Loss of nucleolar organizer regions during chromosomal evolution in the South American cricetid Graomys griseoflavus

Graomys griseoflavus is a South American phyllotine rodent having a remarkable Robertsonian polymorphism which may have produced reproductive isolation between 2n=42−41 and 2n=38−34 karyomorphs. Analysis of nucleolar organizer region (NOR) locations both by silver staining (Ag-NOR) and in situ hybridization revealed that 2n=42 individuals exhibit highly variable Ag-NOR patterns, while specimens of the 2n=38−34 karyomorphic group showed a single Ag-NOR pattern. The latter animals underwent two NOR deletions in reference to the 2n=42 karyomorphs, one of which would be the consequence of a Robertsonian fusion and the other would be produced by the unequal crossing-over mechanism. The differential NOR homogenization supports the hypothesis that G. griseoflavus karyomorphs are evolving separately towards the acquisition of separate species status.

NOR location can be studied by both silver-staining (Ag-NOR) and in situ hybridization (ISH) with ribosomal DNA (rDNA) probes.The first method only detects transcriptionally active rDNA genes, while the second method identifies genes irrespective of their functional state.Reduction of Ag-NOR number in related species or in variants of the same species might be explained by loss or inactivation of rDNA genes.To decide between these alternatives, evidence from in situ hybridization with rDNA probes is needed.
In this work we have analysed NOR location patterns in karyomorphs of Graomys griseoflavus (except 2n = 41) by both Ag-NOR and in situ hybridization.We found that the 2n = 38-34 karyomorphs group underwent two NOR deletions in reference to the 2n = 42 karyomorph; they comprised the paracentromeric NORs of chromosomes 7 and 16.We correlated this phenomenon with the chromosomal evolution and speciation processes in this taxon.

Chromosome preparations and Ag-NOR staining
Air-dried chromosome spreads were obtained from bone marrow cells; to enhance cell proliferation, all specimens were previously treated with inlradermic yeast suspension.Chromosome identification was carried out by G-banding (Zambelli, Vidal-Rioja & Wainberg, 1994).Ag-NOR staining was performed according to Howell and Black (1980).Figure 1 represents Ag-NOR-bearing chromosomes and the site variants found in pairs 7, 8, 11 and 15.
rDNA in situ hybridization The probe employed was human rDNA cloned pAl (Erickson et al., 1981).The insert (7.3 Kb) includes 5.8S and 28S genes and the first and second internal transcribed spacers.The probe was labeled with biotin-14dATP with the BioNick kit (Gibco-BRL).
The biotinylated DNA was eluted in a Sephadex G50 column.An aliquot of each fraction was spotted onto a nitrocellulose strip, and the labeled fractions were detected with streptavidin-alkaline phosphatase conjugate (Blue Gene kit, Gibco-BRL).Slides with metaphase spreads were pretreated and hybridized according to Pardue (1985), except when otherwise described below.After formamide denaturation of chromosomal DNA, the slides received 50 #1 of denatured hybridization mixture containing 1.5 #g/ml biotinylated probe, 50% formamide, 10% dextran sulfate, 4xSSC.Annealing was performed in a humid chamber at 37°C overnight, spreads were then rinsed three times in 0.2xSSC and afterwards blocked with

Ag-NOR patterns
The 2n = 42 karyomorph Specimens with 2n = 42 showed a marked variation in the location of Ag-NORs (Figure 1).Polymorphic variants were found for pairs 7, 8, 11 and 15.On the other hand, in the same individuals, pairs 3 and 16 showed a constant paracentromeric position.
In different individuals the Ag-NOR in pair 7 may show three alternative patterns: i) homozygotes for Ag-NORs interstitially positioned; ii) heterozygotes with interstitial Ag-NORs in one homologue and two NORs (one interstitial and the other paracentromeric) in the other; or iii) homozygotes for two NORs (Figures 1  and 2a).
Regarding pairs 8 and 11, two contrasting patterns can be described: individuals with both homologues carrying paracentromeric Ag-NORs and others with Ag-NOR absence (Figure 1).Pair 15 may also present three forms of Ag-NOR pattern: i) long-arm telomeric Ag-NORs in both members of the pair (homozygote); ii) one homologue with two NORs (one on the telomere of the long arm and the other in paracentromeric position) and the other homologue without NORs (two-NOR heterozygote); iii) absence of Ag-NORs in both homologues (Figures 1 and 2b).Table 2 summarizes

The 2n = 38-34 karyomorphic group
A single Ag-NOR location pattern was found in all specimens of the 2n = 38-34 karyomorphic group.This pattern comprises five Ag-NOR-bearing chromosomes with a total of ten Ag-NORs.In this karyomorphic group, chromosome pairs 15 and 17 are absent and replaced by a large submetacentric pair RF 15 -17, with Ag-NORs placed at the telomere of the long arm of the 15 moiety.The other four pairs of NORs are found in the interstitial region of pair 7 and in the paracentromeric regions of 3, 8 and 11 (Figures 1 and  2c).

rDNA in situ hybridization
To identify chromosomal sites of rDNA clusters independently from their transcriptional activity, in situ hybridization (ISH) with rDNA probes was carried out in the two karyomorphic groups studied.ISH signals were shown to be coincident with Ag-NOR patterns in all animals studied.Due to its easy identification and highly frequent polymorphism in 2n = 42 specimens, the ISH pattern of pair 7 was closely analyzed.Using this approach, a lack of paracentromeric signal after ISH was observed on pair 7 of 2n = 38 and 2n = 36 animals (Figure 3).

