Genome Reduction in a Hemiclonal Frog Rana esculenta From Radioactively Contaminated Areas

Corresponding author: Alexander E. Vinogradov, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave., 4, St. Petersburg 194064, Russia., aevin{at}mail.cytspb.rssi.ru (E-mail)

Communicating editor: W. STEPHAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

A decrease in genome size was found in the hemiclonal hybridogenetic frog Rana esculenta (R. ridibunda x R. lessonae) from areas of radioactive contamination that resulted from the Chernobyl fallout. This genome reduction was of up to 4% and correlated with the background level of gamma-radiation (linear regression corresponded on average to -0.4% per doubling of radiation level). No change in genome size was observed in the coexisting parental species R. lessonae. There was no correlation between genome size and body mass in R. esculenta froglets, which have metamorphosed in the year of the study. The hemiclonal forms may become a suitable object for study on biological significance of individual DNA sequences (and of genome size as a whole) because mutant animals with deletions in a specified genome can arise after a low radiation dose. The proneness to genetic damage makes such forms also a prospective bioindicator of radioactive (and possibly other mutagenic) pollution with the effects of genetic damage conveniently and rapidly monitored by DNA flow cytometry.

THE hemiclonal (hybridogenetic, non-Mendelian) frog Rana esculenta L. arose from the original hybridization of R. ridibunda Pall. x R. lessonae Cam. (BERGER 1968 ). In gametogenesis of this hybrid, one of the parental genomes is selectively eliminated before premeiotic DNA synthesis, and the remaining genome undergoes a compensatory reduplication followed by a "normal" meiosis, which results in gametes containing only one of the parental genomes (UZZELL et al. 1980 ; TUNNER and HEPPICH 1981 ; VINOGRADOV et al. 1990 , VINOGRADOV et al. 1991 ). The hybrid is recreated in each generation by backcrossing to that parental species whose genome was eliminated. The DNA content of the R. ridibunda genome is greater than that of R. lessonae by 16%, which allows us to identify a set of genomes in somatic and germinal cells in any specimen of this hybridogenetic complex by means of DNA flow cytometry (VINOGRADOV et al. 1990 , VINOGRADOV et al. 1991 ). The direction of elimination shows a gene-dosage effect (in triploids that genome is eliminated that is in the minority) and is supposed to be determined by the ratio of activities of elimination- related factors in both genomes, which on average (over all the populations studied) is in favor of the ridibunda genome (VINOGRADOV et al. 1990 , VINOGRADOV et al. 1991 ). Both genomes are expressed in somatic cells of a hybrid (UZZELL et al. 1980 ). Other hybridogenetic complexes are known: e.g., fishes of the genus Poeciliopsis (LESLIE and VRIJENHOEK 1978 , LESLIE and VRIJENHOEK 1980 ; QUATTRO et al. 1991 ) and insects of the genus Bacillus (GIORGI 1992 ).

The hemiclonal genomes of R. esculenta and Poeciliopsis fishes are known to accumulate deleterious recessive mutations (LESLIE and VRIJENHOEK 1978 , LESLIE and VRIJENHOEK 1980 ; GRAF and POLLS-PELAZ 1989 ). Such accumulation is expected in clonal genomes that always occur in heterozygous conditions without recombination ("sheltered" genomes). This was originally proposed for the evolution of the Y chromosome (MULLER 1914 ; NEI 1970 ) and confirmed experimentally (RICE 1994 ). Furthermore, large decreases in DNA content (up to 50%) were found in the sperm of R. esculenta males from radioactively contaminated areas (VINOGRADOV et al. 1990 ), which suggests that genome reduction could appear in zygotes of this hybridogenetic form.

In the present investigation, frogs of the R. esculenta-complex from the areas of radioactive contamination that resulted from the Chernobyl fallout were studied by means of DNA flow cytometry.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Frogs were collected in August 1994 in the Bryansk region (Byelorussia) from three places with different background levels of radioactive contamination: environs of Sennoe village (-radiation was 220 µR/hr), environs of Petryatinka village (-radiation was 60 µR/hr), and, near the national nature reserve Bryansky Les, a control point without contamination (-radiation was 15 µR/hr). The map is shown in Figure 1. Genome size was determined by means of DNA flow cytometry in blood cells as described previously (VINOGRADOV et al. 1990 , VINOGRADOV et al. 1991 ) and presented in relation to the genome of R. temporaria (RT-index), which was used as an internal standard. Triplicate repeats of staining and measurements of cell samples from the individual animals gave SEs < 0.1%.

View larger version (14K): In this window In a new window Download PPT slide   Figure 1. Map of the radioactively contaminated area where frogs were collected. (For position of Gomel on the large-scale map see Daily Telegraph World Atlas, 24, F5.)

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

We found a statistically significant decrease in genome size (of up to 4%) in R. esculenta from the radioactively contaminated points (Figure 2). All the populations were of the most common, diploid L-E type (i.e., R. esculenta coexists with R. lessonae, and the lessonae genome is eliminated). The genome size of the coexisting parental R. lessonae was not changed (Figure 2). The mean genome size of R. esculenta was log-linearly correlated with the background level of -radiation (Figure 3). The linear regression corresponded on average to -0.4% per doubling of radiation level.

