Additionally, endopolyploidization is related to life cycle. Endopolyploidy is very frequently found in annual and biennial species and also in some perennial herbs whereas it seems to be absent in wooden species.
Occurrence of endopolyploidy in seed plants is determined genetically
The tight relationship between taxonomic position and endopolyploidization we found, and as already proposed by Tschermak-Woess (1956), D’Amato (1964), Nagl (1976) and Olszewska & Osiecka (1982), suggests a genetic determination of a species’ capacity to endopolyploidize. Nevertheless, families belonging to closely related clades may differ completely with respect to this feature, for instance, Solanaceae and Lamiaceae, Fagaceae and Cucurbitaceae and Rosaceae and Fabaceae.
Within the analysed Fabaceae, Sophora japonica (the only one without endopolyploidy among the tested Fabaceae) belongs to the tribe Sophoreae consisting of mainly wooden tropical and subtropical species whereas the other species belong to the tribes Vicieae, Trifolieae and Phaseoleae of which the members are predominantly herbs of temperate climates. Likewise, within the tested Poaceae, the three species with similar endopolyploidy pattern Triticum aestivum, Secale cereale and Hordeum vulgare belong to the Pooideae, whereas Zea mays and Oryza sativa with higher and without endopolyploidization, respectively, are assigned to two different subfamilies.
Further results on endopolyploidization in species, which we analysed for different purposes but not in detail with regard to endopolyploidy, confirm our results that the degree of endopolyploidization is family specific or at least very similar for closely related species. The upper leaves of five species of Lamiaceae (Glecoma hederacea L., Lamium purpureum L., Ocimum basilicum L., Galeobdolon luteum Huds. and Betonica officinalis L.), one species of Asteraceae (Bellis perennis L.), two Ranunculaceae (Ranunculus ficaria L. and Caltha palustris L.) and two Rosaceae (Potentilla reptans L. and Fragaria viridis[Duchesne] Weston) showed no endopolyploidization as expected for these species according to our results in Table 1. Leaves of 10 species of Brassicaceae (five Arabidopsis species besides A. thaliana, Teesdalia nudicaulis[L.] R. Br., T. coronopifolia[J.P. Bergeret] Thell., Cardamine amara L., Capsella rubella Reuter, Erysimum pulchellum J. Gray) and Allium scorodoprasum L. (Alliaceae) were endopolyploid as one could expect pursuant to our results.
Two species show that there are also exceptions to a family's predominant endopolyploidization behaviour: Erysimum helveticum Jacq. (Brassicaceae) did not show endopolyploidy in upper leaves whereas Fragaria vesca L. (Rosaceae) showed endopolyploidy in leaves to a low degree.
The degree of endopolyploidization differs between the organs of endopolyploid species
These differences do not reflect the different ages of organs but rather their ontogenetic appearance (lower versus upper) and are apparently genetically fixed. The level of endopolyploidization does not increase within an organ once it is fully developed (De Rocher et al. 1990; Gilissen et al. 1993). Assuming a positive correlation between endopolyploidy level and cell volume in plants (Bradley 1954; Melaragno et al. 1993; see below) a further endopolyploidization in fully developed organs is unlikely since it would lead to a further cell expansion.
Although Galbraith et al. (1991) did not find endopolyploidy in flower buds of Arabidopsis thaliana we found high degrees of endopolyploidization in fully differentiated flower organs, except carpels, for A. thaliana and other endopolyploid species. Hence, endopolyploidy is not excluded from floral structures in general to avoid the potential production of polyploid gametophytes as assumed by Galbraith et al. (1991).
