Allium cepa L. Cultivars from Four Continents Compared by Flow Cytometry show Nuclear DNA Constancy

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1 Annals of Botany 85: 351±357, 2000 doi: /anbo , available online at on Allium cepa L. Cultivars from Four Continents Compared by Flow Cytometry show Nuclear DNA Constancy MICHAEL D. BENNETT*{, SPENCER JOHNSTON{, GEORGE L. HODNETT} and H. JAMES PRICE} {Royal Botanic Gardens Kew, Richmond, Surrey TW9 3DS, UK, Departments of {Entomology and }Soil and Crop Science, Texas A&M University, College Station, Texas , USA Received: 27 August 1999 Returned for revision: 23 September 1999 Accepted: 11 November 1999 In 1965 Van't Hof estimated the nuclear DNA amount of an unidenti ed Allium cepa L. cultivar as 2C ˆ pg (Experimental Cell Research 39: 8±58). This value has been adopted by common usage as the main calibration standard for angiosperm DNA C-value estimations. However, di erent cultivars have been used while assuming species DNA C-value constancy. Surprisingly this assumption has never been tested. A. cepa is an outbreeder with telomeric heterochromatic segments, so intraspeci c variation in C-value, possibly correlated with environmental factors as seen in Zea mays L., might be expected. We used laser ow cytometry to compare nuclear DNA amounts in roots of six A. cepa cultivars used as calibration standards or from di erent environments. Tissues from one cultivar, or similar volumes of tissue from two cultivars, were run and the variance between nuclei in 2C peaks compared. Only one shoulderless 2C peak was seen for all pairs of co-chopped cultivars. Thus, no large di erences in C-value between cultivars from di erent environments were found. Moreover, comparing cultivars run singly or as pairs showed no evidence for increased variation in 2C peaks in the latter, and hence of critical di erences in DNA amounts between `Ailsa Craig' and another cultivar. Such variation was insu cient to make their use as alternative calibration standards, or the practice of imputing Van't Hof's original C-value estimate to them, unacceptable for most practical purposes. Given the mechanisms known which can generate genome size variation, the degree of constancy in DNA C-value found seems remarkable. # 2000 Annals of Botany Company Key words: Allium cepa, onion cultivars, calibration standards, DNA C-value constancy, ow cytometry. INTRODUCTION In 1965 Van't Hof published an estimate of the nuclear DNA amount of Allium cepa (2C ˆ pg) carefully obtained using chemical methods. This was later used as a calibration standard by his colleagues from Brookhaven (e.g. Price and Bachmann, 1975), and by other groups including Rees and colleagues at Aberystwyth (e.g. Jones and Rees, 1968), Bennett and Smith (1976) in Cambridge, and Greilhuber (1977) in Vienna. Indeed, Allium cepa has been informally adopted by common usage as the main calibration standard for C-value estimations in angiosperms. At least 143 (ˆ 46.7%) out of 306 original sources of data listed in Bennett et al. (1997) used A. cepa with an assumed 4C DNA amount of 67.1 [or 67.0] pg as a calibration standard. The crop was chosen for this purpose because it is widely grown, easy to obtain as seed or bulbs, and highly amenable to cytology. If Van't Hof's original absolute estimate for A. cepa was inaccurate, then so are all the estimates for other taxa which use it as a calibration standard (Bennett and Smith, 1976). Such estimates would all deviate from reality by a constant proportion, but the relative di erences between taxa would be una ected. However, there is good reason for believing that the absolute 4C value for A. cepa (67.1 pg) measured * For correspondence. Fax 44 (0) , by Van't Hof (1965) is tolerably accurate. Thus, three separate studies using animal nuclei as calibration standards have independently obtained very similar results; namely Arumuganathan and Earle (1991) using Gallus domesticus (65.4 pg), Ulrich et al. (1988) using both Homo sapiens and G. domesticus (67.5 pg), and Dolezel, Sgorbati and Lucretti (1992) using H. sapiens (69.5 pg). The mean 4C DNA estimate for these three studies (67.46 pg) is within 1% of Van't Hof's original estimate of 67.1 pg. This con rmed an earlier conclusion by Greilhuber et al. (1983) that estimates for several animals, including man, obtained assuming Van't Hof's value for A. cepa (1C ˆ 16.75) are