Intraspecific Variation in Genome Size: A Critical Reassessment

Annals of Botany 82 (Supplement A) : 27–35, 1998 Article No. bo980725

Intraspecific Variation in Genome Size : A Critical Reassessment J. G R E I L H U B E R Institute of Botany, UniŠersity of Vienna, Rennweg 14, A-1030 Wien, Austria Received : 4 November 1997

Returned for revision : 17 April 1998

Accepted : 8 May 1998

Genome size differences between angiosperm species can be very large, up to about three orders of magnitude. However, the occurrence and extent of genome size variation below the species level is, according to the case, controversial. It is obvious that some variation due to common chromosome polymorphisms and spontaneous aberrations ought to occur. Non-recognized taxonomic heterogeneity of the material may also be a cause of ‘ intraspecific ’ variation. Such cases of variation are well understood or at least plausible and may be termed ‘ orthodox ’. The ‘ unorthodox ’ types of variation are those that require ad hoc assumptions to explain them, e.g. rapid transposable element spreads or amplification\diminution events in the course of development or reproduction (plastic genome), or are simply non-explainable in the context of the current taxonomy or of the breeding system of the organism (unorthodox intraspecific genome size variation). At present, many students of the topic seem to believe that such unorthodox variation is common. This review attempts to discuss some of the cases of unorthodox variation in angiosperms with emphasis on those that have been re-investigated by the present author. Upon reanalysis, technical shortcomings (e.g. suboptimal performance of the Feulgen reaction or insufficient standardization) are often shown to be the probable cause of unorthodox variation. The ‘ plastic genome ’ seems to be an idea rather than a defendable scientific hypothesis ; intraspecific variation is less frequent than presently thought. Unorthodox genome size variation should be treated with more scepticism. # 1998 Annals of Botany Company Key words : Genome size, plastic genome, flow cytometry, Feulgen, densitometry.

INTRODUCTION A survey of the reviews on genome size variation indicates considerable and continuous interest in the phenomenon of variation below the species level (Nagl, 1979 ; Nagl et al., 1983 ; Greilhuber, 1984 ; Bachmann, Chambers and Price, 1985 ; Bennett, 1985, 1987 ; Cullis, 1985, 1990 a, b ; Walbot and Cullis, 1985 ; Ohri and Khoshoo, 1986 ; Greilhuber and Ehrendorfer, 1988 ; Price, 1988 a, b ; Cionini, 1989 ; Bassi, 1990 ; Cavallini and Natali, 1991). Genome size is a quantitative character of an organism that ought to be more or less constant between individuals of a population as long as there is enough interbreeding to mix up the gene pool. If the population, and therefore the gene pool, is split up (by geographical barriers), genome size differentiation may occur within a taxonomical bona fide species that actually consists of several reproductive communities. An instructive example is Scilla bifolia L. s. l. (Hyacinthaceae), that is treated as one species by Flora Europaea (McNeill, 1980), but has been split up into 18 species (of which 13 are diploids) by Speta (1980) on the basis of relatively inconspicuous characters. Speta (1971) and Svoma (1981) also showed convincingly that the genus Chionodoxa Boiss. is closely allied with these taxa, as also becomes clear from the fertile hybrids between Scilla niŠalis Boiss. and Chionodoxa sardensis (Whittall ex Barr and Sugden) Speta. Consequently, Chionodoxa has been merged into Scilla (Speta, 1971, 1980). Scilla bifolia, treated as one species, shows a two-fold genome size variation, but if split up taxonomically, intraspecific variation diminishes to hardly more than methodological error (Greilhuber, 1979 ; Greil0305-7364\98\0A0027j09 $30.00\0

huber and Speta, 1985). Interestingly, there are no reasonable genome size differences between the crossable species of Scilla and Chionodoxa, S. niŠalis and C. sardensis (l Scilla sardensis), while the differences in flower structure are significant (Greilhuber, 1979). Thus, there is considerable genome size diversification in the absence of remarkable morphological evolution within the taxon Scilla bifolia sensu McNeill (1980), while there is no reasonable genome size difference between those species assigned to the different genera Scilla L. and Chionodoxa Boiss. This example shows that ‘ intraspecific genome size variation ’ can be a taxonomical artifact. It results from insufficient consideration of the boundaries of the actually interbreeding community and from an incomplete or non-existent knowledge of the phytogeographical history of the populations of the taxon. Genome size is a character, and as such it has taxonomic significance as well as any other character and perhaps even more, because its diversification depends on many, perhaps thousands of mutation steps. The taxonomic rank that should be assigned to such cytotypes characterized by genome size is, of course, disputable. As any degree of transition in genome size will occur in nature, from the slight geographic variant that deserves no taxonomic recognition to the clear-cut entity, and from the strongly deviating entity without morphological differences to the minor variant with a good differentiating morphological trait, the question must remain unanswered ; but this is not the subject of this contribution. The occurrence of this type of intraspecific variation is not at all implausible ; it belongs biologically to the same category as interspecific genome size variation (and is indeed incipient interspecific genome # 1998 Annals of Botany Company