The negative correlation of genome size and endopolyploidization may be caused by the correlation between genome size and life cycle and the growth features of large cells
A compensation of a low amount of DNA by endopolyploidization in species with small genomes to enable certain cell functions is unlikely because of the proportionality of DNA content and cell volume of endopolyploid cells that means an equal ratio of gene products and cell volume. A positive correlation between DNA content and cell volume was demonstrated for endopolyploid pith cells of Nicotiana tomentosa with ploidy levels ranging from 4C to 16C (Bradley 1954) and for epidermal pavement cells of Arabidopsis thaliana leaves with a ploidy range from 2C to 16C (Melaragno et al. 1993). Furthermore, the size of trichomes of Arabidopsis thaliana and the number of their branches depend on their ploidy level (Hülskamp, Miséra & Jürgens 1994). Moreover, in Medicago truncatula the reduced amount of transcripts of the gene ccs52 involved in the switch from mitotic cycles to endocycles leads to a decreased level of endopolyploidy and cell size in roots, cotyledons, petioles and hypocotyls (Kondorosi et al. 2000).
However, comparing mean size and mean ploidy levels of mature root cortex cells of 18 ecotypes of Arabidopsis thaliana, Beemster et al. (2002) showed that even among ecotypes of one species the relationship between these two may vary. In the KRP2-mutant of Arabidopsis thaliana, in which cell division is inhibited and endoreduplication is suppressed in older leaves, cells are enlarged in comparison with the wild type in the absence of enhanced endoreduplication (De Veylder et al. 2001). Likewise, overexpression of the Arabidopsis thaliana cell cycle regulator Cdc2a in tobacco led to fewer but larger cells in leaves although the mean endopolyploidy was the same as in wild-type tobacco (Hemerly et al. 1995). Possibly, the point is that there is a close relation between DNA-content and volume of cytoplasm rather than cell volume. The latter may vary to a considerable extent depending on the cell type, organ, ecotype or mutant caused by the vacuole, which contributes to a high degree to the cell volume. Already Tschermak-Woess (1956) emphasized this problem. However, for meristematic cells, which do not yet have a vacuole, Price, Sparrow & Nauman (1973) reported a close relation between genome size and cell volume for 14 herbaceous angiosperms. Furthermore, within each single mutant and accession investigated by Beemster et al. (2002), De Veylder et al. (2001) and Hemerly et al. (1995) the DNA content/cell volume-relation may hold true.
Therefore, as an alternative to a compensation of a low amount of DNA by endopolyploidization (Nagl 1976), considering the correlation between life cycle and endopolyploidization and life cycle and genome size on the one hand and the growth feature of species with large genomes on the other (see below; Grime & Mowforth 1982 and Grime et al. 1985), one might speculate that endopolyploid species combine the advantages of small genomes and large cells to speed up development.
Advantages of a small genome. Annual and biennial plant species preferentially possess small genomes (Bennett 1972
; Vinogradov 2001
) since species with small genomes tend to have short generation times. This seems reasonable on the basis of a positive correlation between genome size on the one hand and somatic cell cycle time, duration of meiosis and pollen maturation on the other (Bennett 1972
). Because of a shorter cell cycle time, plant species with a small genome are expected to grow faster under supporting conditions (e.g. high temperatures) than species with a large genome when growing mainly by cell division (quasi exponentially) and subsequent cell expansion.
Advantages of large genomes. Plant species growing at higher latitude tend to have larger genomes (Levin & Funderburg 1979
; Bennett 1976
). Grime & Mowforth (1982
) and Grime et al. (1985
) could show that this is due to the differential effect of low temperatures on cell division and cell expansion. Although at low temperatures mitosis is often inhibited, this is not the case for cell expansion. Accordingly, investigating ploidy levels and DNA amounts of 12 Tulipa
, 13 Hyacinthus
, and 16 Narcissus
cultivars, Brandham & West (1993
) demonstrated that a similar genome size, in this case a large amount of DNA, due to either a large diploid genome size or acquired by polyploidization, is optimal for evolutionary adaptation of genera, which are not closely related, to environmental conditions that inhibit mitosis but not cell expansion since fewer cells for the same growth rate are needed.
