Heterochromatin Differentiation Between Two Species of the Genus Dociostaurus (Orthoptera: Acrididae)

Heredity 70 (1993) 458-465 Received 21 July 1992 Genetical Society of Great Britain

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

E. RODRIGUEZ INIGO, J. L. B ELLA & C. GARCIA DE LA VEGA* Unidad de Genética, Departamento de BiologIa, Edificio de Biologia, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain

Dociostaurusjagoi 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, MboI 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.

Keywords:chromosomebanding, Dociostaurus, insect cytogenetics, supernumary heterochromatin.

Introduction Soltani (1978) described sharp differences in male genitalia and number of stridulatory pegs. In this paper Constitutiveheterochromatin, usually revealed by we report chromosome differentiation between both C-banding techniques, is a common component of species, mainly involving the heterochromatin content. eukaryotic genomes that presents large variation in Moreover we have assayed several banding techniques content and distribution both between and within to further characterize the different heterochromatic species (John, 1988). Other banding techniques, such regions. Results obtained support two common as fluorochrome staining or digestion with restriction features of heterochromatin: heterogeneity and equi- enzymes, have revealed that heterochromatin may locality. show important differences in composition (heterogeneity) and it is commonly restricted to certain chromosome regions within a given complement Materialand methods (equilocality). In Acridoid grasshoppers, general surveys of Iberian (Santos et al., 1983) and Australian Adultmales of D. jagoi and D. genei collected from (King & John, 1980) grasshoppers demonstrated natural populations in central Spain were dissected and dramatic changes in heterochromatin content and the testes fixed in ethanol:acetic acid (3:1). To carry distribution even between closely related species. D. out the different banding techniques squash prepara- jagoi and D. genei are two species widely distributed in tions were performed in a drop of acetic acid 45 per drylands of south-western Europe, North Africa and cent, the coverslips were removed after freezing in the Middle East. They are commonly found in liquid nitrogen and the slides were then air-dried. sympatry. On morphological grounds these species C-banding was performed according to Lopez- were considered as a single one up to 1978, when Fernández & Gosálvez (1981). The silver impregnation method was that developed by Rufas & Gosálvez *Correspondence. (1982). Fluorochrome banding with 4,6 diamidin 2 Fig. I(a)and(b)Silver-stained diplotenecellsofD.jagoi(a)andgenei(b)showingtwo activeNORsinbothcasesbutwith 4th indecreasingsize(GarciadelaVega,1984). single inmales(XO)anddoublefemales(XX),isthe and threeshort(S9—Sii)pairs.TheXchromosome, grouped inthreelong(L1—L3),fivemedium(M4—M8) chromosomes. Accordingtotheirsizetheycanbe posed of23(males)and24(females)acro-telocentric The Results Mezzanotte etal.(1983). enzymes (REs)wascarriedoutaccordingto Schweizer (1980).Insitudigestionwithrestriction mycin A3(CMA3)wasobtainedaccordingto phenylindole (DAPI),distamycinA(DA)andchromo- pericentromeric bandsalthough thoseofD.geneiarebiggerthanjagoi.Inthis speciestheLibivalenthasaninter- different location(arrows).(c) and(d)C-bandedfirstmeioticmetaphasesofD.jagoi(c) D.genei(d).Everychromosomehas some. stitial band(arrow). Moreovermostoftheautosomes ofD.geneihavedistal supernumerarysegments(arrows). X:sexchromo- St I2' !t chromosome complementofbothspeciesiscom- C RIw U

xl... ,

fret

0) meric C-bands.However,thesebandsarelargerinD. In lents (Fig.ib). ciated tothecentromericregionoftwoshortbiva- bivalent (Fig.ia),whileinD.geneinucleoliareasso- the Libivalent,andcentromereregionofashort remnants appearassociatedtoaninterstitialregionof location isclearlydistinct.InD.jagoithenucleolar observed inbothspecies.Howevertheirchromosome Throughout Ag-NORs C-banding both specieseverychromosomeshowspericentro-

S 0• patterns HETEROCHROMATIN DIFFERENTIATION459 the firstmeioticprophasetwonucleoliare a' - p -. '' eta r 'A e

