New Record Holders for Maximum Genome Size in Eudicots and Monocots

This paper presents the largest genome sizes reported so far for angiosperms: for the monocots Trillium hagae (Melanthiaceae) 2C = 264.9 pg and for the eudicots Viscum album (Santalaceae) with 2C = 205.8 pg. They were found during ongoing measurements of nuclear DNA content in more than 4000 accessions. Moreover, it is demonstrated that both of the values for the largest genome size so far for the monocots of Fritillaria assyriaca and for the eudicots of Viscum cruciatum need to be corrected.


Introduction
Until recently, genome size in angiosperms varied from 0.321 pg in Arabidopisis thaliana (Brassicacae) [1] to 254.8 pg in Fritillaria assyriaca (Liliaceae) [2], an 800-fold range.This genome size is not related to the number of genes (polyploids excluded) but is due to large amounts of repetitive DNA.Also the number of chromosomes is not very relevant as some plants with very high chromosome numbers have low amount of nuclear DNA like Sedum suaveolens with about 2n = 640 and 18.3 pg of DNA (Table 2).This is called the C-value paradox or the C-value enigma [3].Genome size is not very useful at higher taxonomic levels, but is especially of value at the species level [4][5][6][7][8][9].The amount of nuclear DNA is positively correlated with nuclear and cellular volume, whereby mainly cell size is subjected to selection [10].
The smallest angiosperm genomes are found in the Lentibulariaceae with Genlisea margaretae with a 2C DNA value of only 0.129 pg [11].This is less than half the size of the genome of Arabidopsis thaliana with 2C = 0.321 pg [1] and seems to increase the total range from 800 to 1968fold.At the other end of the scale always the monocot Fritillaria assyriaca with 2C = 254.8pg is named as the species with the largest genome size for the angiosperms [2].However, this is based on the wrong assumption [2], as will be demonstrated below, that Fritillaria assyriaca has the largest genome size.The supposed largest eudicot is Viscum cruciatum with 2C = 158.6 pg.The present study presents the highest values reported so far.They were found during ongoing measurements of nuclear DNA content in more than 4000 accessions [4][5][6][7][8][9].They represent a new record at the higher end of the scale of angiosperm C-values.This emphasizes the need for a proper explanation of these very large genome sizes.

Material and Methods
2.1.Plant Material.Trillium hagae material was obtained from the collection of C. Denton, UK.Viscum crassulae was obtained from E. van Jaarsveld of Kirstenbosch Botanical Garden RSA and Viscum minimum of C. Grootscholten Succulent Nursery, The Netherlands.Viscum album was obtained from the Botanical Garden Wageningen, The Netherlands, and also from Arboretrum Trompenburg Rotterdam, The Netherlands, and collected in south Limburg, The Netherlands; Pyrenees, France, and southern Greece.Live material of Fritillaria was contributed by P. Christian, J. de Groot, Hortus Bulborum Limmen, Potterton and Martin, W. Kletzing and M. de Jaeger.Where possible, material of known wild origin was used, and care was taken to ensure correct identification of all materials.

Flow Cytometric Measurement of Nuclear DNA Content.
For the isolation of nuclei, about 1 cm 2 of adult leaf tissue was chopped together with a piece of Haemantus albiflos (Amaryllidaceae) as an internal standard (see below).The chopping was done with a new razor blade in a Petri dish in 0.25 mL nuclei isolation buffer to which 0.25 mg RNase/mL was added [8].After adding 1.  [12]) (1 picogram = 0.978 × 10 9 base pairs [13]) were chosen as primary standard.This yields 2C = 15.9 pg for nuclei of Agave americana.This in turn was used to measure Haemanthus albiflos and resulted in 2C = 76.0 pg.  1).For real high values one must look to Trillium (Melanthiaceae).Especially the trilliums of Asia have high C-values due to their usually high ploidy [14].The new record holder presented here is T. hagae with 264.9 pg (2n = 30).It is an allohexaploid of T. camchatcense (2n = 10) and T. tschonoskii (2n = 20).Another high DNA 2C-value is the Asiatic T. apetalon (2n = 20) with 190.0 pg [5].

The Eudicots : Viscum. Viscum cruciatum is with 2C
= 158.7 pg [17] presented in the list of plant DNA Cvalues [2] as the eudicot with the largest genome size.For V. album a value of 2C = 152.0pg is chosen [18].Clearly it is chosen as "prime estimate" as it is intermediate between the value of Nagl et al. for V. album with 2C = 107.0pg [19] and those of Ulrich et al. with 2C = 181.5 pg [17].It would have been better to choose both values from a single laboratory.Ulrich et al. [17] found the 2C-value of V. album to be higher than those of V. cruciatum and that is confirmed here.Ulrich et al. used several both A-T and G-C specific dyes and from these results calculated the "absolute DNA content."They also used Feulgen for V. album and obtained nearly the same result (184.4 pg).The present results for these species are 2C = 205.8pg for V. album (2n = 20), and 2C = 175.8pg for V. cruciatum (2n = 20) (Table 2).Also V. crassulae with 2C = 164.0pg (2n = 24) and V. minimum with 2C = 124.6 pg (2n = 28) are here presented as new DNA 2C-values (Table 2).

Discussion
Most of the values of Fritillaria in the list of plant DNA Cvalues are from McLeish and LaCour from 1971 [2].They were measured with Feulgen cytometry and based on a personal communication.The standard species is unknown and they might not have used "best practice" as advocated for the Feulgen method [20].The increasing difference of 11-50 pg going from the lowest to the highest value in Fritillaria as presented by McLeish and LaCour compared with the present results (Table 1) suggest problems with the linearity of their measurements.Actually a 2C DNA value of 134 pg was found for four accessions of the diploid F. assyriaca (Table 1).It cannot be excluded that McLeish and LaCour had a tetraploid F. assyriaca.However, it is more likely, they used the plant that is in the trade as F. assyriaca, but actually is the tetraploid F. uva-vulpis with 2C = 203.9pg.Moreover, Rix (1974) [15] has shown that up to that time all plants in commerce as F. assyriaca were a separate species F. uva-vulpis.
The high values of McLeish and LaCour can be explained by the fact that they were obtained after recalibrating their values against F. pallidiflora with 116.7 pg [16].The present results show F. pallidiflora to have only 87.4 pg (Table 1).The combined results of the 2C DNA values of 0.129 pg for Genlisea margaretae [11] with those presented here with 264.9 pg for Trillium hagae increase the range of genome sizes for angiosperms to 2056-fold.
It may be wondered whether the high value found for V. album has anything to do with its parasitic nature.This seems not likely as the related tree parasite Loranthus europaeus has 16.5 pg [18] and Orobanche hederae has 5.5 pg [5].The large genome size seems also not an impediment to growth rate.V. album on an apple tree in my garden reached football size and bear fruits in three years from seed.

Table 2 :
Comparison of genome sizes (2C in pg) in this paper.# = number of accessions.