consistent with a number of independent, absolute DNA content determinations reported for those species. Thus, it is reasonable to assume that absolute DNA estimates for other taxa made using Allium cepa (assuming 2C ˆ pg) as a calibration standard are tolerably accurate, if other aspects of best practice were followed. Another concern regarding the use of Allium cepa as a calibration standard stems from possible variation in C-values between cultivars. Its consequence would be serious as estimates for unknowns would be di erentially a ected by non-constant and unknown proportions, and the relative di erences between species estimates disturbed. Intraspeci c variation in C-values has been reported in many plant species (Bennett, 1985; Greilhuber, 1998) ranging from a few to a few hundred per cent. The cause /00/ $35.00/00 # 2000 Annals of Botany Company

2 352 Bennett et al.ðnuclear DNA Constancy in Allium cepa Cultivars of such variation is hotly disputed, and in many cases is shown to be artifactual in origin (Greilhuber, 1988, 1998). However, intraspeci c variation is clear and undisputed in some species which display variation in the number and size of heterochromatic segments between cultivars. For example, C-values vary by over 30% between lines of Zea mays with di erent knob numbers (Laurie and Bennett, 1985; Rayburn et al., 1985; Poggio and Naranjo, 1991). While Van't Hof's (1965) estimate for Allium cepa (2C ˆ pg) has been widely accepted as a calibration standard, the cultivar used to determine this C-value was not stated, and is unknown (Van't Hof, pers. comm). However, workers have used several di erent cultivars for this purpose on the implicit or explicit assumption that there is no critical variation in C-values between them. Surprisingly this assumption has never been rigorously tested. The three di erent estimates (65.4 pg, 67.5 pg and 69.5 pg, see above) obtained using two animal standards di er by up to 6.3% which might indicate the occurrence of intraspeci c variation in A. cepa. Moreover, A. cepa is an outbreeding species with blocks of heterochromatin at most telomeres on its chromosomes (Stack and Comings, 1979). Thus, it seemed possible that signi cant detectable intraspeci c variation in C-value might commonly occur between its cultivars, such as occurs in other outbreeders, e.g. Secale cereale L. (Bennett, 1985) and Zea mays (Laurie and Bennett, 1985; Rayburn et al., 1985; Tito et al., 1991). Moreover, such variation in Z. mays may correlate with environmental factors such as latitude and altitude (see Table 2 in Poggio et al., 1998). Perhaps C-values of onion cultivars adapted to the low latitudes of India and Texas di er from those of cultivars adapted to the higher latitudes of the UK or Canada. If so, and if locally adapted cultivars are mainly used as calibration standards, it could introduce systematic errors in DNA estimates for the other taxa in di erent locations. We tested these possibilities by comparing several onion cultivars either listed as calibration standards in published papers by international practitioners in the eld, and/or adapted to grow in di erent environments. MATERIALS AND METHODS Selection of plant materials Table 1 lists six A. cepa cultivars compared in the present work. Van't Hof (1965) did not name the cultivar used to estimate the C-value for A. cepa. The British cultivar `Ailsa Craig' was selected because it has been recommended and widely used as a calibration standard for over 24 years (Bennett and Smith, 1976). Bennett and Leitch (1995, 1997) listed several other A. cepa cultivars used as calibration standards by various authors since Two were chosen which have been well used as calibration standards, and are adapted to widely di erent environments, namely `Stuttgarter Riesen' from Germany, and `Nazik Red' from India. `Canterbury Longkeeper' from the South Island of New Zealand was included as a representative from the Southern Hemisphere. Two North American cultivars from di erent latitudes were included, namely `Precedent', recommended for cultivation near the great lakes in Canada and the USA, and `TG-1015Y', grown in south Texas. These cultivars from six countries in four continents include three which have often been used as calibration standards (Table 1), and a range adapted for cultivation at di erent latitudes in North America or Eurasia. Comparing them should indicate if there is critical intraspeci c variation in DNA TA B L E 1. Cultivars of Allium cepa L. compared in the present work, their country of origin and approximate latitude of cultivation, source of supply, and references citing their use as C-value calibration standards Cultivation Cultivar Country Latitude Supplier/Source `Ailsa Craig' UK approx. 