28

Greilhuber—Intraspecific Genome Size Variation

size variation). In my opinion, parts of Scilla autumnalis L. s. l. (or, more appropriately, Prospero Salisb.) belong to the category of morphologically ill- or non-separated entities well distinguished in genome size (Ebert, Greilhuber and Speta, 1996). Apparently nothing reliable is known about the rate of genome size changes in natural populations. However, if one considers the intraspecific stability, for instance, within Scilla bifolia s. str., S. Šindobonensis Speta, and S. kladnii Schur over one thousand kilometres, and the stable interspecific differences even in regions where these species almost come into contact (Greilhuber and Speta, 1978, 1985), it seems that genome size changes require a long time span to develop, or if they are acquired quickly, perhaps in small isolated populations, they may be followed by long periods of stasis. It is well known that there is almost always some degree of chromosomal variation within populations involving duplications and deletions, spontaneous aneuploidy and polyploidy, heterochromatic segments, B-chromosomes and, in special cases, sex chromosomes. Such chromosomal variation will naturally cause some inter-individual DNA content variation. This category of intraspecific genome size variation is easily comprehended and is termed here ‘ orthodox intraspecific variation ’. Heterochromatin-polymorphisms may become geographically fixed and may then characterize taxa having different genome size. Examples of this have been described in the subspecies pair Scilla bithynica Boiss. subsp. bithynica (many large C-bands) and S. bithynica subsp. radkae (Davidoff) Speta (few small C-bands), and in the species pair S. rosenii C. Koch (few small C-bands) and S. koenigii Fomin (many large C-bands) (Greilhuber, 1984). However, 30 years ago (Evans, Durrant and Rees, 1966) there appeared, for the first time, reports on intraspecific DNA content variation that could not be explained by ‘ orthodox ’ events. As ‘ unorthodox ’ events have to be postulated (for instance rapid amplification\deletion of repetitive DNA), I term this category of genome size variation ‘ unorthodox ’. On the one hand there are rapid changes in genome size that are said to occur in connection with developmental events or at least within two generations. For this, the term ‘ plastic genome ’ has become popular. On the other hand there are differences in genome size between individuals of one population or between populations of the same species that are so large that neither taxonomical artifacts nor common chromosomal heteromorphisms can apply, and that are simply non-explainable in the context of the current taxonomy or the breeding system of the organism. For this I will use the term ‘ unorthodox intraspecific genome size variation ’ (naturally there are transitions between ‘ orthodox ’ and ‘ unorthdox ’ intraspecific variation).

THE PLASTIC GENOME Scilla siberica In 1989 a paper by Deumling and Clermont appeared that merits special interest because it presents the first—and