41 a a. isa¶ 4 460 E. RODRIGUEZ IFJIGO ETAL. genei than those found in D.jagoi(cf. Fig. ic and d). reveals bright bands in the centromere regions of D. Moreover seven chromosome pairs (M5—S11) of D. jagoi(Fig. 2a and c), whereas the centromeric C-bands geneimay show large distal heterochromatic blocks of D.geneishow complex composition (cf. Figs id, 2b (Fig. id). At least five of the pairs show polymorphism and d). Thus the proximal region appears bright with for the presence of these supernumerary segments. The DAPI (Fig. 2b), while DA-CMA3 reveals tiny juxta- regions where NORs have been detected also show centromeric bands below those differentially stained accompanying C-bands. This is particularly obvious in with DAPI in most of the chromosomes (Fig. 2d). The the case of the Li pair of D.jagoiwhere an interstitial supernumerary segments present in D.geneiare band is always present (Fig. ic). The C-bands asso- enriched in AT regions, as revealed by the bright bands ciated with the other active NORs of both species are obtained with DAPI. These regions, in turn, appear closely located to the centromeric bands of the corre- negative with DA-CMA3 (cf. Fig. 2b and d). Finally, the sponding chromosomes. heterochromatin associated with active NORs appears bright with DA-CMA3 in both species (Fig. 2c and d). Fluorochrome banding patterns Restrictionendonuclease banding patterns Centromereheterochromatin of both species can be differentiated when fluorochromes specific for AT Chromosomesof both species were digested in situ (DAPI) or GC (Chromomycin A3) DNA base pair with several REs (Tables 1 and 2). Most of the enzymes enriched regions are assayed. Neither fluorochrome employed reproduce C-like banding patterns; that is,

Fig. 2 Fluorochrome banding. (a) and (b) First metaphases of D.jagoi(a) and D.genei(b) stained with DAP1. Both the centro- meres and the distal segments of D.genei(b) appear bright. In D.jagoi(a) C-banded regions show dull fluorescence. Second metaphases of D.jagoi(c) and D.genei(d) stained with DA-CMA3. In D.jagoitwo half-bivalents show bright bands that corre- spond with the active NORs detected with silver staining. Some chromosomes of D.geneishow paracentromeric positive bands. In this species neither the centromeric regions nor the distal blocks show bright fluorescence. HETEROCHROMATIN DIFFERENTIATION 461

Table 1 Characterization of the heterochromatic regions of Table 2 Characterization of the heterochromatic regions of D.jagoi D. genei

NORs CEN CEN

C-banding + + NORs PROX JUXTA SS DAPI - 0 C-banding + + + + DA-CMA3 + 0 DAPI — + 0 + REs DA-CMA3 + 0 + 0 + + A/uI (AG/CT) REs BamHI(G/GATCC) + + Dral (TTT/AAA) + + AluI(AG/CT) + + + - EcoRl(G/AATTC) + + BamHI (G/GATCC) + + + + Haelll (GG/CC) — + Dra! (TTT/AAA) + + + HpaII(C/CGG) — + EcoRl(G/AATTC) + + + Hinfl(G/ANTC) + + HaelIl(GG/CC) — + — + MboI(/GATC) 0 — HpaII(C/CGG) — + + Sau3A (/GATC) 0 — Hinfl(G/ANTC) + + + + MboI (/GATC) 0 — — + NORs: nucleolar organizer regions; CEN: pericentromeric Sau3A (/GATC) 0 — — + regions. +:positive staining; —:negative staining; 0: dull PvuII (CAG/CTG) + + + + staining. NORs: nucleolar organizer regions; CEN: pericentromeric regions. PROX: proximal regions; JUXTA: juxtacentromeric regions: SS: supernumerary segments. chromosome arms are extensively digested except +: staining; 0: dull staining. where constitutive heterochromatin regions occur. positive staining; —:negative However, some of them have provided clear-cut differences between the heterochromatic regions. Thus HaeIII and HpaII, two enzymes containing only G and assume the concerted evolution of similar repetitive C in their targets, digest extensively the G/C enriched DNA sequences in equivalent chromosome arms at regions of both complements, i.e. the heterochromatin similar positions (Schweizer et a!., 1983; Schweizer & associated with NORs as well as the juxtacentromeric Loidl, 1987). A comparative study of heterochromatin bands of several chromosomes in D. genei (Fig. 3a distribution and heterogeneity in 10 species of acridoid andb). grasshoppers showed the strong tendency within a Te isoschyzomeres MboI and Sau3A digest the given complement for the accumulation of hetero- centromeric regions of both species while, to a large chromatin with similar composition at similar sites, extent, they do not affect the distal blocks of D. genei while different sites within a given complement usually (Fig. 3c and d). On the contrary, AluI produces inten- show different compositional properties (John et a!., sive digestion in the supernumerary blocks while its 1985). This regularity is exemplified by the tendency in effect on the centromeric heterochromatin of D. genei these and some other organisms to show differences in and D. jagoi is negligible (Fig. 4). Interestingly, PvuII, a composition between centric and distal C-bands. six-base cutter that contains the target of AIuI, repro- Moreover, different but related species may show duces the C-banding pattern in both species and does distinct heterochromatin composition at equilocal sites not produce intensive digestion in the supernumerary (John eta!., 1985; Schweizer eta!., 1987). segments (Fig. 4a). Our results on the chromosome complements of D. jagoi and D. genei support this position (Tables 1 and 2). In fact, the equilocal distribution extends to some Discussion other characteristics since certain heterochromatin E. Heitzgave the name heterochromatin to those regions within a complement are different, while those chromosome regions that do not undergo decondensa- located in homologous positions behave in identical tion throughout the cell cycle. He also pointed out that manner after distinct treatments. This is well illustrated these regions occupy similar positions in the chromo- in D. genei where centromeric and distal hetero- somes of a given complement, i.e. they show equilocal chromatin are easily distinguishable when chromo- distribution (Heitz, 1933, 1935). Derived models somes are digested with AluI and the isoschyzomeres 462 E.RODRIGUEZ INIGO El AL. e * to 4S S S .' A 'a