558N M. D. Bennett, Royal Botanic Gardens Kew `Nazik Red' India approx. 258N USDA ARS PGRU, Cornell University `Stuttgarter Riesen' `Canterbury Longkeeper' `Precedent' (ˆ`Sunex 1460') Germany approx. 488N Konrad Bachmann Gatersleben New Zealand approx. 438S Brian Murray University of Auckland Ðseed from Blenheim, South Island USA/ Canada approx. 458N Stokes Seeds Inc, Bu alo, New York `TG-1015Y' USA approx. 308N Leonard Pike, Texas A & M University Reference(s) to use as a calibration standard Bennett and Smith, 1976; Li and Liu, 1996 Ohri and Kumar, 1986; Hiremath and Salimath, 1991 Greilhuber, 1988; Baranyi and Greilhuber, 1996 Ð Ð Ð

3 Bennett et al. ÐNuclear DNA Constancy in Allium cepa Cultivars 353 C-value in A. cepa, and if so, whether it is related to latitude. Sample preparation Samples of nuclei for ow cytometry were obtained either from bulbs or young seedlings. Fifty seeds of a known A. cepa cultivar were placed in 25 ml of deionized water at 48C to imbibe for about 24 h. They were then plated out on treble-thickness lter paper moistened with deionized water in a Petri-dish to germinate in the dark either at room temperature (15±248C), or in an incubator at 238C. After 5±10 d either young roots 10±20 mm long, or seedlings (minus the seed coat) with roots 15±20 mm long were excised using a scalpel in a drop of ice-cold Galbraith bu er (Galbraith et al., 1983) on a microscope slide, and transferred to a 5 cm plastic Petri dish containing 2 ml of ice cold Galbraith bu er for chopping. The bu er ( ph 7.2) consisted of, per litre, 4.26 g MgCl 2,8.84 g sodium citrate, 4.2 g 3-[N-morpholino] propane sulfonic acid, 1 ml Triton X-100 and 1 mg boiled ribonuclease A, as described by Galbraith et al., (1983) and Price and Johnston (1996). Bulbs grown in Texas from seed of Allium cepa L. `Ailsa Craig' supplied from stock held at the Royal Botanic Gardens, Kew were suspended above deionized water in darkness at room temperature (15±248C). The base of each bulb, one per beaker, just touched the water which was changed every 24±48 h. After 2±7 d, root tips 5±10 mm long were excised from roots 10±20 mm long as above and chopped in ice-cold bu er for ow cytometry. Occasionally, a segment of leaf tissue about 10 mm long was excised from an etiolated young leaf projecting 20±50 mm from the top of a bulb. The ratio of tissue to bu er and the minimum volume of bu er needed to obtain satisfactory yields of nuclei were determined in preliminary experiments. These showed that three± ve root-tips or one leaf segment from a bulb, or ve±ten roots from seedlings chopped in 2 ml of bu er, typically yielded 2500± nuclei in runs lasting 5± 18 min using 1 ml samples recovered after ltering. Plants of Pisum sativum L. `Minerva Maple' were grown in pots in a growth chamber given 16 h d at 228C and 8 h nights at 138C. Healthy young lea ets were sampled for calibration checks against Allium cepa `Ailsa Craig' using ow cytometry. One or two lea ets were chopped in 2 ml ice-cold bu er without Allium, whereas half a lea et cochopped with Allium tissue as above normally gave similar yields of Pisum and Allium 2C nuclei, and hence similarlysized peaks. Comparisons of nuclear DNA amount DNA content was determined from young leaf or root tissue excised from bulbs or seeds using laser ow cytometry with propidium iodide (PI) as the uorochrome. The healthy young leaf and/or root tissues excised from bulbs or seeds were chopped in 2 ml of ice-cold bu er using a new razor-blade for each sample. The chopped tissues were ltered through a 53 mm nylon lter. One ml of ltrate was pipetted into a microfuge tube to which ice cold propidium iodide was added to a nal concentration of 50 ppm PI. The samples were stored on ice in a dark refrigerator, and analysed after 1 to 8 h. The DNA content for four to 12 replicates for each cultivar, or pair of cultivars, was determined by a protocol adapted from Price and Johnston (1996) using a Coulter Epic Elite ow cytometer (Coulter Electronics, Hialeah, Florida, USA) equipped with a watercooled