination system for 12 beetles. The karyotype for 6 species is not described because of a lack of mitotic and meiotic metaphases. Results confirm that most of the beetle species possess a small amount of heterochromatin and C-positive segments are weakly visible in pachytene stages and weakly or imperceptible in mitotic and meiotic metaphases. In some species with a large amount of heterochromatin, C-bands were observed in the centromeric region in all autosomes and the X chromosome. The Y chromosome does not show C-bands with the exception of Oedemera viridis in which it possesses a small band of heterochromatin.

Evolutionary dynamics of a B chromosome invasion in island populations of the grasshopper Eyprepocnemis plorans.

Four natural populations of the grasshopper Eyprepocnemis plorans in the Mallorca island were analysed for several years revealing the recent invasion of the B1 chromosome from the south-west part of the island (Palma region) towards the north and to the east. In only 10 years, the mean number of Bs in the northern population at Pollença increased from 0.053 to 0.692. Therefore, B chromosome invasion seems to be very rapid and has recently arrived to the north of the island. The south-west (close to Palma) is the most likely point at which B invasion started in the Mallorca Island. Finally, the number of B chromosomes was significantly associated to an increase in chiasma frequency (and thus recombination) in A chromosomes.

Complex structural features of satellite DNA sequences in the genus Pimelia (Coleoptera: Tenebrionidae): random differential amplification from a common 'satellite DNA library'.

The major satellites of the nine species of the subgenera Pimelia s. str. and Amblyptera characterised in this paper are composed of longer monomers (500 and 700 bp) than those described previously in 26 Pimelia s. str. taxa (357 bp, a sequence called PIM357). Sequence analysis reveals partial similarity among these satellites and with the PIM357 monomers. The discrepancy between the phylogeny obtained based on three mitochondrial and two nuclear markers and that deduced from satellite DNA (stDNA) sequences suggests that the different Pimelia satellites were already present in a common ancestor forming what has been called a 'satellite DNA library'. Thus, the satellite profiles in the living species result from a random amplification of sequences from that 'library' during diversification of the species. However, species-specific turnover in the sequences has occurred at different rates. They have included abrupt replacements, a gradual divergence and, in other cases, no apparent change in sequence composition over a considerable evolutionary time. The results also suggest a common evolutionary origin of all these Pimelia satellite sequences, involving several rearrangements. We propose that the repeat unit of about 500 bp has originated from the insertion of a DNA fragment of 141 bp into the PIM357 unit. The 705-bp repeats have originated from a 32-bp direct duplication and the insertion of a 141-bp fragment in inverted orientation relative to a basic structure of 533 bp.

A cytogenetic study of the leaf beetle genus Cyrtonus (Coleoptera, Chrysomelidae).

The chromosomes of ten species of Cyrtonus and the genome sizes of six are surveyed. Among the total of 15 chromosomally studied species, 11 have 2n = 28 chromosomes and a 13 + Xyp male meioformula, three have 2n = 40 and 19 + Xyp and one 2n = 46 and 22 + Xyp. All but one species with 28 chromosomes show only metacentric or submetacentric chromosomes, whereas the species with 40 and 46 chromosomes display some telocentrics or subtelocentrics, that are probably derived from the former by centric fissions. However, since the number of major chromosome arms is strikingly higher in these latter species (NF = 70 and 78) than in the 28-chromosome species (mostly NF = 56), other chromosomal rearrangements such as pericentric inversions or heterochromatin accretions could also be involved. The genome sizes display a narrow range, from 1C = 0.6-1.22 pg, and they are not significantly correlated with the chromosome numbers. Some possible factors implied in the rough chromosomal evolution of Cyrtonus are discussed in relation to a few other genera of the subfamily Chrysomelinae.

Satellite DNA in the elm leaf beetle, Xanthogaleruca luteola (Coleoptera, Chrysomelidae): characterization, interpopulation analysis, and chromosome location.

In this paper the satellite DNA (stDNA) of the phytophagous beetle Xanthogaleruca luteola is analyzed. It is organized in a tandem repeat of 149-bp-long monomers, has an AT content of 59%, and presents inverted internal repeats. Restriction analysis of the total DNA with methylation-sensitive enzymes suggests that this repetitive DNA is not methylated. Analysis of the electrophoretic mobility of stDNA on non-denaturing polyacrylamide gels showed that this stDNA is not curved. In situ hybridization with a biotinylated probe of the stDNA revealed a pericentromeric localization of these sequences in the majority of the meiotic bivalents. We have studied the stDNA of X. luteola from two populations with very distinct geographical origins. The sequence and phylogenetic analysis of monomers from these two populations showed that the repetitive element is conserved within the species. Putative gene conversion tracts are identified when the different monomers of the same population are compared. These results could indicate the existence of processes of homogenization that would extend these mutations to all the satellite repeats.