We speculate that endopolyploid species are able to develop faster than species without endopolyploidy with the same genome size by combining these advantages. In endopolyploid species the corresponding organs may be able to grow faster, since initial growth by cell division is followed by extensive cell expansion, mediated by endopolyploidization. At low temperatures, endopolyploid species with small genomes should grow as fast as non-endopolyploid species with large genomes since they may reach the same cell size by expansion. At temperatures that are optimal for mitosis, the former should grow faster by combining short cell cycles and endopolyploidy-mediated cell expansion. This hypothesis may be tested by systematic investigation of growth of endopolyploid versus non-endopolyploid species of different genome sizes under different conditions.
Fast growth and development of endopolyploid species might get further support since less cell surface material due to larger cells (Barlow 1978), no spindle formation, no chromosome condensation and decondensation, no breakdown and reconstruction of the nuclear envelope is needed and the transcription is maintained during endoreduplication (Nagl 1978). In addition, generation time is shortened by faster meiosis and pollen maturation in species with small genomes.
This conclusion corresponds to the suggestion of Cavalier-Smith (1978) that the most fundamental way organisms adapt to varying r- and K-strategy (which are, according to Grime 1974, 1977 and 1988, analogous to the C-, S- and R-strategies in the plant kingdom) is by evolving particular cell volumes and cell growth rates.
The importance of endopolyploidization for higher growth rate is further indicated by hypocotyls grown in the dark and in light. Hypocotyls of Lupinus albus grown in the dark are four times longer than those grown in light whereas cell number is the same in both (Giles & Myers 1964). This is apparently due to endopolyploidization since DNA content per hypocotyl is increased. In addition, for Pisum sativum (Van Oostveldt & Van Parijs 1975) and Arabidopsis thaliana (Gendreau et al. 1998) a higher endopolyploidization level was found in etiolated than in non-etiolated epicotyls and hypocotyls, respectively. Hence, the potential to grow by endopolyploidization seems to be advantageous for etiolated seedlings since it leads to elongated shoots by fast growth. This might be crucial under natural conditions in order to pass the soil surface for starting photosynthesis.
An advantage of endopolyploidy for fast growth could even explain the different degree of endopolyploidization between organs of the lower and upper parts of a plant since the lower parts often develop during earlier seasons and thus at lower temperatures in temperate climes.
In some species, we found endopolyploidy only to a low degree and not in all organs where it normally occurs. Therefore, in these species endopolyploidization barely serves for a fast development. Identification of individual cells that are actually endopolyploid could provide clues with respect to the function of endopolyploidy within these species, which could be generation of certain morphological structures (Pyke, Marrison & Leech 1991; Kondorosi et al. 2000) or support of high metabolic activity as in suspensor cells (Nagl 1978).
Considering the significance of endopolyploidization for growth and development of plants, the weak correlation of endopolyploidization with genome size and life cycle is astonishing. This might be explained by the superposition of these two correlations by the life strategy of the corresponding species (the strategy a species embarks on to compete successfully for a ecological niche), which includes more ecological features than just the life cycle.
Life strategy may possibly superpose the correlation of endopolyploidization with life cycle and genome size
Twenty-three out of the 26 tested short-lived species revealed endopolyploidy, and all tested bushes and trees did not. This agrees well with the assumed relationship between endopolyploidization and life cycle. Nevertheless, more than one-half of the tested perennial herbs are endopolyploid. Therefore, endopolyploidy is possibly not only important for species with short generation times, which have to complete the whole life cycle from germination to seed production within one season. A fast development mediated by endopolyploidy might also be advantageous for perennial herbs that can exploit convenient growing conditions available only temporarily. In these cases, it is rather life strategy in general than life cycle as a special part of life strategy that drives exhibition of endopolyploidization. Four of the five tested Central European endopolyploid perennial herbs mainly grow at frequently disturbed locations (Trifolium pratense: anthropo-zoogenic heathlands and grasslands; Aquilegia vulgaris, Parietaria officinalis, Urtica dioica: herbaceous vegetation of often disturbed locations; Ellenberg et al. 1992). They have to complete growing, flowering and seed production within a limited period or have to be able to regenerate quickly after having been disturbed. The fifth tested Central European endopolyploid perennial herb, Allium ursinum, exhibiting a relatively high endopolyploidization in addition to its large genome, has to develop quickly before deciduous trees shadow its habitat during late spring and summer. Anemone ranunculoides, which is found in a similar habitat to Allium ursinum and which has a relatively large genome, too, but no endopolyploidy, starts growing and flowering much earlier in the year and stays green until approximately the same time as Allium ursinum.
