Hereditas 139: 13–17 (2003)

Karyotype evolution in South American subterranean rodentsCtenomys magellanicus (Rodentia: Octodontidae): chromosomerearrangements and (TTAGGG)n telomeric sequence localization in2n=34 and 2n=36 chromosomal formsMARTA LIZARRALDE1, ALEJANDRO BOLZAN2 and MARTHA BIANCHI2

1 Centro Austral de in�estigaciones Cientıficas, CADIC, Ushuaia, Tierra del Fuego, Argentina2 Instituto Multidisciplinario de Biologıa Celular IMBICE, La Plata, Bs. As., Argentina

Lizarralde, M., Bolzan, A. and Bianchi, M. 2003. Karyotype evolution in South American subterranean rodents Ctenomysmagellanicus (Rodentia: Octodontidae): chromosome rearrangements and (TTAGGG)n telomeric sequence localization in2n=34 and 2n=36 chromosomal forms. — Hereditas 139: 13–17. Lund, Sweden. ISSN 0018-0661. Received January 3,2003. Accepted July 17, 2003

Ctenomys is the most numerous genus of South American subterranean rodents and one of the most karyotypicallydiverse clades of mammals known. Ctenomys magellanicus is the southernmost species of the group and the only oneliving in Isla Grande de Tierra del Fuego (Argentina). This species presents two chromosomal forms, i.e. 2n=34, and2n=36 (FN=68). Recent studies suggest that genetic divergence between both karyotypic forms resulted from achromosomal speciation process. In order to identify the chromosomal rearrangement involved in the process ofkaryotype evolution in this species, we used chromosome banding techniques and fluorescence in situ hybridization witha telomeric probe to metaphase chromosomes of the two chromosomal forms of Ctenomys magellanicus. Chromosomeanalysis of Giemsa-stained and G-banding preparations showed that Cm34 and Cm36 karyotypes differ in onerearrangement involving chromosomes A9 from Cm34 and B12 and B17 from Cm36. In addition FISH analysis showedthat all of the chromosomes from both chromosomal forms exhibit a telomeric-only distribution pattern of the(TTAGGG)n sequence, indicating that none of the chromosomal forms of Ctenomys magellanicus has true telocentricchromosomes. Our results suggest that a chromosome fission event would have occurred during the process of karyotypeevolution in this species.

Marta Lizarralde, Centro Austral de in�estigaciones Cientıficas, CADIC, CC 92, 9410 Ushuaia, Tierra del Fuego, Argentina.E-mail: martalizarralde@hotmail.com

Ctenomys is the most numerous genus of SouthAmerican subterranean rodents with about 60 speciesdistributed from Peru and Brazil to Tierra del Fuego(Argentina). This is one of the most karyotypicallydiverse clades of mammals known, with diploid num-bers ranging from 10 to 70 chromosomes (REIG et al.1990). For this reason, Ctenomys is very useful toinvestigate the dynamics of chromosome evolution aswell as the process of speciation in mammals. Severallines of evidence suggest that karyotype evolution ofCtenomys involves an increasing trend in both diploidand fundamental numbers (REIG et al. 1990; MAS-

SARINI et al. 1991; ORTELLS 1995; BIDAU et al.1996).

Ctenomys magellanicus is the southernmost speciesof the group and the only one living in Isla Grandede Tierra del Fuego (Argentina). This species presentstwo chromosomal forms, i.e., 2n=34, and 2n=36(KIBLISKY and REIG 1968; GALLARDO 1979;LIZARRALDE 1992). The presence of a constant num-ber of chromosome arms (FN=68) and interstitialC-bands suggests that C. magellanicus underwent achromosome reorganization through numerical andstructural changes (REIG and KIBLISKY 1969; GAL-

LARDO 1991; ALVAREZ et al. 1994). A recent studyshowed that the two chromosomal forms of thisspecies exhibit non significant differences in terms ofgenetic (estimated by allozyme analysis) and morpho-logical differentiation (LIZARRALDE et al. 2001). Thismight indicate a relatively recent karyotypic differen-tiation, suggesting a chromosomal speciation process.

Despite all the studies performed so far, the chro-mosome rearrangements involved in the karyotypeevolution of C. magellanicus are still unknown.Hence, in the present study, we used chromosomebanding techniques and fluorescence in situ hy-bridization with a telomeric probe to metaphasechromosomes of the two chromosomal forms of thespecies in order to determine the possible chromoso-mal rearrangements involved in the karyotype evolu-tion of this species.