hence only—illustrations, by photographs, of genome size plasticity. Tissue of triploid Scilla siberica Haw. in Andr. (2n l 3x l 18 ; 2C l approx. 99 pg) had been brought into culture and within 2 years the chromosome number became diploid, the very large chromosomes became more numerous and smaller and finally tiny. DNA amount was reduced to 20 %, satellite and highly repetitive DNA was lost. While chromosome number variability and aberrations in tissue culture are not uncommon (Bayliss, 1980), such a dramatic loss of sequences was unprecedented in the literature. Novel as these results are, there are various interpretations possible. Those who like the idea of the plastic genome, will take the results of Deumling and Clermont (1989) as definite evidence for the real existence of genome plasticity. Others who are less enthusiastic but would not a priori reject the ‘ plastic genome ’, would perhaps take a neutral position. Scepticists, however, might reject the ‘ plastic ’ interpretation and attempt to explain the results of Deumling and Clermont (1989) as possibly due to a contamination of their plant material with that of one or two alien species (with smaller and more chromosomes), whose bulbs had been confused with those of Scilla siberica and in the period of the investigation outgrew the S. siberica cells (that were probably slower growing because of their large nuclei). Indeed, a careful examination of the paper of Deumling and Clermont (1989) reveals only ambiguous evidence that the tissue cultures had been started and maintained in a monoclonal way. The material had been obtained commercially, and it appears not improbable that initially bulbs of diploid and triploid Scilla siberica had been inadvertently mixed up with those of one or more ornamental species of Hyacinthaceae, as Muscari, Hyacinthella, or even Ornithogalum. The latter interpretation by contamination seems to me much more plausible than the assumption of ‘ genome plasticity ’, but even an implausible idea can be long-lived, provided there are people who like it. Much can be learned from the paper by Deumling and Clermont (1989) about the contemporary ‘ paradigm ’ (Kuhn, 1993) of genome size plasticity. Hedera helix and Sambucus racemosa The first time I felt stimulated to re-investigate a reported case of ‘ genome plasticity ’ concerned ivy, Hedera helix (Ko$ nig, Ebert and Greilhuber, 1987). The ivy is particular by its phase change from juvenile to adult (flowering), where growth form shifts from creeping or climbing, with dorsiventral stems with aerial roots and palmate leaves, to orthotropy without aerial roots, with spiral foliation and rhomboidal leaf form. Kessler and Reches (1977) had estimated DNA content per cell biochemically (8n0 and 6n7 pg DNA in juvenile and adult leaves, respectively) and genome size by reassociation technique (3n62 and 1n85 pg in juvenile and adult leaves, respectively). From this they concluded that ‘ juvenile leaves contain more DNA per cell and per haploid genome and their ploidy seems to be 2N. Upon transition to the mature phase the cellular as well as the genomic DNA level decreases, but their ploidy becomes 4N ’. From their melting and reassociation experiments they concluded that ‘ polyploidization upon phase transition

Greilhuber—Intraspecific Genome Size Variation results from a selective amplification of sequences rather than from endoduplication of the entire genome ’. On the other hand, Scha$ ffner and Nagl (1979 ; see also Nagl, 1979 ; Nagl et al., 1983) found less DNA in juvenile than in adult meristems, on average 3n6 and 6n2 pg, respectively. Although they did not address the discrepancy with the results of Kessler and Reches (1977), they too postulated a kind of differential replication of DNA (71 % DNA plus that is integrated into the adult genome, but has to be deleted before the emergence of a new juvenile ivy plant). Surprisingly, juvenile nuclei had more heterochromatin than adult nuclei and it was postulated that during the phase change towards the adult form the heterochromatin does not participate in replication (i.e. there is differential replication of euchromatic DNA). Large as the reported 1n71-fold difference between the phases is, other authors (Polito and Alliata, 1981 ; Ko$ nig et al., 1987) were unable to reproduce it. Moreover, a difference in heterochromatin content between phases could not be confirmed, either by volumetric measurement of condensed and diffuse chromatin in interphase (Polito and Chang, 1984) or by measurement of C-banded metaphase chromosomes (Ko$ nig et al., 1987). Nagl and co-workers also reported phenomena similar to that in ivy in other plants : ‘ floral DNA ’ (elevated genome size during floral induction) in Rhoeo spathacea (Sw.) Stearn, in Scilla decidua Speta (actually S. niŠalis) and in Sambucus racemosa L. There is one conclusion emerging from the negative results of Ko$ nig et al. (1987) in ivy : if authors find 1n71-fold variation and there is actually none, something must be wrong with their methods. Further indications for this are the following : for instance, the name Scilla decidua (an endemic of Anatolia that has never been commercially available) was chosen by Frisch and Nagl (1979) and Nagl, Frisch and Fro$ lich (1979) because their DNA determination in their material (2C about 13n6 pg in root tips) suggested this identification (compare Greilhuber, 1979). However, it is highly probable that these authors had purchased S. niŠalis (which appears to be the only species of the alliance that one can buy in shops, but as S. bifolia), a species having much less DNA (2C about 8n2 pg) than S. decidua (Greilhuber, 1979). An almost 1n7-fold error of measurement seems to have been made by Frisch and Nagl (1979). Later, Greilhuber (1988 a) found no evidence for ‘ floral DNA ’ in Sambucus racemosa, but reported the occurrence of condensable tannins in meristems that can induce severe stoichiometric errors (see below). Flax, Linum usitatissimum The ‘ plastic genome ’ has a long history. Flax (Linum usitatissimum L.) was the first example (Evans, Durrant and Rees, 1966 ; Evans, 1968 a, b ; Durrant and Jones, 1971 ; Joarder et al., 1975) and is maintained up to the present as a valid case of that phenomenon that has even found its way into a genetics textbook (Alberts et al., 1986). When a ‘ plastic ’ genotype (abbreviated Pl), e.g. ‘ Stormont Cirrus ’, is grown under certain environmental conditions (warm temperature) in a glasshouse and fertilized either with phosphorus or nitrogen, small (S) and large (L) plants, so-