Why do not all species growing in often disturbed habitats exhibit endopolyploidy since it appears to be so advantageous (e.g. the tested Asteraceae and Lamiaceae)? A second factor, namely a species’ nutritive demand, may favour endopolyploidization of species of the same life cycle type to different degrees. Ellenberg et al. (1992) divided the seed plant species of Central Europe in respect of this feature into nine classes. This classification reveals that most of the tested endopolyploid Central European species have higher nutritive demands than most species without endopolyploidy. Eight endopolyploid herbaceous perennial species are at least considered to be more abundant in nitrogen-rich habitats than in mediocre habitats. Trifolium pratense (Fabaceae) is not characterized by Ellenberg et al. (1992) in respect to its nutritive demands but an adequate nitrogen supply can be assumed as in general for this family because of nitrogen-fixing endosymbionts in root nodules. Arabidopsis thaliana is the only tested endopolyploid Central European species with low nutritive demands. On the other hand, four of the tested Central European species without endopolyploidy are more abundant in nitrogen-poor habitats (if one accepts Lactuca serriola as ancestor of L. sativa and therewith the classification of L. serriola). This is also true for Aquilegia vulgaris, which exhibits endopolyploidization only to a low extent. Anemone ranunculoides is the only tested herbaceous perennial Central European species without endopolyploidization but high nutritive demands.
When endopolyploid species indeed may develop faster than species without endopolyploidization (also Melaragno et al. 1993 discussed a more rapid growth by endopolyploidization) it seems to be understandable that they preferentially occur in habitats that require a fast completion of growth, flowering and seed production and support fast development by optimal nutrient supply. Apparently, in such habitats, the ability of endopolyploid species to make use of supporting conditions by fast development enables them to compete successfully with species without endopolyploidy. Investigation of genome size, endopolyploidization and phenology of species that are more or less restricted to a given habitat could reveal whether endopolyploidization is of ecological significance under defined growth conditions.
Similar life strategies of species of one family might cause the strong correlation between endopolyploidy and family affiliation
Within many plant families species tend to grow in similar habitats (Hodgeson 1986; Ellenberg et al. 1992). For instance, in Central Europe, arable farmland is the habitat richest in species of Brassicaceae, Chenopodiaceae and Solanaceae (Ellenberg et al. 1992), families, which revealed in our experiments very high degrees of endopolyploidization. On the other hand, wood is the habitat richest in species of Ranunculaceae, Rosaceae and rich in Lamiaceae, families, which show low or no endopolyploidization. Similar ecological preferences of species of the same family imply that it is rather the predominant life strategy of a family than the family affiliation itself that causes the strong correlation between the factor ‘family’ and endopolyploidization. If this would be the case, species that differ from the predominant life strategy of a family should also differ from a family's main endopolyploidization behaviour. In fact, this is true for three of the tested species, namely, Aquilegia vulgaris (Ranunculaceae), Duchesnea indica (Rosaceae) and Sophora japonica (Fabaceae). If one considers the relationship between life strategy and endopolyploidization and the apparent absence of a close relation between phylogeny and endopolyploidization it seems likely that the former favours endopolyploidy within closely related species.