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Origins, colonization, and lineage recombination in a widespread perennial soybean polyploid complex.

Polyploidy is a dominant feature of flowering plant genomes, including those of many important crop species, implying that polyploidy confers evolutionary advantages on plant species. Recent molecular studies suggest that polyploids often originate many times from the same progenitor diploids. For this to provide a broader genetic base for a polyploid species, there must be lineage recombination in the genomes of polyploids having different origins, and this has rarely been documented in recently formed wild polyploid species. Glycine tabacina, a wild relative of soybean, forms a widespread polyploid complex in Australia and the islands of the Pacific Ocean. In a sample of 40 G. tabacina plants, DNA sequence variation at one homoeologous histone H3-D locus identified three alleles, each also found in Australian diploid Glycine species. These data agree with our previous studies of chloroplast DNA variation in suggesting that this polyploid has originated several times. Both the origins of the polyploid and several independent dispersals from Australia to oceanic islands appear to have occurred within the last 30,000 years. The distributions of histone alleles, chloroplast haplotypes, and alleles at two isozyme loci were uncorrelated, and 20 multilocus genotypes were found among the 40 plants sampled. Extensive lineage recombination is thus hypothesized in the polyploid, involving migration and occasional outcrossing in this predominantly inbreeding species. The combination of multiple origins with gene exchange among lineages increases the genetic base of a polyploid and may help explain the wide colonization of polyploid G. tabacina relative to its diploid progenitors.

Journal Article↗

Evolutionary consequences of changes in species' geographical distributions driven by Milankovitch climate oscillations.

We suggest Milankovitch climate oscillations as a common cause for geographical patterns in species diversity, species' range sizes, polyploidy, and the degree of specialization and dispersability of organisms. Periodical changes in the orbit of the Earth cause climatic changes termed Milankovitch oscillations, leading to large changes in the size and location of species' geographical distributions. We name these recurrent changes "orbitally forced species' range dynamics" (ORD). The magnitude of ORD varies in space and time. ORD decreases gradual speciation (attained by gradual changes over many generations), increases range sizes and the proportions of species formed by polyploidy and other "abrupt" mechanisms, selects against specialization, and favor dispersability. Large ORD produces species prone neither to extinction nor gradual speciation. ORD increases with latitude. This produces latitudinal patterns, among them the gradient in species diversity and species' range sizes (Rapoport's rule). Differential ORD and its evolutionary consequences call for new conservation strategies on the regional to global scale.

Biological Evolution↗

Chromosome analysis in X-irradiated primary cells of a human squamous cell carcinoma by fluorescence in situ hybridization.

Chromosomes in primary tumour cells derived from a squamous cell carcinoma of the gingival mucosa were analysed. The ploidy of individual chromosomes and the frequencies of reciprocal translocations in unirradiated and irradiated cells were assessed by the method of fluorescence in situ hybridization. Hybridization probes for whole chromosomes 1, 2, 3, 4, 9 and 12 were used. The analysis was carried out in early (1st-3rd) passage cells and for comparison, in late (25th-30th) passage cells. A radiation-induced polyploidy of all analysed chromosomes was observed in irradiated (D = 4 Gy) early as well as late passage cells, with the exception of chromosome 4 in late passage cells. Ploidy of chromosomes 1 and 4 was studied at lower doses (1-3 Gy), too. Polyploidy was observed for doses >2 Gy. Some of the analysed chromosomes showed a high 'spontaneous' translocation frequency in both types of cells. The frequencies of radiation-induced translocations were similar in early and late cells and these frequencies were not always proportional to the length of the chromosomes studied. The radiosensitivity of individual chromosomes was not correlated with the level of spontaneous translocation frequency.

Carcinoma, Squamous Cell↗

Sequence diversity in the tetraploid Zea perennis and the closely related diploid Z. diploperennis: insights from four nuclear loci.

