Unidad de Genetica. Departamento de Biología. Facultad de Ciencias.
Universidad Autónoma de Madrid. 28049 Madrid, Spain.
Correspondence should be addressed to: Jose M. Rubio, PhD.
Servicio de Parasitología. Centro Nacional de Microbiología. Instituto de Salud Carlos III, Cra. Majadahonda Pozuelo Km2,5. Majadahonda, 28220 Madrid, Spain.
Submitted for publication: January 1999
Keywords: Hybrid zone, Chorthippus parallelus, NOR, Speciation
Chorthippus parallelus (Zetterstedt) is a very common meadow grasshopper distributed through Europe, comprising, at least, two subspecies Chorthippus parallelus parallelus widespread through Western Europe and Chorthippus parallelus erythropus endemic of the Iberian peninsula During the ice age these subspecies were separated and diverged in allopatry in zones more temperate in the South of Europe and North of Africa. In the post glacial age both subspecies contacted along the Pyrenees where they formed a hybrid zone.
Some chromosomal information exists on Chorthippus parallelus parallelus populations and in Chorthippus parallelus erythropus populations on the hybrid zone in Pyrenees between both subspecies but nothing has been reported on the chromosomes of Chorthippus parallelus erythropus in isolated populations.
The results show that the populations of Chorthippus parallelus erythropus of the Sistema Central in Spain have important cytogenetical differences with respect to the populations close to the hybrid zone previously studied mainly in the number of Nucleolus Organiser Region (NOR) and in heterochromatin composition
Thus, the study of the hybrid zone should be accompanied with information not just from Chorthippus parallelus parallelus isolated populations but also from Chorthippus parallelus erythropus isolated groups. The Sistema Central populations of Chorthippus parallelus erythropus may be a good system to study the evolution of these subspecies in absence of gene flow towards Chorthippus parallelus parallelus.
Chorthippus parallelus (Zetterstedt) is a very common meadow grasshopper distributed through Europe, comprising, at least, two subspecies Chorthippus parallelus parallelus widespread through Western Europe and Chorthippus parallelus erythropus endemic of the Iberian peninsula.
Faber (1) distinguishes between both subspecies based on some morphological differences as the number of pegs and as the colour of the hind tibiae, which is red in Chorthippus parallelus erythropus. Additional information made by Butlin and Hewitt (2, 3) from a hybrid zone in the Pyrenees showed that both subspecies differ in other important morphological characters such as in certain enzyme loci, as well as in certain ethology characters, such as the courtship song. In the last few years molecular markers were used in order to differencied both subspecies, but these are small (4, 5, 6) or there are not differences (7, 8). The main differences, found between both subspecies, are restricted to the Sex-chromosome in the heterochromatin localisation and the presence or absence of a nucleolus organiser region (NOR) (9, 10).
During the ice age these subspecies were separated and diverged in allopatry in zones more temperate in the South of Europe and North of Africa. In the post glacial age both subspecies expanded into Europe. Chorthippus parallelus parallelus expanded through all Europe as far as the Pyrenees including the main island of the Great Britain but not Ireland island and its distribution is continuous, instead Chorthippus parallelus erythropus is restricted to the meadow mountain in the Iberian Peninsula and there is not contact between the different populations (4, 5, 11). Both subspecies contacted along the Pyrenees where they formed a hybrid zone ( 2, 3, 6, 9, 12).
Some chromosomal information exists on Chorthippus parallelus parallelus populations (13, 14, 15, 16, 17, 18), on Chorthippus parallelus erythropus Pyrenees populations (19), on the hybrid zone between both subspecies in Pyrenees (9, 10, 20) and in laboratory hybrids (21) but nothing has been reported on the chromosomes of Chorthippus parallelus erythropus in isolated populations, except for the amount of heterocromatin (22). Although comparison of french populations with museum specimens from Central Spain does not show differences in respect to morphological characters (2), it could be interesting to study the possible variation on cytogenetical characters as in molecular markers (4, 5, 6).
The Chorthippus parallelus erythropus populations, at the moment the ice were restricted to the high mountains, leave the low land and move up the mountains, losing the contact and the possible gene flow between the different populations, localised on the Sistema Central (central Spain), the Sistema Cantabrico (North-West of Spain), the Sierra Nevada (South Spain) and the Pyrenees (Noth-East), and star to diverge between them, at the same time that the Pyrenees populations meet with the populations of Chorthippus parallelus parallelus and make a hybrid zone.