a b

Fig.3 Restriction endonuclease banding patterns. (a) and (b) First meiotic metaphases of D. jagoi (a) and D. genei (b) after digestion with HaeIII and stained with Giemsa. Most of the chromatin is resistant to this enzyme. Clear-cut differentiation due to extensive digestion (arrows) is obtained in those regions that are positive to CMA3 staining. (c) and (d) First meiotic metaphases of D. jagoi (c) and D. genei (d) after digestion with Sau3A and stained with ethidium bromide. In both cases the centromeric heterochromatin (c) is extensively digested, while the distal blocks (H) of D. genei are resistant to the attack of this endonuclease.

MboI and Sau3A (see Fig. 4). Since these enzymes pro- tains G and C, e.g. HaeIII (Gosálvez et at., 1987; duce extensive digestion in particular regions, i.e. MboI Lopez-Fernandez et at., 1989), nor revealed with spe- and Sau3A in the centromeric regions and AtuI in the cific banding techniques such as the N-banding (Fox & supernumerary segments, we can assume that in both Santos, 1985); (ii) both species show pericentromeric cases those C-bands must contain highly repetitive heterochromatin which is different both in size and DNA sequences which include the corresponding DNA base composition. D. jagoi does not show any target. Similar results where repetitive sequences are response after DA-CMA3 or DAPI, while D. genei detected after digesting chromosomes with REs have with bigger C-bands shows positive response after been reported in both plant and animal species (see DAPI in all these regions and DA-CMA3 positive references in Lopez-Fernandez et at., 1991). response in some of them. Interestingly, in both species On the other hand, the combined use of fluoro- the restriction enzymes assayed produce similar chrome banding and in situ restriction endonuclease results. Thus, in spite of being different, they still share digestion shows large heterogeneity among presumed the targets for some of the REs, i.e.: MboI and Sau3A; similar C-banded regions (see Table 1 and 2). Thus: (i) (iii) this situation differs from that found in the distal the heterochromatin associated with the NORs seems supernumerary heterochromatic segments which are to be similar in both species. It shows DA-CMA3 posi- exclusive to D. genei. In this case the heterochromatin tive response indicating GC richness, which is a seems to be quite different from that situated in the common feature of this kind of heterochromatin in dis- pericentromeric regions of both species. This may tinct species of plants and animals (Deumling & Greil- mean a distinct origin possibly because of an amplifica- huber, 1982; John et at., 1985; Schweizer et at., 1983; tion of sequences resident in these distal areas of D. Gosálvez et at., 1987; Lopez-Fernandez et at., 1989). genei. This situation coincides with the hypothesis of However, these regions are neither always digested telomeric regions as C-band initiation sites (Greilhuber with restriction endonucleases whose target only con- & Loidl, 1983; Schweizer & Loidl, 1987). HETEROCHROMATIN DIFFERENTIATION 463 0 a p I' - As Ts A 1i'f$ 'as t p. e ,0 Y C)- 0• 0 ,

Fig.4 Restriction endonuclease banding patterns. (a) and (b) First meiotic metaphases of D.geneiafter digestion with PvuII (a) and AluI (b) and stained with Giemsa. PvuII reproduces the C-banding in this species. Both centromeric and distal segments are resistant to this enzyme. However, A/uI, whose target is included in that of PvuII, provokes extensive digestion of the heterochromatic segments (arrows). (c)—(h) selected bivalents of D. genei after digestion with Sau3A (c)—(e) and AluI (f)—(h) to show the complementary effect of these enzymes on the heterochromatin of D. genei. (c) and (f) phase contrast; (d) and (g) ethidium bromide staining; (e) and (h) Giemsa staining.