Polyploidy has been an extremely common phenomenon in the evolutionary history of angiosperms. Despite this there are few data available to evaluate the effects of polyploidy on genetic diversity and to compare the relative effects of drift and selection in polyploids and related diploids. We investigated DNA sequence diversity at four nuclear loci (adh1, glb1, c1, and waxy) from the tetraploid Zea perennis and the closely related diploid Z. diploperennis. Contrary to expectations, we detected no strong evidence for greater genetic diversity in the tetraploid, or for consistent differences in the effects of either drift or selection between the tetraploid and the diploid. Our failure to find greater genetic diversity in Z. perennis may result from its relatively recent origin or demographic factors associated with its origin. In addition to comparing genetic diversity in the two species, we constructed genealogies to infer the evolutionary origin of Z. perennis. Although these genealogies are equivocal regarding the mode of origin, several aspects of these genealogies support an autotetraploid origin. Consistent with previous molecular data the genealogies do not, however, support the division of Zea into two sections, the section Zea and the section Luxuriantes.

Alleles↗

Cell cycle checkpoint function in bladder cancer.

BACKGROUND: Cell cycle checkpoints function to maintain genetic stability by providing additional time for repair of DNA damage and completion of events that are necessary for accurate cell division. Some checkpoints, such as the DNA damage G1 checkpoint, are dependent on p53, whereas other checkpoints, such as the decatenation G(2) checkpoint, are not. Because bladder transitional cell carcinomas (TCCs) often contain numerous chromosomal aberrations and appear to have highly unstable genomes, we analyzed cell cycle checkpoint functions in a panel of TCC lines. METHODS: Cell cycle arrest was induced in normal human fibroblasts (NHF1-hTERT) and normal human uroepithelial cells (HUCs), and TCC lines and checkpoint functions were quantified using flow cytometry and fluorescence microscopy. The inducers and checkpoints were ionizing radiation (i.e., DNA damage) (G1 and G2 checkpoints), the mitotic inhibitor colcemid (polyploidy checkpoint), or the topoisomerase II catalytic inhibitor ICRF-193 (decatenation G2 checkpoint). Four of the five TCC lines expressed mutant p53. RESULTS: HUCs had an effective G1 checkpoint response to ionizing radiation, with 68% of cells inhibited from moving from G1 into S phase. By contrast, G1 checkpoint function was severely attenuated (<15% inhibition) in three of the five TCC lines and moderately attenuated (<50% inhibition) in the other two lines. NHF1-hTERT had an effective polyploidy checkpoint response, but three of five TCC lines were defective in this checkpoint. HUCs had effective ionizing radiation and decatenation G2 checkpoint responses. All TCC lines had a relatively effective G2 checkpoint response to DNA damage, although the responses of two of the TCC lines were moderately attenuated relative to HUCs. All TCC lines had a severe defect in the decatenation G2 checkpoint response. CONCLUSION: Bladder TCC lines have defective cell cycle checkpoint functions, suggesting that the p53-independent decatenation G2 checkpoint may cooperate with the p53-dependent G1 checkpoints to preserve chromosomal stability and suppress bladder carcinogenesis.

Carcinoma, Transitional Cell↗

Characterization of human zona pellucida glycoproteins.

The human egg may only be fertilized by one spermatozoon to prevent polyploidy. In most mammals, the primary block to polyspermy occurs at the zona pellucida (ZP). Little is known of the human ZP and the changes occurring following fertilization to prevent polyploidy. Using antibodies directed against synthetic peptides predicted from the human ZP2 and ZP3 cDNA, we identified ZP3 as a 53-60 kDa glycoprotein and ZP2 as a 90-110 kDa glycoprotein in prophase-I oocytes. Characterization of the ZP from metaphase II arrested eggs (inseminated-unfertilized and fertilized-uncleaved), shows no visible modification of ZP3, but demonstrates that ZP2 undergoes limited proteolysis in the amino terminal domain, to a 60-73 kDa species, denoted ZP2p, which remains linked to the proteolysed fragments by intramolecular disulphide bonds. A lack of ZP2 proteolytic activity in acrosomal supernatants is consistent with an oocyte origin for the protease. The ZP2-specific protease may be released during cortical granule exocytosis which occurs during meiotic maturation and following sperm-egg fusion as part of the block to polyspermy. Since mouse ZP2 acts as a secondary sperm receptor, it is possible that intact ZP2 binds a secondary egg binding protein, whereas cleaved ZP2 does not, suggesting a possible mechanism for the block to polyspermy.

Acrosome Reaction↗

Induction of chromosomal aberrations and spindle disturbances in Chinese hamster epithelial liver cells in culture by pyrene and benzo[a]pyrene quinones.