Sequential FISH analysis with rDNA genes and Ag-NOR banding in the lady beetle Olla v-nigrum (Coleoptera: Coccinellidae).

We have characterized the meiosis of Olla v-nigrum by standard analysis, performed a NOR study using NOR banding, FISH of rDNA genes and sequential FISH/AgNOR analysis, and adapted the FISH methodology to Coccinellidae. The chromosome number determined at metaphase I was n = 9 + Xyp. At zygotene it was possible to identify the sex vesicle which presented a deeply stained heteropycnotic block. Chromosome X is much larger than the y and the two combine, forming a "parachute" in metaphase I. FISH analysis using a probe of rDNA genes 18S, 28S and 5.8S of D. melanogaster was used to map the genes in the sex vesicle. The NOR band showed high gene activity in this region. These results were confirmed using sequential FISH/Ag NOR analysis. The data obtained for Olla v-nigrum agree with the classical hypothesis raised to explain the type of sex chromosome association in a parachute format (Xyp) as being due to the presence of nucleolar material. The chromosome number and parachute configuration during metaphase I in this species agree with the basic karyotype of most Coleopterans. The major adaptation of the FISH method was the simultaneous denaturation and hybridization that permitted preservation of chromosome morphology, an essential factor when the chromosomes are small.

Sequence, secondary structure and phylogenetic analyses of the ribosomal internal transcribed spacer 2 (ITS2) in the Timarcha leaf beetles (Coleoptera: Chrysomelidae).

Internal transcribed spacer 2 (ITS2) sequences of the nuclear rDNA in forty-seven specimens (thirty-four species) of the leaf beetle genus Timarcha have been studied. Timarcha ITS2 (523 bp on average) share some sequence features with other Chrysomeloidea relatives (Chrysolina, Diabrotica and Bruchus) but have no clear similarity with any other arthropod ITS2 sequences. Interspecific divergences are in the range 0.002-0.166, and 0.124-0.206 in the comparisons between subgenera. No evidence of intragenomic divergent ITS2 sequences has been found. Secondary structures are concordant with the four-domain model proposed for vertebrates and yeast, but differs from those proposed for dipterans. Phylogenetic analysis of the ITS2 data confirms the results of a previous study based on mitochondrial sequences, as the basality of the Metallotimarcha subgenus and the absence of phylogenetic support for the Timarchostoma subgenus.

Nested cladistic analysis, phylogeography and speciation in the Timarcha goettingensis complex (Coleoptera, chrysomelidae).

The Timarcha goettingensis complex is a monophyletic assemblage of closely related leaf beetles (Chrysomelidae), distributed from the north half of the Iberian Peninsula to Central Europe. Oligophagy, mountainous habitat and apterism are factors which are assumed to promote speciation in these beetles. We have used cytochrome oxidase subunit II mitochondrial DNA genealogies obtained from 31 sampling localities and a nested geographical distance analysis to assess the population structure and demographic factors explaining the geographical distributions of the mtDNA haplotypes in the T. goettingensis complex. The results show that there is a significant association between genetic structuring and geography. Inferences about the historical population processes in the species complex are discussed, being in general in accordance with contiguous range expansions and past fragmentations. The use of the cohesion species concept approach suggests the existence of several systematic ranks among the different T. goettingensis populations, which is in part supported by ecological traits such as trophic selection and altitudinal distribution.

The evolutionary history of the genus Timarcha (Coleoptera, Chrysomelidae) inferred from mitochondrial COII gene and partial 16S rDNA sequences.

The apterous genus Timarcha consists of three subgenera and more than 100 species in its Palearctic distribution, with specialized feeding on few plant families. Fifty-four sequences sampled from 31 taxa of the genus plus three outgroup leaf beetles were studied for their complete cytochrome oxidase II (COII) and a fragment of 16S rDNA mitochondrial genes, representing a total of about 1200 bp. Phylogenetic analyses using maximum-parsimony and distance methods for each gene separately and for the combined data set gave compatible topologies. The subgenus Metallotimarcha consistently appears in a basal position and is well differentiated from the remaining Timarcha, but no clear monophyletic grouping of Timarchostoma and Timarcha s. str. subgenera can be deduced from our analysis. Calibration of the molecular clock has been done using the opening of the Gibraltar Strait after the Messinian salinity crisis (about 5.5 MYA) as the biogeographic event causing disjunction of two particular taxa. Accordingly, the COII evolutionary rate has been estimated to be of 0.76 x 10(-8) substitution/site/year in Timarcha. Relation between phylogeny and host-plant use indicates widening of trophic regime as a derived character in Timarcha.