29

called genotrophs, could be obtained. The quantitative characters in these genotrophs remained heritable, if certain conditions were met with (cf. Cullis, 1977). S and L came to differ 1n16-fold in genome size, L becoming 1n1-fold larger than the original and S smaller by 0n94-fold. However, reversions and changes to a smaller genome size did occur when the plants were grown outside for some generations. A difference in chromosome size in allotetraploid LiS crosses was not seen (Evans, 1968 b), and is not expected to be. Schweizer (1980) compared genome size in root tips of S and L genotrophs with stable cytofluorometry using DAPI and chromomycin A3. Interestingly, although he could not confirm a larger genome size in L, the values were even larger in S with both fluorochromes, so that a bias due to GC content can be ruled out. A statistical analysis of his data conducted recently (Greilhuber and Schweizer, unpubl. res.) shows that the difference between L and S, though not so small, was actually non-significant. Recently, we compared the genotroph lines (plastic, L and S) independently with Feulgen densitometry (Greilhuber and Schweizer, unpubl. res.). In eight experiments involving more than 1000 root tip nuclei (metaphase nuclei in side view, as the mean absorbance per scanning point of pro- or telophase nuclei was deemed too low to yield reliable estimates) no significant difference between S and L was found, and Pl was on average about 2 % higher than both (marginally significant, as the difference turned out in any of the eight tests). Commercial linseed had a genome size very similar to that of ‘ Stormont Cirrus ’. These results (to be published separately) suggest alternative explanations for the ‘ plastic genome ’ in flax. As the 2C value of flax was determined as 1n12 pg (standards : Allium cepa 33n5 pg, Pisum satiŠum 8n84 pg, 2C, see Greilhuber and Ebert, 1994), and is therefore low, a 1n16-fold Feulgen absorbance variation without true DNA variation would not appear improbable if one considers the differences in the environments causing growth differences and possibly also condensation differences in nuclear chromatin. Differences of that size order between nuclei on a single slide are not uncommon in Feulgen preparations (where they are considered to be random technical and not real DNA content differences). There is another technical point that should be considered : Evans (1968 a) mentioned thick cell walls and high oil content (or tannins ?) in root tips of flax as the reason why he chose to measure shoot tip 4C interphase nuclei (Evans et al., 1966). However, root tips from germinating seeds (as measured by Schweizer, 1980, and myself) do not have thick cell walls or cytoplasmic inclusions such as oil-bodies. It appears possible that the physiological conditions of the plant tissues used for measurement by Evans et al. (1966) and Evans (1968 a, where the cytological methods are described), and by those who did not find a difference, had an influence on the Feulgen-DNA data variation irrespective of real DNA content. Moreover, Evans (1968 a) and probably also his cooperators used hot hydrolysis which is more susceptible to variation than the cold hydrolysis (Fox, 1968) used in my laboratory. [It should also be noted that a 10 % genome size difference was found between Linum satiŠum cultivars ‘ Dakota ’ and ‘ Mandarin ’ by Evans et al. (1966). This difference should be independently tested, as it could provide

30

Greilhuber—Intraspecific Genome Size Variation

T     1. Comparison of genome size estimates in two particular lines of Helianthus annuus, independently published by two different research laboratories Genome size (pg) Helianthus annuus lines RHA 271 HA 89 Ratio HA 89\RHA 271

Michaelson et al. (1991) 7n21 6n96 0n97 (n.s.)

