J. L. Bella

Correlation between constitutive heterochromatin and restriction enzyme resistant chromatin in Arcyptera tornosi (Orthoptera)

Fixed mitotic chromosomes of A. tornosi have been analysed by means of C-banding, DA-DAPI and Chromomicin A3 fluorescence, as well as by digestion in situ with Alu I, Hae III, Hinf I and Hind III restriction endonucleases. From the results obtained at least nine types of chromatin can be distinguished in A. tornosi, Some C-band positive areas (constitutive heterochromatin) which show a characteristic fluorescence pattern are digested by specific endonucleases, whilst others are undigested. C-band negative areas (euchromatin) are digested by some restriction endonucleases but not by others. Regions digested are supposed to contain highly repetitive DNAs. It is noteworthy, however, that the heterochromatin associated with NORs is not attacked by any of the enzymes we used, while regions believed to contain AT-rich DNA (DA-DAPI positive) are digested by Hae III that cleaves the GG↓CC base sequence target.

The origin of the Chorthippus parallelus hybrid zone: chromosomal evidence of multiple refugia for Iberian populations

A study of the variation in pattern and frequency of constitutive heterochromatin and nucleolar organizing regions of the X chromosomes of male Chorthippus parallelus grasshoppers in 25 populations within the Iberian peninsula requires us to revise our interpretation of the biogeography and evolutionary history of this species. Hybridization between the subspecies Cp erythropus and Cp parallelus, previously only known from populations in the Pyrenean cols, is shown to extend at least 400 km further into north‐west Spain. A novel X‐chromosome variant is described that appears to be close to fixation in 18 populations, mainly from the centre and south of Spain. Our findings indicate a possible independent origin for each of three distinct, nonderivative X variants present in Spain: the northern Cp erythropus and Cp parallelus variants, and a central‐southern Cp erythropus variant. The first two are distinguished by interstitial and distal C bands, respectively, whereas the central‐southern form has neither. This central‐southern form is probably the current representative of the ancestral Iberian X variant. The pattern of variation supports the hypotheses of multiple refugia for Iberian populations and that more hybrid zones exist between these chromosomal variants.

Chromosomal differentiation in Helianthus annuus var. macrocarpus : heterochromatin characterization and rDNA location

The 2n = 34 chromosomes of the inbred line HA89, and the Flamme and Mirasol hybrids of Helianthus annuus var. macrocarpus possess centromeric heterochromatin as established by Giemsa C-banding. This heterochromatin can not be differentiated by fluorochromes such as DAPI or Chromomycin A3, with selective affinity for specific DNA base pairs. This situation probably results from either a balanced AT/GC composition of the involved repeat or the existence of alternating repetitive sequences of opposite base pair composition in these heterochromatic areas. However, there is also heterochromatin associated with the secondary constrictions on three pairs of chromosomes. This heterochromatin appears to be GC-rich according to its response to the fluorochrome treatments, thus indicating heterochromatin heterogeneity in H. annuus. Silver staining reveals the existence of active NORs associated with these secondary constrictions. In situ hybridization with an rDNA probe confirms these results and makes the existence of other inactive rDNA sites unlikely. These results are relevant to evolutionary and breeding studies on sunflowers.

Meiotic imbalance in laboratory-produced hybrid males of Chorthippus parallelus parallelus and Chorthippus parallelus erythropus

The grasshopper Chorthippus parallelus has two quite distinct subspecies, which meet along the Pyrenees forming a hybrid zone. Using silver staining we show that on the French side Cp. parallelus has three nucleolar organizer regions, on the L 2 , L 3 and X chromosomes, while on the Spanish side Cp. erythropus has only two NORs, on the L 2 and L 3 . Laboratory F1 hybrid males show reciprocal differences in the expression of NORs. When a Cp. erythropus is female parent the male progeny show four active NORs in mitotic cells and two silver precipitates in meiotic cells, as expected. But when a Cp. parallelus female donates the X with a NOR, her male offspring have a variable disrupted nucleolar expression. Some NORs are not expressed and extra sites of cryptic rDNA are revealed. Meiosis is more disturbed in this latter F1 cross with higher levels of polyploidy, but both Fls show around 90% spermatid abnormality. Such variation in rDNA expression is also found in individuals collected from the hybrid zone, and the role of this disturbance in affecting fitness is discussed.

A hybrid zone between two subspecies of Chorthippus parallelus. X-chromosome variation through a contact zone

Two grasshopper subspecies, Chorthippus parallelus parallelus (Cpp) and Chorthippus parallelus erythropus (Cpe), meet along the Pyrenees where they hybridize and produce a hybrid zone. A contact zone located in the Col de Portalet has been analyzed for the distribution of chromosome markers on the sex (X) chromosome in pure and hybrid populations. C‐banding allowed us to distinguish both pure subspecific forms and recombinant forms, and to demonstrate their different frequencies through the contact zone. Interestingly, the distal C‐band (P) on the X chromosome that characterizes Cpp individuals occurs at very high frequency through the zone and then drops sharply, close to where pure Cpe populations are found. A novel interstitial C‐band (H) has been found, probably derived from that characterizing Cpe individuals (E). This marker band is only present in hybrid populations. These data are discussed in the light of the dynamics of the hybrid zone.