Regioisomers of pyrene and benzo[a]pyrene quinones were tested for their ability to induce structural and numerical aberrations and spindle disturbance in Chinese hamster epithelial liver (CHEL) cells in culture. All quinones tested were clastogenic. Pyrene-1,8-quinone (P-1,8-Q) and benzo[a]pyrene-3,6-quinone (BP-3,6-Q) induced strikingly high levels of triradials. In addition, dicentrics and ring chromosomes were very common in BP-3,6-Q-treated cultures. Isomers of these compounds, pyrene-1,6-quinone (P-1,6-Q) and benzo[a]pyrene-1,6-quinone (BP-1,6-Q), induced unobtrusive patterns of chromosomal aberrations. We suspect that the P-1,8-Q and BP-3,6-Q moieties bound to the DNA were still reactive, and formed crosslinks and/or underwent redox cycling leading to high local concentrations of reactive oxygen species. In addition, P-1,8-Q and BP-3,6-Q induced c-mitoses, hyperdiploidies and polyploidies, in particular endoreduplications. These effects were not seen with the other two test compounds, or they were only detected at the highest concentrations used, which were strongly cytotoxic (c-mitoses with P-1,6-Q, polyploidies with BP-1,6-Q).

Animals↗

In vivo cytokinesis blocked micronucleus assay with carbendazim in rat fibroblasts and comparison with in vitro assays.

A successful in vivo application of the cytokinesis blocked micronucleus assay for the detection of aneuploidy induced by carbendazim (CARB) was carried out in the granuloma pouch assay. This was performed in two ways: (i) in vivo exposure of the skin fibroblasts to cytochalasin B (cytB) and CARB, by simultaneous injection of both substances into the pouch; (ii) in vivo exposure to CARB followed by in vitro culturing of the fibroblasts in the presence of cytB. Only the first assay was successful. Injection of cytB (with or without the test compound) into the pouch resulted in the induction of binucleate cells in vivo, up to a maximum of 5% at 1 mg cytB/pouch. After injection of CARB (0-50 or 0-10 mg/pouch) and cytB (1 mg) into the pouch, aneuploidy was determined in the isolated binucleate fibroblasts by fluorescence in situ hybridization with a general centromeric probe and combinations of chromosome-specific probes (19p + 19q, 4q + Yq). With all probes, the induction of chromosome loss and/or non-disjunction by CARB was very pronounced; at 10 mg CARB/pouch the total malsegregation frequency of chromosomes 4, 19 and Y was approximately 300/1000 binucleate cells. In an in vitro cytokinesis block assay with CARB (0-2.5 microg/ml) in primary skin fibroblasts the induced aneuploidy frequencies were as high as observed in the in vivo assay. The use of two probes for chromosome 19, which enabled the scoring of chromosome breaks in addition to aneuploidy, revealed no significant induction of chromosome breaks by CARB. The frequency of polyploid mononucleate and binucleate cells was decreased after CARB treatment, in both the in vivo and in vitro assays. However, in an additional in vitro assay without cytB a major induction of polyploidy from 2.5 microg/ml CARB and above was observed, showing that cytB may interfere with polyploidy induction.

Animals↗

Griseofulvin induces mitotic delay and aneuploidy in bone marrow cells of orally treated mice.

Griseofulvin (GF) is a fungicide drug well characterized for its aneugenic activity in vitro. In vivo strong evidence of aneuploidy and polyploidy induction has been obtained in germ cells, especially in oocytes. More controversial are the data on the aneugenicity of griseofulvin in somatic cells. In this paper we provide evidence that GF induces non-disjunction and cell cycle delay in bone marrow cells of orally treated mice. Adult female mice were administered olive oil suspensions of 200, 666 or 2000 mg/kg GF by gavage and killed 18 or 24 h later. To minimize animal-to-animal variation in the absorption and distribution of GF, mice were fasted from the time of GF administration to the time of killing. Two hours before treatment the animals were s.c. implanted with a bromodeoxyuridine tablet to obtain differential chromatid staining and to determine the number of divisions after GF treatment for each metaphase. Mitostatic effects of GF were assessed by the relative proportions of first, second and third generation metaphases and the average generation time (AGT) method. A statistically significant increase with respect to the control AGT value was observed after treatment with 666 and 2000 mg/kg, suggesting that GF, as already shown in meiosis, interfered with cell cycle progression. Hyperploidy was scored in second generation metaphases. Eighteen hours after treatment, the frequencies of hyperploid cells were significantly (P < 0.05) higher in all GF-treated groups than in their matched control group. The effect was not dose-dependent. No further increase in aneuploidy was observed at 24 h, suggesting that cells overcoming mitotic arrest did not have a higher rate of non-disjunction. No induction of polyploidy was demonstrated. We conclude that GF induces mitotic delay and aneuploidy in mouse bone marrow and suggest that the protocol used to formulate the gavage suspensions and the after-treatment fasting of the animals enhanced the bioavailability of GF to bone marrow cells.