View larger version (33K): In this window In a new window Download PPT slide   Figure 2. Population histograms of genome size. Background level of -radiation: Sennoe, 220 µR/hr; Petryatinka, 60 µR/hr; Bryansky Les, 15 µR/hr (control point without contamination).

View larger version (21K): In this window In a new window Download PPT slide   Figure 3. The relationship between mean genome size (with SE bars) of R. esculenta and background level of -radiation in the populations studied. The mean genome size of R. esculenta from Bryansky Les (control point, 15 µR/hr) is set at 100%. The linear equation is Y = 101.7 (± 0.15) - 1.4X (± 0.08), which corresponds to -0.4% of genome size per doubling of -radiation level. Dashed lines, confidence limits; dotted lines, prediction limits (both for P = 0.95).

The most plausible explanation for genome reduction in R. esculenta (and the absence of such reduction in the coexisting parents) is that these frogs resulted from fertilization with gametes having deletions in the clonal ridibunda genome (of up to 8%). The R. esculenta males, which produced sperm with ridibunda genome (in accordance with their population type) but had part of sperms with the decreased DNA content, were found in the L-E population from a radioactively contaminated area (VINOGRADOV et al. 1990 ). The decrease was much greater then (up to 50%), and it is unlikely that zygotes resulted from fertilization with those sperms would be viable. The -radiation level was also much higher in the previous study, conducted exactly 1 yr after the fallout (1.1 mR/hr; environs of Babichi village, Byelorussia, 130 km W-SW from the area of the present study). No change in genome size was then found in gametes and somatic cells of coexisting R. lessonae, nor in somatic cells of R. esculenta (born before the fallout), which suggests that the preferential damage occurred at gametogenesis in the clonal ridibunda genome. Such damage should then accumulate according to the expectation for the sheltered genome condition (MULLER 1914 , MULLER 1964 ; NEI 1970 ; LESLIE and VRIJENHOEK 1978 , LESLIE and VRIJENHOEK 1980 ; RICE 1994 ).

Owing to the increased vulnerability of a genome, its accumulation of genetic damage under a sheltered condition, and the outcome of its change in genome size, hemiclonal forms may become a prospective bioindicator of radioactive (and possibly other mutagenic) pollution with the effects of genetic damage convenient for rapid registration with DNA flow cytometry.

There was no correlation between genome size and body mass in R. esculenta froglets, which had metamorphosed in the year of the study, although body masses of animals from the point with the highest radiation level (Sennoe) seemed to be more scattered (Figure 4). This suggests that deletions of up to 4% of total DNA (probably, of up to 8% in the clonal ridibunda genome) were not connected with fitness reduction (because the main adaptive goal of the froglets is to gain weight). Even if the revealed deletions did not involve the coding DNA, the question arises about the biological significance of changes in genome size (or the role of redundant, noncoding DNA). The absence of correlation between genome size and froglets weight suggests the neutralist interpretation for the existence of redundant DNA (PAGEL and JOHNSTONE 1992 ; CHARLESWORTH et al. 1994 ; ELDER and TURNER 1995 ). However, the possibility cannot be excluded that the effect of genome reduction would be expressed later, at a low-metabolic stage of overwintering, in accordance with the adaptive (ecophysiological) concept of redundant DNA (VINOGRADOV 1995 , VINOGRADOV 1998 ). Note that, because selection pressure is not effective in the sheltered clonal genome, there should be a mutation pressure for the observed reduction in genome size. The same pressure occurs in the case of the Y chromosome, which is usually significantly reduced compared to its counterpart (reviewed by MORELL 1994 ), up to the XO condition in some species. If such reduction is a typical net result of mutation pressure, then accumulation of redundant DNA in larger genomes should be explained by selection pressure.

View larger version (14K): In this window In a new window Download PPT slide   Figure 4. Plot of body mass vs. genome size for R. esculenta froglets, which had metamorphosed in the year of the study (collected in August). , Bryansky Les; , Petryatinka; , Sennoe.

The hybridogenetic forms could become a suitable object for research on biological significance of individual genomic elements (and of genome size as a whole) because mutants with deletions in a specified genome can arise after a low-dose mutagenic action, probably not complicated by the heavy damage of the rest of the genome. The latter was the main objection to the conclusion of the study on radiation-induced deletion mutants in Drosophila (WU et al. 1989 ), the only work where the association of deletions in satellite DNA with fitness reduction was demonstrated (ELDER and TURNER 1995 ). The parent, in which no deletions of comparable size occur, can be used as a control for even this low-dose mutagenic action.

	ACKNOWLEDGMENTS

The authors are grateful to Robert C. Vrijenhoek and an anonymous reviewer for helpful comments. This work was supported by a grant from the Russian Foundation for Basic Research (to A.V.).

Manuscript received August 10, 1998; Accepted for publication December 14, 1998.

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