Distribution of Wolbachia infection in Chorthippus parallelus populations within and beyond a Pyrenean hybrid zone.

Two subspecies of the meadow grasshopper Chorthippus parallelus meet in a secondary hybrid zone in the Pyrenees. We have recently detected Wolbachia infection in this extensively studied species. C. p. parallelus (Cpp) and C. p. erythropus (Cpe) harbour bacteria from the B and F supergroups, but they differ noticeably in the incidence and type of infection present in their populations. We can distinguish different regional infection patterns that are associated with the distribution of pure and hybrid C. parallelus individuals. The northern pattern of low-level infection characterizes Cpp populations, whereas the southern pattern of high-level infection affects Cpe locations. These patterns converge in the hybrid zone and generate a third infection pattern featuring an extremely high degree of co-infection with the two Wolbachia types in hybrids. The distribution of Wolbachia among the studied populations encourages us to consider the possibility that this bacterium has a significant influence on the origin, maintenance and dynamics of this hybrid zone, given the reproductive alterations that are often associated with Wolbachia.

Low levels of chromosomal differentiation between the grasshoppers Chorthippus brunneus and Chorthippus jacobsi (Orthoptera; Acrididae) in northern Spain

The grasshopper species Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae) form a hybrid zone in northern Spain. These species probably diverged while isolated in southern refugia during one of the recent ice ages, and are clearly distinguished by morphology and male calling song. However, in contrast to other Chorthippus taxa that form hybrid zones in Europe, these two species cannot be reliably distinguished on the basis of characteristics of the karyotype such as heterochromatin banding patterns and composition, as revealed by C-banding and fluorochrome staining. Silver staining also reveals the presence of two autosomal nucleolar organiser regions (NORs) in both species. However, differentiation between C. brunneus and C. jacobsi was revealed on the X chromosome using fluorescent in situ hybridisation (FISH). C. brunneus individuals showed additional rDNA sequences on the X chromosome that were not observed in any C. jacobsi individuals. These sequences are not transcribed, indicating either mutational silencing of an ancestral NOR on the X chromosome, or the transposition of non-functional sequences from the autosomes. The implications of these results for the evolution of NOR number in Chorthippus are discussed.

Wolbachia infection in the Chorthippus parallelus hybrid zone: evidence for its role as a reproductive barrier.

Abstract Chorthippus parallelus parallelus (Cpp) and Chorthippus parallelus erythropus (Cpe) are two grasshopper subspecies whose distributions overlap in the Pyrenees, where they form a hybrid zone at those points where the topography and their ecological requirements allow. This hybrid zone is a consequence of secondary contact between endemic Iberian (Cpe) and continental European populations (Cpp) that expanded after the most recent glaciations from the refugia in which they had diverged genetically in allopatry. The morphological, physiological, genetic and behavioral differences between these subspecies (and their natural and laboratory-reared hybrids) have been intensively studied in recent years, for which reason this hybrid zone is considered a singular model in evolutionary biology. These studies reveal a complex pattern of factors involved in the origin, structure and maintenance of the hybrid zone. They offer an excellent panorama of evolution “in action”. Wolbachia is a genus of obligate endosymbiont bacteria that induce changes in the reproduction of arthropods and nematodes. In previous studies we have reported the existence of these bacteria in individuals and populations of Chorthippus parallelus. Now we show that they produce a significant reproductive barrier in this hybrid zone, implying, as proposed elsewhere, that Wolbachia may be involved in speciation phenomena.

Heterochromatin differentiation between two species of the genus Dociostaurus (Orthoptera: Acrididae)

Dociostaurus jagoi and Dociostaurus genei are two acridoid grasshoppers which show large differences in heterochromatin content. The use of several banding techniques, including fluorochrome staining and restriction endonuclease digestion, provides further information on the characteristics of the constitutive heterochromatin regions revealed with the C-banding method. Thus both species show GC-enriched bands accompanying active nucleolar organizers which differ in location. Because of its bright response to DAPI and CMA3, the centromeric heterochromatin seems to have a bipartite nature in terms of DNA composition in D. genei, while it shows dull staining with both fluorochromes in D. jagoi. However, two endonucleases, Mbol and Sau3A, extensively digest these regions in both species. The supernumerary heterochromatic segments present only in D. genei seem to be AT-rich and are extensively digested with Alul. These results reveal heterogeneity in the distinct C-banded regions which, in turn, are equilocally distributed in both chromosome complements.

Chromosomal localization of telomeric sequences in three species of Akodon (Rodentia, Sigmodontinae)

The distribution of the vertebrate telomeric sequence T2AG3 in three species of the rodent genus Akodon was examined by FISH with a peptide nucleic acid probe. In addition to the expected telomeric hybridization, non-telomeric signals were observed in the three species. In A. dolores, centromeric signals were visible in two of the four biarmed autosome pairs featuring Robertsonian polymorphism, indicating the retention of at least part of the telomeric sequences during the fusion process, and an interstitial signal of lower intensity was observed in the short arm of another. In A. boliviensis, a strong signal was observed near the centromeric end of the first chromosome pair. The first pair of A. azarae (homologous to the first pair of A. boliviensis) showed a similar but markedly amplified signal, and a subcentromeric signal in the X chromosome corresponding to a heterochromatic region; additionally, interstitial signals of lower intensity were present in one to four chromosomes in the majority of cells examined.