Cavallini et al. (1986) 6n82 9n20 1n35 (P

0n001)

a means of evaluating the accuracy of the DNA measurements by Evans and co-workers]. The flax genotrophs provide a doubtful instance of a plastic genome phenomenon that has nevertheless found favour for 30 years. There is a need to resolve this issue. As methods to compare genome sizes with higher accuracy than ever before are now available (Dolez3 el et al., 1998), I suggest that an international board of teams should investigate the DNA changes in flax with flow cytometry. Sunflower, Helianthus annuus Another quite recent example of the plastic genome is found in sunflower, Helianthus annuus L. Cavallini et al. (1986) found 1n58-fold genome size variation between lines and 1n16-fold DNA content in genomes of embryos from the periphery of the flower head compared with those from the centre. They later suggested an amazing process to compensate for this variation in the next generation (Cavallini et al., 1989). Cavallini et al. (1996), while introducing the reader to the idea that these nuclear DNA changes in H. annuus are due to variations in the degree of methylation and amount of repetitive DNA, write : ‘ Evidence is accumulating in the literature that intraspecific changes in genome size due to quantitative variation in certain nuclear DNA fractions can occur, particularly in plants. Therefore, the view that fluid domains may exist in plant genomes, in addition to more stable portions, is gaining ground in the scientific community ’. Indeed, Price and Johnston (1996) showed that light quality can strongly influence genome size in the sunflower : a high proportion of far-red results in reduction of genome size (measured from leaves) by more than 50 %. However, opinions diverge among those working on sunflower. Michaelson et al. (1991) could not confirm a genome size difference between the lines RHA 271 and HA 89, in which Cavallini et al. (1986) found 1n35-fold variation (Table 1), but found similar variation in other lines. It should be mentioned that the American workers did their flow measurements mostly with leaf material that showed strong intra-plant variation. For instance, Michaelson et al. (1991), who found 1n32-fold variation between lines and up to 1n19-fold variation within lines, reported up to 1n48-fold variation between leaves of a single plant. They also figured a clear negative correlation between measured leaf DNA content and the age of the plant, from which a newly expanded leaf had been taken for

measurement. Such a result leads one to suspect stoichiometric errors in dye binding, because it is known that secondary plant metabolites such as polyphenols and even low-molecular weight substances such as catechin and quercetin (a frequent flavonoid) can reduce the intensity of Feulgen staining (Greilhuber, 1988 a, 1997) and is suspected to influence fluorochrome binding as well (see below).

The role of stoichiometric errors Methodological errors that cause non-stoichiometric binding of the chromophore are often responsible for variations in measurement data that may be interpreted as indicating genome size variation or fluctuation. For instance, hot hydrolysis (1  HCl, 60 mC) is not recommended because the staining optimum only lasts a very short time (see Fox, 1968). However many authors today just use hot hydrolysis, or, if cold hydrolysis is done (5  HCl, correctly applied at 20n0 mC for 60 min), the molarity of the hydrochloric acid is not adjusted nor is the temperature kept constant (room temperature is often given, whatever this may be). Some authors (e.g. Cavallini and co-workers) use pectinase-macerated squashes on slides for subsequent Feulgen staining (pectinase may contain DNases). Greilhuber and Ebert (1994) have shown that such slides deteriorate during storage, and after 3 months show highly erratic staining behaviour and are effectively spoiled. These are probably only some of the many sources of faulty data that could easily be avoided by careful and prudent experimentation. From the viewpoint of genome plasticity and intraspecific variation there is one other type of stoichiometric error that seems to be particularly important and influential. I have termed this sort of technical flaw ‘ self-tanning error ’ (Greilhuber, 1988 b), because there is reduced and distorted Feulgen staining (and probably also fluorochrome staining) when tannins or similar polyphenols are present in the cells and tissues to be analysed. In ŠiŠo these substances are localized in vacuoles and they occur frequently in meristems. After cell death and after most protocols of fixation these substances permeate and soak the tissue. The cells tan themselves. As is typical for tannins, these compounds bind strongly to proteins and particularly firmly to chromatin, as tannin staining tests with vanillin sulphuric acid show. Unfortunately, quantitative cytochemical reactions for DNA are severely disturbed by these metabolites. Greilhuber (1986) found that formaldehyde is able to polymerize tannins of the condensable type in situ, i.e. in the vacuole, where they are solidified and thereafter do not interfere with quantitative staining of the nuclei because they cannot move in the cell. After the common acetic alcohol fixtion, tannins are not seen in the meristems, but they are ubiquitous. Formaldehyde mixed with alcohols and acid (e.g. FAA and FPA ) is not suitable to avoid self-tanning, because the &! vacuole membrane is quickly destroyed and tannins soak the cell before being polymerized (this is a critical remark upon the results of Mellerowicz, Riding and Greenwood, 1995, who found seasonal DNA content fluctuation in cambial nuclei of conifers). The disturbing effect of tannins