Administration, Oral↗

The incidence of chromosomal abnormalities in frozen-thawed mouse oocytes after in-vitro fertilization.

Cryopreservation of mouse oocytes induced a high rate of atresia. Frozen oocytes observed immediately after thawing did not exhibit any alteration in the frequency of chromosomal abnormalities, aneuploidy or polyploidy. After in-vitro fertilization attempts, the cleavage rate of frozen-thawed mouse oocytes was decreased. Cytogenetical observations of inseminated eggs also confirmed this decrease in fertilization rate. First and second cleavages were delayed compared to fresh controls but subsequent development to the 4-cell stage was not altered. Freeze-thawing increased the incidence of chromosomal abnormalities in inseminated oocytes but this only concerned the frequency of triploidy and not monosomic or trisomic aneuploidy. The increase in triploidy seemed to be largely due to the presence of digynic embryos. Second polar body retention seemed to be mainly responsible for this high rate of polyploidy.

Aneuploidy↗

Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation.

Regulation of transcriptional responses in growth-arrested human cells under conditions that promote potentially lethal damage repair after ionizing radiation (IR) is poorly understood. Sp1/retinoblastoma control protein (RCP) DNA binding increased within 30 min and peaked at 2-4 h after IR (450-600 cGy) in confluent radioresistant human malignant melanoma (U1-Mel) cells. Increased phosphorylation of Sp1 directly corresponded to Sp1/RCP binding and immediate-early gene induction, whereas pRb remained hypophosphorylated. Transfection of U1-Mel cells with the human papillomavirus E7 gene abrogated Sp1/RCP induction and G(0)/G(1) cell cycle checkpoint arrest responses, increased apoptosis and radiosensitivity, and augmented genetic instability (i.e., increased polyploidy cells) after IR. Increased NF-kappaB DNA binding in U1-Mel cells after IR treatment lasted much longer (i.e., >20 h). U1-Mel cells overexpressing dominant-negative IkappaBalpha S32/36A mutant protein were significantly more resistant to IR exposure and retained both G(2)/M and G(0)/G(1) cell cycle checkpoint responses without significant genetic instability (i.e., polyploid cell populations were not observed). Nuclear p53 protein levels and DNA binding activity increased only after high doses of IR (>1200 cGy). Disruption of p53 responses in U1-Mel cells by E6 transfection also abrogated G(0)/G(1) cell cycle checkpoint arrest responses and increased polyploidy after IR, but did not alter radiosensitivity. These data suggest that abrogation of individual components of this coordinate IR-activated transcription factor response may lead to divergent alterations in cell cycle checkpoints, genomic instability, apoptosis, and survival. Such coordinate transcription factor activation in human cancer cells is reminiscent of prokaryotic SOS responses, and further elucidation of these events should shed light on the initial molecular events in the chromosome instability phenotype.-Yang, C.-R., Wilson-Van Patten, C., Planchon, S. M., Wuerzberger-Davis, S. M., Davis, T. W., Cuthill, C., Miyamoto, S., Boothman, D. A. Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation.

Cell Cycle↗

Bistable Mutation-Selection Equilibria and Violations of Fisher's Theorem in Tetraploids: Insights from Nonlinear Dynamics.