Characterization and chromosome location of satellite DNA in the leaf beetle Chrysolina americana (Coleoptera, Chrysomelidae).

This paper is the first record of the satellite DNA of the specialized phytophagous genus Chrysolina. The satellite DNA of Chrysolina americana is organized in a tandem repeat of monomers 189 bp long, has a A + T content of 59.6% and presents direct and inverted internal repeats. Restriction analysis of the total DNA with methylation sensitive enzymes suggests that this repetitive DNA is undermethylated. In siti hybridization with a biotinylated probe of the satellite DNA showed the pericentromeric localization of these sequences in all meiotic bivalents. The presence of this repetitive DNA in other species of the genus was also tested by Southern analysis. The results showed that this satellite DNA sequence is specific to the C. americana genome and has not been found in three other species of Chrysolina with a different choice of host plants than in the former.

Mitochondrial DNA phylogeny and the evolution of host-plant use in palearctic Chrysolina (Coleoptera, Chrysomelidae) leaf beetles.

The genus Chrysolina consists of specialized phytophagous leaf-beetles (Coleoptera, Chrysomelidae) with feed on several plant families. There is no explicit phylogenetic hypothesis available for this genus, which includes 65 subgenera and more than 400 species with a wide distribution. We obtained 839-bp sequence data from the 16S rDNA and cytochrome oxidase subunit I (COI) mitochondrial genes. Thirty Chrysolina taxa representing eight host-plant affiliations, two species of the closely related genus Oreina, and two outgroups were sampled. These data sets were used separately and combined to obtain the mitochondrial cladogram of the group using maximum-parsimony and maximum-likelihood criteria. The results were compared to current proposals for Chrysolina systematics that are based on morphological, ecological, and karyological data. The trees obtained were in the most part congruent with the proposed ancestral association of Chrysolina to Lamiaceae based on chromosome number in several lineages. A minimum of five host-plant switches from the ancestral state inferred at the family level and two at the subclass level suggests the absence of parallel evolution of beetles and their host plants. Another switch leading to oligophagy at the family level was deduced to have occurred in the lineage of the subgenus Chrysolina s.str.

Polymorphic curvature of satellite DNA in three subspecies of the beetle Pimelia sparsa.

The curvature of the monomeric repeats of satellite DNAs from three subspecies of the beetle Pimelia sparsa (Coleoptera, Tenebrionidae) has been analysed. Evidence of curvature was inferred from their retarded migration in native polyacrylamide gels, which was confirmed by direct electron microscopy visualisation. Sequence-comparison analysis, which included sequence alignments and modelling studies, was used to reveal the patterns of local bending and curvature. The effects of the minor-groove-binding drugs distamycin and berenil on the curvature were assayed, and analysed with respect to their (A+T)-rich sequence preferences. Since our study deals with satellite DNAs from closely related organisms (three subspecies), we correlated the differences in sequence, and also the high similarity conserved in the (A+T)-rich regions, with the changes in the patterns of curvature.

Conservation of satellite DNA in species of the genus Pimelia (Tenebrionidae, Coleoptera).

Satellite DNA has been characterized in six allopatric species from the genus Pimelia: P. interjecta, P. integra, P. variolosa and P. baetica, inhabiting Iberian Peninsula, and P. elevata and P. criba, endemic to Balearic Islands Ibiza and Mallorca, respectively. All species show the presence of a single satellite DNA of a basic monomer length of 357 bp and A+T content of 69%, comprising a considerable amount of the genome (39%-45%, corresponding to about 4.5 x 10(5) copies per haploid genome). The sequence analysis of 22 cloned repeats reveals very high intra- and interspecific sequence similarity. Phylogenetic analysis separates the satellite sequences into two clusters, each comprising clones from three species exclusively. Within the clusters, satellite clones are not grouped species-specifically, except those of P. integra where species-diagnostic nt substitutions are detected with a pattern that could be produced by gene conversion. Such high sequence conservation could be related to preservation of satellite DNA curvature, resulting in a higher order helical structure, proposed to act as a specific protein binding domain.

Tenebrio obscurus satellite DNA is resistant to cleavage by restriction endonucleases in situ.