MATERIAL AND METHODS

Seventy specimens of C. magellanicus belonging toboth chromosomal forms (2n=34, and 2n=36)from 11 isolated populations living in the Isla Grandede Tierra del Fuego (Argentina) were studied. Data

M. Lizarralde et al.14 Hereditas 139 (2003)

about locations where animals were captured werereported elsewhere (LIZARRALDE et al. 2001). Skullvoucher specimens of all of the animals studied weredeposited in the Collection at the Ecogenetic’s Labo-ratory (CADIC-CONICET, Tierra del Fuego,Argentina).

Mitotic chromosomes were prepared directly frombone marrow cells after in vivo colchicine treatment.The specimens were yeast-injected 24 h before sac-rifice (LEE and ELDER 1982). Routine karyotypicanalysis was performed to preparations stained with5% Giemsa solution. In order to identify possiblerearrangements, metaphases were G-banded accord-ing to the method of SEABRIGHT (1971). C-bandingwas performed following the technique of SUMMER

(1972). According to their relative length, chromo-somes were classified as large, medium, or small, asindicated in the ideogram of C. magellanicus previ-ously reported by REIG and KIBLISKY (1969). Kary-otype description was made according to MASSARINI

et al. (1991), who distinguished the chromosomecomplement in other species of Ctenomys, in gono-somes, and two groups of autosomes, i.e. ‘‘A’’biarmed, and ‘‘B’’ telocentrics.

In order to detect the presence of (TTAGGG)nrepeats, fluorescence in situ hybridization (FISH) wasperformed to metaphase chromosomes with a Cy3-conjugated peptide nucleic acid (PNA) pantelomericprobe obtained from DAKO Corporation (Califor-nia, USA). FISH was performed according to theinstructions provided by the supplier. Briefly, after 10min pretreatment with formaldehyde and a solutioncontaining proteinase K, the sample DNA was dena-tured at 80°C for 4 min under a coverslip in presenceof the Cy3-conjugated probe. Hybridization (1 h atroom temperature) was followed by two washes usingsolutions provided in the kit. Afterwards, slides were

mounted in an antifade reagent containing DAPI(4�,6-diamidino-2-phenylindole) as counterstain. Sig-nals were observed using a Carl Zeiss (Germany)epifluorescence microscope equipped with an HBO100 mercury lamp and filters for DAPI and Cy3(Chroma Technology, USA). Ektachrome film, ASA400 (Eastman Kodak Company, Rochester, NY) wasused for photography.

RESULTS

Chromosome analysis of Giemsa-stained prepara-tions showed two different karyotypes for C. magel-lanicus, one with 2n=34 (Cm34, n=28 individuals)and another one with 2n=36 (Cm36, n=42 individ-uals). Both karyotypes were similar to the ones previ-ously reported (REIG and KIBLISKY 1969;GALLARDO 1979; LIZARRALDE 1992).

Cm34 karyotype (Fig. 1) includes 16 autosomalpairs of ‘‘A’’ biarmed chromosomes of large to smallsize, A11 pair carrying a secondary constriction. Onthe other hand, Cm36 karyotype (Fig. 2) is basicallythe same as Cm34, except for the following features:(1) it has only 15 ‘‘A’’ pairs of large to small size and2 ‘‘B’’ pairs (B12 medium-sized, and B17 pair is thesmallest from Cm36 complement); (2) A10 chromo-some pair carries a secondary constriction. Sexualchromosomes are identical in both karyotypes. X is alarge metacentric, and Y a medium subtelocentric(Figs. 1 and 2).

Fig. 3 illustrates the pattern of G-banding homolo-gies between the two chromosomal forms. This pat-tern shows that both karyotypes differ in onerearrangement involving A9 chromosome fromCm34, and B12 and B17 from Cm36. Long (p) andshort (q) arms of A9 chromosome are homologous tochromosome pairs B12, and B17, respectively.

Fig. 1. Conventional karyotype of Ctenomys magellanicus 2n=34.

Chromosome e�olution in Ctenomys magellanicus 15Hereditas 139 (2003)

Fig. 2. Conventional karyotype of Ctenomys magellanicus 2n=36.

Fig. 3. G-banding pattern comparison between Cm34 and Cm36. Numbers under the chromosomesindicate the pair number in each chromosomal form.