Polyploidy and whole genome duplication (WGD) are widespread biological phenomena with substantial cellular, meiotic, and genetic effects. Despite their prevalence and significance across the tree of life, population genetics theory for polyploids is not well developed. The lack of theoretical models limits our understanding of polyploid evolution and restricts our ability to harness polyploidy for crop improvement amidst increasing environmental stress. To address this gap, we developed and analyzed deterministic models of mutation-selection balance for tetraploids under polysomic (autotetraploid) and disomic (allotetraploid) inheritance patterns and arbitrary dominance relationships. We also introduced a new mathematical framework based on ordinary differential equations and nonlinear dynamics for analyzing the models. We find that autotetraploids approach Hardy-Weinberg Equilibrium 33% faster than allotetraploids, but the different tetraploid inheritance models show little differences in mutation load and allele frequency at mutation-selection balance. Our model also reveals two bistable points of mutation-selection balance for dominant alleles with biased mutation rates over a wide range of selection coefficients in the tetraploid models compared to bistability in only a narrow range for diploids. Finally, using discrete time simulations, we explore the temporal dynamics of allele frequency and fitness change and compare these dynamics to the predictions of Fisher's Fundamental Theorem of Natural Selection. While Fisher's predictions generally hold, we show that the bistable dynamics for dominant mutations fundamentally alter the associated temporal dynamics. Overall, this work develops foundational theoretical models that will facilitate the development of population genetic models and methodologies to study evolution in empirical tetraploid populations.

Fisher&#x2019;s Fundamental Theorem↗

Photosynthesis in Fescue : III. RATES OF ELECTRON TRANSPORT IN A POLYPLOID SERIES OF TALL FESCUE PLANTS.

Photosystem I electron transport activity has been found to be considerably higher in a decaploid tall fescue (Festuca arundinacea Schreb.) genotype as compared to a common hexaploid genotype. The decaploid genotype also displayed a higher photosystem whole chain (Photosystem II plus Photosystem I uncoupled) activity, suggesting a connection between polyploidy and increased electron transport activity. However, when a polyploidy series of tall fescue, ranging from diploid to decaploid with several different genetic isolates at each ploidy level, was examined in natural growth conditions, no effect of increasing genome content on electron transport and photophosphorylation was found. These results suggest that a gene component of one of the genomes involved may be responsible for the increased activity rather than simply the total chromosome content.

Journal Article↗

Chromosomal anomalies in first-trimester miscarriages.

BACKGROUND: It is well known that a large proportion of first-trimester spontaneous abortions is caused by chromosomal disorders. The present study represents a unique material and the success rate of the karyotyping was high. METHODS: Chromosomal analysis from chorionic villus sampling of 259 of 304 consecutive first-trimester miscarriages in the Uppsala County, Sweden is presented. RESULTS AND CONCLUSIONS: An abnormal karyotype was found in 61% of the cases. Autosomal trisomies were most frequently detected (in 37% of the karyotyped samples), followed by polyploidies (9%) and monosomy X (6%). Cases with an extra sex chromosome constituted approximately 5% of the karyotyped abortions, with a remarkable high frequency of 47,XXY (3.4%), that is approximately 40 times greater the prevalence of Klinefelter syndrome among live birth. Similar to autosomal chromosome abnormalities, present finding indicates that the majority of sex chromosome abnormalities do not survive to term. Autosomal trisomies and an extra X-chromosome in males (47,XXY) were associated with an advanced maternal age, whereas monosomy X as well as polyploidy changes seems to be inversely related to the age of the mother. The single most common aberration was trisomy 16, which was found in 14% of the chromosomally abnormal abortions.

Abortion, Spontaneous↗

Hybrid origin and differentiation of two tetraploid Achillea species in East Asia: molecular, morphological and ecogeographical evidence.