31

Greilhuber—Intraspecific Genome Size Variation in Feulgen staining is now well known (Greilhuber, 1986, 1988 a, b ; for an extended discussion see Greilhuber, 1997). Its role in flow cytometry can approximately be estimated only from occasional side-notes of authors (e.g. Bharathan, Lambert and Galbraith, 1994), but phenolics in general seem to be important in some cases (M. B. Schro$ der, pers. comm.). Curiously enough, there are still authors (Berlyn, Royte and Anoruo, 1990), who deny the role of tannins in Feulgen staining and reject the use of formaldehyde even though it has so often been shown to be a suitable fixative (e.g. Greilhuber, 1988 a). Greilhuber (1986), who reported reduced Feulgen staining due to tannins, is said by Berlyn et al. (1990) to have fixed his material (root tips of Pinus) for too long (namely 24 h), which is considerably longer than the 1 h Carnoy fixation used in their studies, and this extended fixation (and not the tannins) could have contributed to the decreased staining. This is an unsatisfactory argument. Greilhuber (1986) also fixed his material for 30 min only (in methanol-acetic acid, to be accurate, but this makes no difference), and some processes were carried out with no fixation. (It should be noted that the universally defective results of Berlyn and co-workers in conifers are paradigmatic as the outcome of a scientific misconception that has been strongly influenced by the idea of the plastic genome ; see Greilhuber, 1986, 1988 a, b, 1998, for discussion).

INTRASPECIFIC GENOME SIZE VARIATION Collinsia verna A typical example of ‘ unorthodox intraspecific genome size variation ’ is Collinsia Šerna Nutt. (Scrophulariaceae), in which an almost four-fold genome size variation in conspecific samples from three midwestern states in the United States of North America has been reported by Greenlee, Rai and Floyd (1984). Neither geographic nor morphological correlations with DNA content were found. Fortunately (as I am tempted to say), results were

subsequently published by Greenlee and Rai (1986) showing that in a cross between C. Šerna with 28n6 pg (4C) and C. heterophylla with 10n6 pg (4C) meiotic metaphase I bivalents did not show reasonable asymmetries. As it is well known that with an almost three-fold genome size difference of the parents such bivalents should be strongly asymmetrical (e.g. Brandham, 1983 ; see also Greilhuber, 1995), it becomes clear that the DNA content determination by Greenlee et al. (1984) is highly questionable. Greenlee and Rai (1986) considered differential condensation of the meiotic chromosomes as an explanation, but this argument is difficult to support. Greenlee et al. (1984) applied a Carnoy-type fixative, meiotic squashes were hydrolysed and stained, the type of hydrolysis was not specified. It fits with the results of Greenlee et al. (1984) that one of the authors subsequently found a nearly three-fold genome size variation in the mosquito, Aedes albopictus (Skuse) (Kumar and Rai, 1990). Attempts by other authors to reproduce the results by Greenlee et al. (1984) have been frustrated as no original research material was available (Bennett and Leitch, 1995), so the case of Collinsia should not be cited uncritically in future. Pea, Pisum sativum Pisum satiŠum L. is another plant in which considerable intraspecific genome size variation has been reported, among others by Guerra (1983), Cavallini and Natali (1990, 1994) and Cavallini et al. (1993) using Feulgen densitometry, and by Arumuganathan and Earle (1991) using flow cytometry. Pisum satiŠum is a variable taxon that has been classified into three or more subspecies, it is adapted to various climates all over the world, it occurs in different states of cultivation, it has been under human selection for hundreds of years, and it is an inbreeder. All these conditions are compatible with some intraspecific genome size variation, so variation in Pisum satiŠum is not necessarily unexpected. Data by Cavallini and co-workers deserve special attention as these authors found not only up to 1n29-fold genome size variation between commercial cultivars of vegetable peas,

T     2. Feulgen densitometric results in Šarious Pisum sativum cultiŠars published by CaŠallini and Natali (1990 ; data sets 1–4), CaŠallini et al. (1993 ; data set 5), and CaŠallini and Natali (1994 ; data set 6) Data set number and fixative Pisum line Espresso Generoso Dolce provenza Proteo Vip Subsp. arŠensis Senatore 5075 Rondo Telefono Progress 9