In summary, our results show the existence of clear King, 1983). Although some authors have proposed karyotypic differentiation between both species that these differences could be involved in species revealed not only by the four different classes of divergence (Nagl, 1978), this assumption has been heterochromatin found (see Tables 1 and 2), but also criticized (John & Miklos, 1979; John & King, 1983). by the existence of extra heterochromatic segments In our case the karyotypic differences between D. genei distally placed in D. genei. This is particularly interest- and D. jagoi show that the chromosomal divergence ing since these grasshoppers are commonly found in occurred during their speciation. After extensive sympatric populations and their exophenotypic sampling and chromosome characterization carried characteristics are roughly similar (Soltani, 1978). out in different Spanish populations we have not yet Previous reports on chromosome characteristics of found any hybrid individuals. these species (Santos et al., 1983; Navas-Castillo et al., Unfortunately information on the chromosome 1986) did not take into account the taxonomic differ- characteristics of both species in other regions of their ences and have been erroneously attributed to D. genei geographical distribution is scarce. Soltani (1978) when they agree with the data here reported for D. reported the presence of "terminal heterochromatic jagoi. segments" in D. genei and also in D. jagoi from Israel. There are many other examples where closely This could mean that the supernumerary heterochro- related species with very similar karyotypes differ to a matin is present in both species in the easternmost large extent in the heterochromatin content (John & region of their range. However, this author does not 464 E. RODRIGUEZ IFJlGO ETAL. provide information on the methods employed nor GOSALVEZ, i. BELLA, J. L. AND HEWITF, G. M. 1988. Chromosomal photographic evidence of his observations. Thus fur- differentiation in Podisma pedestris: a third race. Heredity, ther analyses are needed to confirm these results. 61, 149—157. The heterochromatin differentiation shown by D. GOSALVEZ, J., BELLA, J. L., LOPEZ-FERNANDEZ, C. AND MEZZANOTrE, R. 1987. Correlation between constitutive hetero- jagoi and D. genei is similar to that described in the chromatin and restriction enzyme resistant chromatin in grasshopper Podisma pedestris. In this case, the two Arcyptera tornosi (Orthoptera). Heredity, 59, 173—180. Alpine races which form a hybrid zone (for review see GREILHUBER, J. AND LOIDL, .1983.On regularities of C-band- Hewitt & Barton, 1980) show pericentromeric ing patterns and their possible cause. In: Brandham, P. E. heterochromatin in all the chromosomes as well as and Bennett, M. D. (eds) Kew Chromosome Conference II, large distal heterochromatic segments in the medium George Allen & Unwin, London, pp. 344. sized and sex chromosomes. These heterochromatic HErI-z, E. 1933. Die somatisone heteropynose bei Drosophila regions seem to be GC-enriched in terms of DNA base melanogaster und ihre genetisone bedentung. Biol. Z. pair composition (Westerman & Hewitt, 1985). How- Zellforsch, 20, 237—287. ever, the same species in the Pyrenees does not show I-IEITZ, E, 1935. Die herkunft der chromozentren. Planta, 18, 517—6 35. distal heterochromatin and the pericentromeric hetero- HEWITF, G. M. AND BARTON, N. H. 1980. The structure and main- chromatin is split into two narrow bands which differ in tenance of hybrid zones as exemplified by Podisma base pair richness (Gosálvez et al., 1988). Since viable pedestris. In: Blackman, R. L., Hewitt, G. M. and Ash- hybrids between Pyrenean and Alpine individuals were burner, M. (eds) Insect cylogenetics, Blackwell Scientific obtained in the laboratory, these populations are con- Publications, Oxford, pp. 149—170. sidered to belong to distinct chromosomal races (Bella JOHN, B. 1988. The biology of heterochromatin. In: Verma, R. etal., 1990, 1991). S. (ed.) Heterochromatin, Cambridge University Press, Thus the existence of similar chromosomal differen- Cambridge, pp. 1—147. tiation in systems of dissimilar taxonomic categories JOHN, B. AND KING, M. 1983. Populations cytogenetics of supports the view that the chromosomal differences Atractomorpha similis I. C-band variation. Chromosoma, here described were not causally involved in the 88, 57—6 8. JOHN, B. 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