Achillea (Asteraceae-Anthemideae) offers classical models for speciation by hybridization and polyploidy. Here, we test the suspected allotetraploid origin of two species, Achillea alpina and Achillea wilsoniana between phylogenetically distinct lineages in East Asia. A total of 421 AFLP bands from 169 individuals and 19 populations of five 2x- and two 4x-species were obtained. The data set was analysed with a newly developed model that accounts for polyploidy and assumes lack of recombination between the parental chromosome sets (i.e. disomic inheritance). A. alpina and A. wilsoniana then appear to be allotetraploids between Achillea acuminata-2x (sect. Ptarmica) and Achillea asiatica-2x (sect. Achillea). The two 4x-species share 44% and 48% of their AFLP bands with A. acuminata-2x, and 39% and 38% with A. asiatica-2x, respectively. Eight plastid haplotypes (A-H) were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses. A. alpina-4x and A. wilsoniana-4x share haplotype F only with A. asiatica-2x. This is consistent with the hybrid origin(s) involving the latter as the maternal ancestor. This result corroborates our previous DNA sequence data, where A. alpina-4x and A. wilsoniana-4x are also placed close to A. asiatica-2x. Morphology, ecology, and amplified fragment length polymorphism (AFLP) profiles of the two 2x-species are distinct, whereas the two 4x-species, grouped as A. alpina aggregate, form a nearly continuous link between them. Considering all evidence, this 4x-aggregate is regarded as the product of a hybridization between genetically distant 2x-ancestors limited to China and adjacent areas: one A. acuminata-like, and the other A. asiatica-like. The allopolyploid A. alpina agg. exhibits considerable morphological variation and ecological flexibility, and has expanded throughout eastern Asia and to northern North America, far beyond the ranges of their presumed 2x-ancestors.

Achillea↗

Use of genomic history to improve phylogeny and understanding of births and deaths in a gene family.

Polyploidy events have played an important role in the evolution of angiosperm genomes. Here, we demonstrate how genomic histories can increase phylogenetic resolution in a gene family, specifically the expansin superfamily of cell wall proteins. There are 36 expansins in Arabidopsis and 58 in rice. Traditional sequence-based phylogenetic trees yield poor resolution below the family level. To improve upon these analyses, we searched for gene colinearity (microsynteny) between Arabidopsis and rice genomic segments containing expansin genes. Multiple rounds of genome duplication and extensive gene loss have obscured synteny. However, by simultaneously aligning groups of up to 10 potentially orthologous segments from the two species, we traced the history of 49 out of 63 expansin-containing segments back to the ancestor of monocots and eudicots. Our results indicate that this ancestor had 15-17 expansin genes, each ancestral to an extant clade. Some clades have strikingly different growth patterns in the rice and Arabidopsis lineages, with more than half of all rice expansins arising from two ancestral genes. Segmental duplications, most of them part of polyploidy events, account for 12 out of 21 new expansin genes in Arabidopsis and 16 out of 44 in rice. Tandem duplications explain most of the rest. We were also able to estimate a minimum of 28 gene deaths in the Arabidopsis lineage and nine in rice. This analysis greatly clarifies expansin evolution since the last common ancestor of monocots and eudicots and the method should be broadly applicable to many other gene families.

Amino Acid Sequence↗

Duplication and DNA segmental loss in the rice genome: implications for diploidization.

* Large-scale duplication events have been recently uncovered in the rice genome, but different interpretations were proposed regarding the extent of the duplications. * Through analysing the 370 Mb genome sequences assembled into 12 chromosomes of Oryza sativa subspecies indica, we detected 10 duplicated blocks on all 12 chromosomes that contained 47% of the total predicted genes. Based on the phylogenetic analysis, we inferred that this was a result of a genome duplication that occurred c. 70 million years ago, supporting the polyploidy origin of the rice genome. In addition, a segmental duplication was also identified involving chromosomes 11 and 12, which occurred c. 5 million years ago. * Following the duplications, there have been large-scale chromosomal rearrangements and deletions. About 30-65% of duplicated genes were lost shortly after the duplications, leading to a rapid diploidization. * Together with other lines of evidence, we propose that polyploidization is still an ongoing process in grasses of polyploidy origins.

Biological Evolution↗

The HSR chromosome 1 in the house mouse, Mus musculus, from regions of the Urals polluted by mutagens.

The incidence of the HSR chromosome 1 was examined in Mus musculus musculus from 12 localities of the Middle and South Urals and Northwestern Siberia. The HSR chromosome was found in 7 localities, its frequency varying from 5 up to 18%. The HSR incidence was shown to correlate with the mean pooled frequency of aneuploidy and polyploidy in bone marrow, but not with the mean proportion of cells carrying structural chromosome aberrations. In mice heterozygous for HSR the rate of aneuploidy and polyploidy was significantly higher than in animals with standard karyotype, on average by a factor 2.35. HSRs tend to be more abundant in localities adjacent to sites of radiation incidents with contamination mainly by 90Sr.

Animals↗