1 (3 : 1)

2 (F)

3 (3 : 1)

4 (F)

5 (3 : 1)

6 (3 : 1)

128n9 124n9 119n5 118n0 115n6 111n7 109n8 108n1 106n9 100n0

124n0 115n2 120n2 109n1 113n4 113n8 106n5 105n3 104n4 100n0

c. 138n7 c. 129n6

c. 124n6 c. 115n6

127n7 123n1

128n7 124n5

116n9

118n0

112n3 110n8 c. 114n4

c. 105n5

c. 100n0

c. 100n0

108n5 100n0

106n3 100n0

Data have been normalized taking Pisum satiŠum ‘ Progress 9 ’ (l ‘ Nano Progress ’ of Cavallini and Natali, 1990 ; see Baranyi and Greilhuber, 1996) as 100 %. Data from a hydrolysis curve (data set 5) and an optical density curve (data set 6) have been estimated from Figures and are approximate.

32

Greilhuber—Intraspecific Genome Size Variation

T     3. Flow cytometric and Feulgen densitometric comparison of genome size in Pisum sativum cultiŠars by Baranyi and Greilhuber (1996) P. satiŠum cultivars

DAPI

Feulgen

Espresso Generoso Progress 9 Kleine Rheinla$ nderin

99n1 100n0 100n0

101n8 100n0 100n4

Zea mays ‘ Sundance ’ was standard for DAPI measurements. The data are normalized taking ‘ Progress 9 ’ as 100 %.

T     4. Comparison of genome size in commercial Pisum sativum cultiŠars, purportedly differing up to 1n29-fold (CaŠallini et al., 1990 ; Feulgen densitometry), by Baranyi and Greilhuber (1996) using ethidium bromide and DAPI flow cytometry

Soybean, Glycine max

Baranyi and Greilhuber (1996) Pisum satiŠum cultivars Espresso Generoso Senatore Rondo Telefono Progress 9

EB 100n6 100n9 99n0 101n3 100n0

DAPI, with ‘ Kleine Rheinla$ nderin ’ co-chopped Unimodal, Unimodal, Unimodal, Unimodal, Unimodal,

CV CV CV CV CV

1n46 % 1n47 % 1n80 % 1n33 % 1n44 %

problems with the procedures used. The consequences of this are far-reaching : until doubts about the methods used by Cavallini and colleagues can be dispelled, great care should be exercised in using their results. Naturally, the data on genome size variation in Helianthus annuus (Cavallini et al., 1986, 1989, 1996), Festuca arundinacea Schreb. (Ceccarelli, Falistocco and Cionini, 1992 ; Frediani et al., 1992 ; Ceccarelli et al., 1993), Dasypyrum (Cremonini et al., 1994 ; Frediani et al., 1994), and Vicia faba L. (Ceccarelli et al., 1995 ; Minelli et al., 1996) also have to be considered with caution and should not be cited uncritically, as the same technical procedure (involving pectinase and hot hydrolysis) has always been applied. Furthermore, it now appears that the molecular data related to genome size variation published by these authors might also have to be viewed with due caution.

Cavallini and Natali (1990) Feulgen 128n9 111n7 108n1 106n9 100n0

Data are normalized taking ‘ Progress 9 ’ as 100 %. In material (obtained from the I. F. G. Bari), that was co-chopped with ‘ Kleine Rheinla$ nderin ’, unimodal peaks were also obtained with P. satiŠum subsp. arŠense, and the cultivars Dolce Provenza, Senatore, Rondo, and Telefono, that purportedly deviate from ‘ Progress 9 ’ 1n25-fold, 1n12-fold, 1n08-fold and 1n07-fold, respectively, according to Cavallini and Natali (1990).

but also a correlation with nucleotypic characters such as cell size, early growth and degree of endopolyploidy in cotyledon cells. Scrutiny of the data of Cavallini and Natali (1990, 1994) and Cavallini et al. (1993) reveals that the general distribution of DNA variation in pea lines was found not less than six times in separate experiments (Table 2). Greilhuber and co-workers have re-analysed Pisum satiŠum for genome size variation with Feulgen densitometry and flow cytometry (Greilhuber and Ebert, 1994 ; Baranyi and Greilhuber, 1995, 1996) and have paid special attention to the Italian cultivars of Cavallini and co-workers (Baranyi and Greilhuber, 1996). Surprisingly, no reasonable evidence emerged for statistically significant genome size variation in Pisum satiŠum on a world-wide scale (in contrast to other wild pea taxa, see Baranyi, Greilhuber and Swiecicki, 1996). In particular, the Italian lines were of identical genome size, irrespective of the method of measurement applied (Tables 3 and 4). With respect to these results, the consistently correlated variation in pea genome size data and their correlation with nucleotypically influenced parameters found by Cavallini and Natali (1990, 1994) and Cavallini et al. (1993) are difficult to explain. However, it is safe to conclude that their reported DNA variation could be due to