laser tuned at 514 nm and 500 mw. Fluorescence emission at 4615 nm was detected with a photomultiplier screened by a long pass lter. The ow cytometer was tuned to produce minimal variance with Coulter Flow-Check 2 uorospheres (Coulter Corporation, Miami, Florida, USA) and mean uorescence of A. cepa `Ailsa Craig' 2C nuclei between 370 and 410. The following approach was used to test whether cultivars di er in DNA C-values. First, nuclei of single cultivars were run and the coe cient of variation (CV) among 2C nuclei was noted. Second, nuclei from pairs of known co-chopped cultivars were run together and the corresponding statistic noted for 2C nuclei. When pairs of cultivars were co-chopped, equal volumes of tissue from each cultivar were used. If two cultivars di er greatly in C-values (i.e. 45%) ow cytometry should show separate 2C peaks. Given only a small di erence in C-value (i.e. 42%) the shape of a 2C peak should be detectably broader for the mixtures than for either cultivar run alone (Hopping, 1994). Thus, if two cultivars di ered even so little in C-value, the variance for the 2C peak should be higher for a co-chopped mixture than for either single cultivar. The statistic chosen for this comparison, given by the ow cytometer printout was the half peak CV comparing similarly sized populations of 2C nuclei. Both the coe cients of variation and arc sine root transformed CVs were compared using the orthogonal contrast statement from PROG GLM in v6.12 SAS (SAS Institute, Inc. Cary, NC, USA). RESULTS The material and methods used gave acceptable yields of nuclei for ow cytometry for all the A. cepa cultivars studied. However, long runs, usually min, were needed to acquire estimates for populations typically of about 5000 nuclei. Figure 1A and B shows typical plots for two Eurasian cultivars run alone, while Fig. 1C shows a typical plot for the same two cultivars co-chopped and run together. Figure 2 shows a similar comparison for two American cultivars run alone (A and B) or together (C). Fluorescence pro les for root-tip nuclei typically had a much larger 2C than 4C peak with little or no evidence of an 8C or higher peak (Figs 1 and 2). The C-value status of these peaks was initially veri ed in a few preliminary comparisons with peaks for nuclei from Pisum sativum lea ets, whose pro le and 2C values were known from our previous work (e.g. Johnston et al., 1999), run separately or together using the same FC setting. The uorescence peaks from young leaf tissue from bulbs had a di erent pro le. The 2C peak was largest, but a 4C peak was relatively larger than in root tissue. Other smaller peaks gave clear evidence of 8C and 16C populations (data

4 354 Bennett et al.ðnuclear DNA Constancy in Allium cepa Cultivars FIG. 1. Relative red uorescence of PI-stained nuclei of `Ailsa Craig' (A) and `Nazik Red' (B) run alone, and (C) together. FIG. 2. Relative red uorescence of PI-stained nuclei of `TG-1015Y' (A) and `Precedent' (B) run alone, and (C) together. not shown). The 8C peaks may provide a useful calibration standard with pg for estimating unknown DNA amounts in species with high C-values in the range 100± 200 pg. However, a drawback with the use of such leaf tissue was a tendency for material in the sample to clog and slow the ow, requiring cleaning and resetting of the cytometer. This problem also occurred using roots, but to a much lesser degree. Nevertheless, the long durations of runs, combined with an unpredictable need to clean and reset the FC, made it impossible to compare all the present materials in one experiment, or sometimes to complete all the replicates intended on a single day. The critical test was between known cultivars run individually or together in pairs in the same experiment, so sensitivity was not precisely standardized between all experiments. Thus the photomultiplier was set to give 2C peaks for `Ailsa Craig' around 400 (Table 2) or 370 (Table 4), but arbitrarily at a higher range around 450 (Table 3) in expt 4 with `Canterbury Longkeeper'. Most importantly, given the main reason for this study (i.e. the possibility that cultivars of A. cepa may have critical di erences in C-value), no evidence of two separate 2C peaks was seen in any of the six combinations of two cochopped cultivars examined, i.e. `Ailsa Craig' plus each one of the ve other cultivars studied, or `TG-1015Y' plus `Precedent' (Tables 