The pattern of C-bands has been previously de-scribed (GALLARDO 1991; ALVAREZ et al. 1994).As expected, most chromosomes of both C. magel-lanicus chromosome forms showed no C-bands

(REIG et al. 1992). Only 3 pairs, A4, A14, and sexchromosomes showed distinct C+ bands, whereasY chromosome was entirely heterochromatic (datanot shown).

M. Lizarralde et al.16 Hereditas 139 (2003)

FISH analysis showed telomeric signals only at theterminal regions of chromosomes from Cm34 andCm36 karyotypes. All telomeres were clearly labeled(Fig. 4a and b). It is interesting to note that eventhose chromosomes appearing as telocentrics withGiemsa staining or G-banding, displayed four telom-eric signals, two at each end, indicating that eachchromosome has two arms. Thus, the distributionpattern of the telomeric sequence (TTAGGG)nstrongly suggests that none of the chromosomalforms of C. magellanicus has true telocentricchromosomes.

DISCUSSION

Although karyotypes of C. magellanicus has beenpreviously published (REIG and KIBLISKY 1969;GALLARDO 1979; LIZARRALDE 1992), this is the firstreport describing both the G-banding and the distri-bution pattern of the telomeric sequence(TTAGGG)n in the two chromosomal forms of thisspecies.

Chromosome analysis of Giemsa-stained and G-banding preparations showed that Cm34 and Cm36karyotypes differ in one rearrangement involvingqA9.B12 and pA9.B17. In addition, FISH analysisshowed that all of the chromosomes from both chro-mosomal forms exhibit a telomeric-only pattern ofdistribution of the (TTAGGG)n sequence. Althoughdetailed molecular studies will be required to deter-mine whether the telomeric sequence is at the extremeterminus of each telomere from each chromosome, insitu data indicate that probably none of the chromo-somal forms of C. magellanicus have true telocentricchromosomes, as described by DARLINGTON (1939).

From our present data, it is possible to assume thatthe type of chromosomal rearrangement involved inthe karyotype evolution of C. magellanicus would beeither a Robertsonian fusion (ROBERTSON 1916) or achromosome fission event. The process of Robertso-nian fusion is generally believed to occur by one oftwo possible means, centromeric fusion (HSU et al.1975) or reciprocal translocation in the close vicinityof the centromere (WHITE 1973). The key event dur-ing these processes is a breakage that may occur atdifferent points within the pericentric region. Bothprocesses actually lead to the loss of chromosomematerial from the rearranged chromosomes. How-ever, our present data do not reveal any loss ofchromosome material in none of the chromosomalforms of C. magellanicus. In addition, we foundneither telomeric signals nor C bands either at thepericentric or centromeric region of A9 chromosomepair of Cm34, therefore, it seems unlikely that aRobertsonian fusion event involving B pairs of Cm36could have occurred during karyotype evolution ofthis species.

Taking into account the above observations, itseems more likely to hypothesize that a centric fissionevent in A9 pair from the Cm34 chromosomal form,producing B12 (formerly qA9) and B17 (formerlypA9) pairs and giving rise to the karyotype 2n=36,has occurred during the process of karyotype evolu-tion in C. magellanicus. According to this view, 2n=36 karyotype should have derived from 2n=34chromosomal form through a chromosomal fissionevent. Telomeres in the short arms of chromosomesB12 and B17 (as detected by FISH) could have arisende novo as a result of telomerase activity.

Fig. 4. In situ localization of the telomeric sequence(TTAGGG)n on the metaphase chromosomes of Ctenomysmagellanicus 2n=34 (a) and 2n=36 (b). Arrowheads indi-cate the location of B12 and B17 chromosome pairs.

Chromosome e�olution in Ctenomys magellanicus 17Hereditas 139 (2003)

Our hypothesis is in good agreement with previ-ously reported data on the chromosome evolution(ORTELLS 1995) and RPCS satellite studies in Cteno-mys, suggesting that chromosomal rearrangementsinvolved in chromosomal evolution of C. magellani-cus would have been primarily chromosome fissionsaccompanied by RPCS significant expansion (SLAM-

OVITS et al. 2001).Undoubtedly, further studies will be necessary to

confirm or disregard our hypothesis on karyotypeevolution of C. magellanicus. These studies are cur-rently in progress in our laboratory.

Acknowledgements – This work was supported by grantsfrom CONICET (PIP 4306/96) and CIC of Argentina. Wewish to thank Dr. Nestor Bianchi for his helpful commentsabout the manuscript and Julio Martın Escobar for techni-cal assistance.

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