Satellite DNA from the mealworm beetle, Tenebrio obscurus, is composed of 344 bp long monomers of high AT content (68%), and represents 15% of the total DNA. In situ hybridization reveals the positions of the satellite on the pericentromeric heterochromatin of all T. obscurus chromosomes. To compare restriction enzyme (RE) effects with those on naked DNA, fixed chromosomes were digested with REs having recognition sites in most of the satellite monomers, and also with enzymes having target sites present only partially, or very rarely in the satellite units. All enzymes produce similar C-like banding patterns showing heterochromatin resistance to digestion regardless of the enzyme used. In situ nick translation suggests the inability of REs to cleave satellite DNA rather than the inefficient extraction of DNA fragments. DNA in heterochromatin was only extensively digested when the chromosomes were preincubated with proteinase K, indicating that accessibility of REs to DNA is increased by the removal of chromosomal proteins. This is in contrast to recently obtained results in Tenebrio molitor, where cleavage of satellite DNA is equally efficient in both fixed chromosomes and in naked DNA. The satellite DNAs of the two congeneric species differ in their AT content, and their primary and higher order structure, which could influence both heterochromatin structure and the accessibility of REs to satellite DNA.

Localization of tandemly repeated DNA sequences in beetle chromosomes by fluorescent in situ hybridization.

In situ hybridization to chromosomes and nuclei of Tenebrio molitor shows the massive presence of a species-specific satellite DNA in all chromosomes and six sites of rDNA in mitotic chromosomes. These sites are located in two autosomal pairs and in the X and Y chromosomes. In a related species, Misolampus goudoti, in which two different families of highly repetitive DNA have been previously characterized, one family is located in centromeric regions of all chromosomes with the exception of chromosome Y, while the other repeated DNA family is present both in centromeric and distal regions of all chromosomes. rRNA genes in this species are present in a medium-sized autosomal pair only. These results show that molecular cytogenetics can be applied to coleopteran chromosomes and open the way for a physical mapping of DNA sequences in these organisms. The results also provide insights into the type of meiotic association of the X and Y chromosomes in Coleoptera and the distribution of repeated DNAs within the genome of these insects.

Satellite DNA and heterochromatin of the flour beetle Tribolium confusum.

The chromosomes of Tribolium confusum have conspicuous bulks of pericentromeric constitutive heterochromatin. The amount of heterochromatin measured by C-banding in metaphase chromosomes is estimated to be 40-45%. It is composed of an A + T rich DNA according to the distamycin A/diamidinophenylindol staining of chromosomes. Restriction analysis of isolated T. confusum genomic DNA shows that this species has a satellite DNA that constitutes about 40% of the genome. Cloning and sequencing experiments reveal a monomer length of 158 base pairs and a copy number of 5.77 x 10(5) per haploid genome. Its sequence is A + T rich (73%), with direct and inverted repeats, one of them with a possibility of forming stable cruciform structure. The abundance, monomer length, and the mutation rate are similar to those found in other satellite families from different species of Tenebrionidae, but no sequence homology has been found among them. No retarded mobility of satellite DNA, characteristic for molecules with sequence-induced curvature, has been detected by electrophoresis on nondenaturing polyacrylamide gels. In situ digestions with restriction enzymes and in situ hybridization show that this satellite DNA is located in pericentromeric positions of all chromosomes coinciding with C-bands.

Characterization of the heterochromatin of the darkling beetle Misolampus goudoti: cloning of two satellite DNA families and digestion of chromosomes with restriction enzymes.

The darkling beetle Misolampus goudoti Er. has 58% of C-banded chromosome material. In this paper we deal with the study of the heterochromatin of this insect both by molecular and cytogenetical methods. Two different satellite DNA families have been characterized in Misolampus goudoti by agarose gel electrophoresis of EcoRI and PstI restriction fragments, respectively. The EcoRI family is composed of a monomeric unit of 196 bp (64.3% A-T rich) DNA sequence, representing about 120,000 copies per haploid genome. The presence of frequent intermediate-size satellite variants and an internal direct repetition of 61 bp in the EcoRI repetitive main monomer suggest that the evolution of this satellite proceeded by unequal crossing-over, occurring both within and between the 196 bp unit. Another highly repetitive sequence, defined by digestion of genomic DNA with PstI, has a more complex unit of 1.2 kb with about 70,000 copies per haploid genome. In situ digestion of M. goudoti chromosomes with restriction enzymes shows a non-specific chromosome DNA extraction from pericentromeric positions with EcoRI and chromosome specific extraction of DNA with PstI and HinfI. This is discussed in relation to the chromosomal location of both satellites.