Among the species showing intraspecific genome size variation, the ones of particular biological interest are those in which genome size can be correlated with parameters of the environment that are important for the adaptation of the organism. In maize, significant variation in C-value (up to 40 %, caused at least in part by heterochromatic knob material) is correlated with maturity group assignment, i.e. those races having smaller genomes can occur in more northerly regions, where the growth period is shorter (Laurie and Bennett, 1985 ; Rayburn et al., 1985 ; Rayburn, Benzinger and Hepburn, 1989 ; Rayburn and Auger, 1990). This behaviour is predicted by the nucleotype theory (Bennett, 1972), since smaller genomes have shorter nuclear cycles, and more rapid development could occur. Similarly to maize, lower genome size has been found also in Glycine max (L.) Merr. (soybean) cultivars adapted to higher latitudes where the growth period is shorter (Graham, Nickell and Rayburn, 1994). This correlation and the high genome size variation in general, reported in the literature on G. max, prompted Greilhuber and Obermayer (1997) to re-analyse the cultivars in which Graham et al. (1994) had found 1n15-fold variation. Nineteen of the 20 cultivars investigated by Graham et al. (1994) were available and were analysed with Feulgen densitometry and ethidium bromide and DAPI flow cytometry. Interestingly, no reproducible genome size differences between these cultivars were found with either technique, and there was no significant correlation with maturity group. It was seen that the statistical significance of the correlation stated by Graham et al. (1994) resulted from only one exceedingly low value in their data set for the early-maturing cultivar ‘ Maple Presto ’. If this cultivar is removed from the analysis, the significance level of the correlation with maturity group became worse than 0n05. The discrepancy between the results of Graham et al. (1994) and Greilhuber and Obermayer (1997) highlights a serious methodological problem : standardization. Greilhuber and Obermayer (1997) consequently conducted internal standardization of every run by co-chopping test- and standard material, while Graham et al. (1994) relied on external standardization,

Greilhuber—Intraspecific Genome Size Variation calibrating the instrument daily when starting the analysis. It is probable that the omission of internal standardization caused the higher variation in the results of Graham et al. (1994). However, this cannot explain the positive correlation with maturity groups that they report. Meanwhile, Rayburn et al. (1997) reported 1n12-fold variation in 90 Chinese soybean accessions, again using external standardization. It is possible, though not yet proven, that this variation may again be technical in origin.

CONCLUSION Genome size diversification is an important process during speciation in plants and in cases precedes noticeable phenotypic differentiation. It is a useful parameter for estimating cryptic taxonomic differentiation within species or aggregates, and techniques to measure genome size constitute a novel tool for biodiversity research and may even have importance for conservation activities. Rapid methodologies for measuring plant genome size are now available (flow cytometry, video-based densitometry). Therefore, it is not surprising that more and more plant scientists are engaged in measuring DNA amounts, among them many botanists who previously restricted themselves to the study of chromosome number and form, and who now want to utilize these methods for refined taxonomic, evolutionary and phylogenetic studies. However, it is clear that much suspect or demonstrably wrong data have been and continue to be accumulating in the literature, as shown by the present survey of those published results on infraspecific DNA content variation, which could be reanalysed by myself and co-workers. The situation has really become a serious problem in this research field. Especially if one considers the publications advocating the ‘ plastic genome ’, Paul Feyerabend’s principle of ‘ anything goes ’ (Feyerabend, 1993) comes to mind. High-quality studies are therefore urgently needed, methodological standards have to be set and suspect reports have to be subjected to the test of reproducibility whenever possible. The techniques for accurate genome size determination are available, and they work with high inter-laboratory reproducibility (Dolez3 el et al., 1998), provided certain methodological rules are followed.

A C K N O W L E D G E M E N TS Support by the Austrian Research Foundation (project P9593-BIO) is gratefully acknowledged.

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