MATERIALS AND METHODS
Two hundreds adult males of Chorthippus parallelus erythropus were collected from natural populations in the Sistema Central in Spain. Testes were removed and fixed in 3:1 ethanol-acetic acid.
Air dried chromosome preparations were made by squashing tissues in 45% acetic acid, freezing them in liquid nitrogen and removing the coverslip with a razor blade. The Giemsa C-banding method was carried out according to Rubio and Ferris (23) and the silver staining method was performed according to the method described by Rufas and Gosálvez (24) adding, in some cases, a pre-treatment with double standard saline citrate solution to obtain a better resolution in the position of the active NORs (25). Fluorochrome banding with chromomycin A3 (CMA3) was obtained according to Schweizer (26, 27) and Bella et al. (28).
The male chromosome complement of Chorthippus parallelus comprises 16 autosomes of different size plus one X chromosome. The three long pairs are submetacentric, while the reminder, including the X, display terminal centromeres (9, 10, 13).
The C-banding pattern of the Sistema Central populations did not show differences in respect to that described for the Pyrenees population of Chorthippus parallelus erythroppus. All the chromosomes present pericentromeric heterochromatin, additionally, terminal C-bands are present in all the autosomal acrocentric chromosomes and in the shorter arms of L2 and L3 chromosomes, most of these bands being polymorphs. Interstitial bands are found in both arms of the L3 chromosome, in the M4 chromosome and in the X chromosome, which no present terminal heterochormatin like happens on the Chorthippus parallelus parallelus subspecies.
A 3% of individuals from Sistema Central populations of Chorthippus parallelus erythroppus carry extra chromosomes which also have been described on Pyrenees populations.
Silver staining can be used on meiocytes of males to localise the nucleolus active in previous interphases, with the pre-treatment of double standard saline citrate solution before silver staining it is possible observe the NOR (Nucleolus organiser region) as two small dots on the chromosomes.
The Chorthippus parallelus parallelus showed three different sites on the genome where produce rRNA. These NORs were localised near to the centromere on the short arm of L2 chromosome, on the short arm on the L3 chromosome in a distal position and in the X chromosome, also in distal position in relation with the C-band that this chromosome present in this region. Chorthippus parallelus erythroppus from Pyrenees present the same autosomal NORs but the NOR on the X chromosome is absent (9). The in situ hybridisation, with a probe of ssrDNA, shows the same localisation that the silver staining and corroborated that the absence of NOR on the sex chromosome of Chorthippus parallelus erythroppus is due to the absence of rDNA genes instead of a differential expression of the genes (data not shows).
The populations of Chorthippus parallelus erythroppus in the Sistema Central present a number variable of NORs, the two autosomal NORs are kept but the 50% of the individuals present a third NOR, this was not observed on the Pyrenees populations where just less of 5% of the individuals present a third NOR localised on the megameric chromosome M6 like the populations of Chorthippus parallelus parallelus. This supplementary NOR is not always on the same chromosome, 18% of the individuals present it on the megameric chromosome M6, a 6% on the M4, a 3% on the S8, the smaller chromosome of the complement and on the sexual chromosome (22%), where is more frequently observed, and where was associated with the interstitial heterochromatic band observed with C-banding (Figure 1).
The CMA3, fluorochrome which stain specifically to GC-rich DNA, revealed differences on the chromosomes of Chorthippus parallelus erythroppus in the Sistema Central with respect of the Chorthippus parallelus erythroppus in Pyrenees. While all the heterochromatin present in the Pyrenees populations of both subspecies were light positive and show to be partially rich in GC DNA base pairs (9), the heterochromatic regions of the Chorthippus parallelus erythroppus in the Sistema Central were strong positive at the CMA3 mainly the centromeric zones of some small chromosomes (M6, M7 and S8). Moreover, on the shorter arm of the three long chromosomes pairs the positive heterochromatin at CMA3 was bigger than ones of Chorthippus parallelus erythroppus in Pyrenees. Further, on the M4 chromosome of Chorthippus parallelus erythroppus in the Sistema Central appeared two small dots at the middle of the chromosome CMA3 positive. This band-CMA3 positive was more evident when this M4 chromosome had an active NOR. The sexual chromosome showed also two small positive dots at the CMA3 corresponding to the C-band and the positive centromere (Figure 2).