Discussion
It was recently proposed that a non-random sequence of Robertsonian fusions, starting from 2n = 42 karyomorph, led to the current karyotypic differentiation of the cricetid Graomys griseoflavus (Zambelli, Vidal-Rioja & Wainberg, 1994).The present investigation adds further evidence for the chromosomal speciation process occurring in this taxon.
The major finding revealed 2n = 42 individuals exhibiting variable Ag-NOR patterns on pairs 7, 8, 11 and 15.Consequently, in ten 2n = 42 animals analyzed, the range of Ag-NORs was 8-13 with variable chromosomal distributions (Table 2).Contrarily, a single Ag-NOR pattern with a constant number of 10 NORs was shown across the entire 2n = 38-34 karyomorphic complex.Only the NOR loss of pair 16 may be ascribed to the occurrence of Robertsonian fusions.Pair 16 is part of RF16-18, where fusion resulted in deletion of the paracentromeric NOR.Both silver staining and ISH failed to detect NOR material in the centromere of the RF16-18 chromosomes.The loss of EG250 major satellite in the centromere of those Robertsonian chromosomes supports the above assumption (Zambelli & Vidal-Rioja, 1995).
Another Ag-NOR loss in the 2n = 38-34 group may be presumed at the paracentromeric site of pair 7 (present in seven out of the ten 2n = 42 individuals analysed).ISH of rDNA on chromosomes 7 of 2n = 38 and 2n = 36 animals shows no signal at paracentromeric position, although it is present at the interstitial location.This result would support the notion that the absence of paracentromeric Ag-NOR signal on chromosome 7 of 2n = 38-34 specimens is not due to functional inactivation but to structural deletion.
In the light of recent findings, caveats would be advisable on the role of Robertsonian rearrangements as the only cause of variability in NORs appearance.In the course of chromosome evolution of four species of Microtidae, for instance, NORs seem to have been either gained or lost; yet some of these events occurred without apparent relation with chromosome rearrangements.It was then suggested that rDNA genes may be able to transpose from one chromosome to another (Schubert & Wobus, 1985;S~nchez et al., 1990), although, so far, in this rodent taxon there is no evidence supporting this hypothesis.
Variability in NOR numbers could also be attributed to unequal crossing-over during concerted evolution of rDNA (Smith, 1976;Ohta, 1980;Arnheim et al., 1980;Arnheim, 1983;Seperack, Slatkin & Arnheim, 1988).According to this model, the number of rDNA copies fluctuates until finally reaching its homogenization within a given taxon.This process may result in an amplification or deletion of the sequences involved, depending on selective pressures undergone by the population.
The invariable Ag-NOR location pattern exhibited by 2n = 38-34 individuals might be derived from 2n = 42 ancestral karyomorphs having the Ag-NOR pattern currently observed in 2n = 38-34 animals.Since none of the ten 2n = 42 individuals (obtained from three different localities) showed the 2n = 38-34 Ag-NOR pattern, we consider that this hypothesis is not very probable.An alternative explanation, however, arises if we refer to the unequal crossing-over model.It might be possible that the genome reorganization that caused RF15-17 and RF16-18 and triggered the 2n = 42/38 chromosome differentiation also induced unequal crossing-over events in germ line cells of 2n = 38 animals.Chance or selection may have favoured afterwards loss of the paracentromeric 7 NOR (as ISH results suggested) and constancy of the paracentromeric 8 and 11 NORs.Consequently, since 2n = 37-34 karyomorphs derive from 2n = 38 in a downward sequence, all specimens of the 2n = 38-34 complex share the same Ag-NOR pattern.
The differential NOR homogenization gives additional evidence that Graomys griseoflavus karyomorphs are evolving separately.This finding, together with cytogenetic and molecular data and the inability to interbreed (Zambelli, Vidal-Rioja & Wainberg, 1994;Zambelli & Vidal-Rioja, 1995) support the hypothesis that a clear differentiation exists between 2n = 42 and 2n = 38-34 karyomorphs, which would be relevant to the acquisition of separate species status.

Figure 3 .
Figure 3.Comparison of rDNA locations by Ag-NOR (N) and ISH (I).Chromosome 7 (a) and RF15-17 (b) in a 2n = 36 individual; notice the absence of paracentromeric ISH signal in chromosome 7. Chromosome 15 (c) with two NORs in a 2n = 42 individual; notice the triple NOR association among two NOR 15 and homozygous paracentromedc NOR 16.

Table 2 .
Ag-NOR pattern and total NOR number found in each 2n = 42 animal analysed.Variable NOR sites are indicated in the table.They correspond to chromosomes 7, 8, 11 and 15.Hm: homozygote; Ht: heterzygote; Hmi: homozygote for interstitial NOR; Hrn2NOR: homozygote for 2 NOR; Hmt: homozygote for telomerie NOR; a: absence of NOR