2±5). Similarly, no repeatable evidence of a shoulder on the peak containing 2C nuclei was noted in any replicate for any one of these combinations (Figs 1 and 2). Thus, no evidence for large di erences in C-value, such as commonly occur between cultivars of Zea mays from di erent environments, was found for any of the present A. cepa materials grown in di erent regions and environments. As noted above, comparisons of the shapes of peaks for populations of 2C nuclei for pairs of cultivars run singly or together should provide a telling test for small but still unacceptably large di erences in C-values between cultivars used as calibration standards. The orthogonal contrasts showed no signi cant di erences in CVs for nuclei of 2C peaks for singletons and co-chopped pairs of cultivars including some from di erent latitudes where di erences might have been expected (Figs 1 and 2; Tables 2±5). Thus, the mean CVs (Table 5) for six mixtures (2.25) and six single cultivars (2.20) were virtually identical (F 1,56 ˆ 0.71;

5 Bennett et al. ÐNuclear DNA Constancy in Allium cepa Cultivars 355 TA B L E 2. Mean red uorescence from PI-stained 2C nuclei of three cultivars of A. cepa used as calibration standards, chopped alone, or in pairs including `Ailsa Craig' Cultivar(s) `Ailsa Craig' `Nazik Red' `Stuttgarter Riesen' `Ailsa Craig' `Nazik Red' `Ailsa Craig' `Stuttgarter Riesen' Replicate Mean CV Mean CV Mean CV Mean CV Mean CV Experiment Experiment Mean TA B L E 3. Mean red uorescence from PI-stained 2C nuclei of `Ailsa Craig' and `Canterbury Longkeeper' chopped alone or together Cultivar(s) `Ailsa Craig' `Canterbury Longkeeper' `Ailsa Craig' `Canterbuty Longkeeper' Replicate Mean CV Mean CV Mean CV Experiment Ð Ð Ð Ð Ð Ð Ð Ð Experiment 4 5 Ð Ð 452.3* * Ð Ð 452.8* * 2.19 Mean * * 2.30 (405.5) (421.8) * The mean red uorescence for 2C peaks obtained in expt 4 re ects a FC sensitivity giving values uniquely above those obtained for these and other cultivars within the normal range of settings in other experiments or days. Consequently, means for values omitting this anomalous setting are also given (in brackets) to aid comparison. TA B L E 4. Mean red uorescence from PI-stained 2C nuclei of three cultivars of A. cepa chopped alone, or in pairs Cultivar(s) `Ailsa Craig' `TG-1015Y' `Precedent' `Ailsa Craig' `TG-1015Y' `Ailsa Craig' `Precedent' `TG-1015Y' `Precedent' Replicate Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Experiment Ð Ð 2 Ð Ð Ð Ð Experiment Ð Ð Ð Ð Ð Ð Mean

6 356 Bennett et al.ðnuclear DNA Constancy in Allium cepa Cultivars TA B L E 5. Mean CV for 2C peaks in six A. cepa cultivars chopped alone or co-chopped in pairs Single cultivars CV Paired cultivars CV `Ailsa Craig' 2.36 (6) `Ailsa Craig' `Nazik Red' 2.11 (6) 2.39 (2) 2.53 (4) 2.42 (12) `Nazik Red' 1.94 (6) `Ailsa Craig' `Stuttgarter Riesen' 1.99 (6) `Stuttgarter Riesen' 1.79 (6) `Ailsa Craig' `TG-1015Y' 2.16 (6) `TG-1015Y' 2.35 (5) `Ailsa Craig' `Precedent' 2.35 (4) `Precedent' 2.68 (6) `TG-1015Y' `Precedent' 2.30 (5) `Canterbury Longkeeper' 2.34 (4) `Ailsa Craig' `Canterbury Longkeeper' 2.30 (6) Mean 2.25 (6) 2.20 (6) The number of replicates are given in parentheses for each mean. P ). Surprisingly, on average, the 2C peaks for mixtures were just as sharp as for single cultivars. The mean CVs (Table 2) of 2C peaks for `Ailsa Craig' (2.36) from the UK (approx. 558N) and `Nazik Red' (1.94) from India (approx 258N) were not signi cantly di erent from their cochopped mixture (2.11) (F 1,24 ˆ 0.05; P ). Similarly, the mean CVs (Table 4) of 2C peaks for `TG-1015Y' (2.35) from the southern USA (approx. 308N) and `Precedent' (2.68) from the Great Lakes (approx. 458N) were not signi cantly di erent from that of their cochopped mixture (2.30) (F 1,23 ˆ 0.56; P ). Thus, the present work provides no evidence of a latitudinal cline for DNA C-value in A. cepa, unlike that reported for Zea mays (Laurie and Bennett, 1985; Rayburn et al., 1985). DISCUSSION As noted previously (Bennett and Smith, 1976; Bennett and Leitch, 1995, 1997) various workers have used several di erent cultivars of Allium cepa as calibration standards on the explicit or implicit assumption that no critical variation in DNA C-value exists between them. However, this assumption had never been widely tested, although some comparisons of cultivars from the same region showed no di erence (Waldherr, 1992). The present work