Two major cytogenetic differences were found between both subspecies. The first one was in the C-band of chromosome sexual, while in Chorthippus parallelus parallelus there is a distal C-band besides of a centromeric band, in Chorthippus parallelus erythropus (Pyrenees populations) that band is replaced by one interstitial band (9, 10). The population of Chorthippus parallelus erythropus in the Sistema Central maintain this difference.
The second main difference was the presence of a third NOR on the populations of Chorthippus parallelus parallelus. In both subspecies appear two NORs; one on the L2 chromosome, near the centromere, and other on the short arm of the L3 chromosome. Besides the Chorthippus parallelus parallelus show a third NOR on the sexual chromosome, associated with the distal heterochromatic band, which is never present on the populations of Chorthippus parallelus erythropus in Pyrenees, in some occasions both subspecies present another NOR on the megameric chromosome M6. In the Sistema Central the Chorthippus parallelus erythropus shows a high variation on the number of NORs. The two NORs localised on the autosomes are present in all the individuals but a 50% of individuals present a third located on different chromosomes, including a 22% on the sexual chromosome.
Since one would expect natural selection to control the number of ribosomes in a cell and hence the number of copies of rDNA sequences in the NORs, the X-NOR difference represents a significant genome modification. If the two subspecies required roughly the same number of ribosomes (9, 29) the subspecies Chorthippus parallelus erythropus should find a solution to this problem. Two alternatives would be possible. First, the rDNA sequences contained in three NORs in Chorthippus parallelus parallelus should be contained in two NORs in Chorthippus parallelus erythropus (9). The other solution would be having a new NOR. This last process may be taking place within some individuals from the Sistema Central populations. Moreover, if this new NOR is on an autosome these individuals will present six NORs in three pair of autosomes while in Chorthippus parallelus parallelus only have two on two pair of autosomes and other in the sexual chromosome, this may be a great waste for the carrier individual. For this reason, it is possible that this event appear in the population at low frequency (9%). The best solution would be to place this new NOR on the sexual chromosome that occurs in 22% of the individuals of Chorthippus parallelus erythropus in the Sistema Central. It could be that this new NOR was a reactivation of the old one. But the first result obtained with the hybridisation in situ shows that on the sexual chromosome of Chorthippus parallelus erythropus of Pyrenees there are not any copies of rDNA.
Individuals with extra chromosome which increase the mean chiasmata frequency as happens in the Pyrenees populations, would seem a faster evolution for reaching a better genetic combination for the subspecies taking less time. However, this process in grasshopper usually is join at the presence of supernumerary segments on the smaller chromosomes of complement (30, 31) and not for extra chromosomes (13, 32), moreover, this last system is easier to remove since these extra chromosomes are more unstable that the heterochromatic segments.
Differences in the quantity and location of DNA rich in GC base pairs should be other example of possible isolated evolution of these populations of the Sistema Central front the Pyrenees populations.
Recent works suggest that the french colonization by the Chorthippus parallelus parallelus was originate from a Balkan expansion (4, 5), not from the Iberian refugee (2, 3). Then, the common ancestor of the two subspecies lived more than one glacial cycle ago, and the refugial populations have probably been diversing in isolation for five or six glacial cycles (4, 11). In the same way, it is possible than the different Chorthippus parallelus erythropus populations were isolated between them in different glacial cycles, like probably happend with the populations near the hybrid zone which show a close genotype relation with the north populations of Chorthippus parallelus parallelus (6) instead to be close related with other Chorthippus parallelus erythropus populations. Moreover, it could be that the gene flow between Chorthippus parallelus parallelus and Chorthippus parallelus erythropus from Pyrenees is elevate and this produces a higher differentiation between both populations of Chorthippus parallelus erythropus than the populations of Chorthippus parallelus involved in the hybrid zone in Pyrenees. Specially, if the genes of Chorthippus parallelus erythropus are less advantageous by the hybrid than the genes of Chorthippus parallelus parallelus as it may be occurring in the hybrid zones of Pyrenees (10, 29, 33).
The populations of Chorthippus parallelus erythropus in Pyrenees have important cytogenetical differences with respect to the Sistema Central populations as it could not be deduced from morphological characters (2, 34). Thus, the study of the hybrid zone (6, 9, 12, 29, 35, 36) should be accompanied with information of Chorthippus parallelus parallelus isolated populations (7, 8, 11, 34, 37, 38) but also with Chorthippus parallelus erythropus isolated groups. The Sistema Central populations of Chorthippus parallelus erythropus may be a good system to study the evolution of these subspecies in the absence of gene flow towards Chorthippus parallelus parallelus.
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