compared six cultivars from widely di erent geographical locations and adapted to di erent environments, including two from Europe (`Ailsa Craig' and `Stuttgarter Riesen') and one from India (`Nazik Red') previously used as calibration standards. Comparing 2C curves for known pairs of cultivars run either singly or together showed no evidence of increased variance in the latter, and hence no evidence for critical di erences in nuclear DNA amounts between the test cultivars. Thus the present results for A. cepa parallel previous work by Bennett and Smith (1976) which found no critical di erences between cultivars of Vicia faba L., and of Hordeum vulgare L. Moreover, it supports the extensive recent results by Greilhuber and colleagues showing little or no signi cant variation between cultivars of Pisum sativum (Greilhuber and Ebert, 1994; Baranyi and Greilhuber, 1995, 1996) and Glycine max (L.) Merr. (Greilhuber and Obermayer, 1997). Clearly a tendency towards a global constancy for C-values within many species is not uncommon. To obtain perfect, absolute C-values the ideal calibration standard should exhibit no variation between individuals. In the real world of biodiversity and error variation this ideal is approached by using best practice to minimize real and error variation, and to produce acceptable C-value estimates whose accuracy re ects need and resources. In order to minimize error due to biodiversity in A. cepa it is desirable to use just one cultivar of the species as a calibration standard. Moreover, to minimize the possibility of variance due to environment, it is best to use seed from a large population grown in one place and harvested at one time. The use of such seed of A. cepa `Ailsa Craig', distributed widely for this purpose since 1976, has presumably tended to minimize such errors, and is still best practice to approach the ideal. Nevertheless, the present work indicates that best practice would not be seriously prejudiced by using the other cultivars compared in the present work. Thus, it is now reasonable to presume that their past use to estimate DNA C-values for other species caused no unacceptable errors. It is not suggested that no intraspeci c variation in DNA C-value occurs between individuals or cultivars in A. cepa. Vosa's observation (1973) that most chromosomes were heteromorphic for C-band patterns in an Allium species suggests otherwise. Rather we note that the level of such variation between `Ailsa Craig' and ve other cultivars compared in the present work was not so large as to make their use as alternative calibration standards unacceptable, or to render the practice of imputing to them the C-value originally estimated for an unidenti ed cultivar of A. cepa by Van't Hof (1965) untenable, for most practical purposes. Recent results for 14 A. cepa cultivars including `Stuttgarter Riesen' estimated by Feulgen microdensitometry or ow cytometry using propidium iodide strongly support this view (Baranyi and Greilhuber, 1999). Thus, their assumption of 2C ˆ for `Stuttgarter Riesen' as a calibration standard gave 2C DNA estimates ranging from only to for the 13 other cultivars. The variation in C-values between the present onion cultivars was clearly small, perhaps surprisingly so, given the very di erent environments in which they are cultivated. Clearly mechanisms exist which can rapidly generate

7 Bennett et al. ÐNuclear DNA Constancy in Allium cepa Cultivars 357 considerable variation in DNA C-value within individuals and even in individual cells. In view of the molecular mechanisms now known which can generate variation in genome size (Kubis et al., 1998; Sanmiguel and Bennetzen, 1998), the degree of C-value constancy found in many species, including A. cepa, is remarkable, and needs explanation. Indeed, it is arguable that such constancy would not be expected without some mechanism(s) to select for constancy (or against drift) in C-value, which thereby controls variation in DNA C-value back towards some encoded norm for each species. Were it not so, the frequency and extent of intraspeci c variation in DNA amount would surely be much larger, and the observed degree of species DNA constancy would present a new C- value paradox. Genome size is widely perceived as free to vary, changes being undetected and uncorrected by internal control mechanisms. 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