Genome sizes of 227 accessions of Gagea (Liliaceae) discriminate between the species from the Netherlands and reveal new ploidies in Gagea

Nuclear genome size, as measured by flow cytometry with propidium iodide, was used to investigate the relationships within the genus Gagea (Liliaceae), mainly from the Netherlands. The basic chromosome number for Gagea is x = 12. The inferred ploidy in the Dutch and German accessions varies from diploid to decaploid. Consequently there is a large range of genome sizes (DNA 2C-values) from 14.9 to 75.1 pg. Genome sizes are evaluated here in combination with the results of morphological observations. Five species and the hybrid G. × megapolitana are reported. Apart from 14 diploid G. villosa, six plants of G. villosa with an inferred tetraploidy were found. For the 186 Dutch accessions investigated 85 turned out to be the largely sterile G. pratensis (inferred to be pentaploid). Inferred tetraploid and hexaploid G. pratensis were found in 30 and 20 localities, respectively. In one locality an inferred decaploid (10×) plant was found that could represent a doubled pentaploid G. pratensis. An inferred decaploid G. pratensis was never reported before. The genome size of Gagea × megapolitana from Germany fitted with its origin as a cross between the two hexaploids G. pratensis and G. lutea. Gagea spathacea from the Netherlands was inferred to be nonaploid as was recorded from plants across Europe. The aim of the study was to use flow cytometry as a tool to elucidate the taxonomic position of the Dutch Gagea.


Background
The genus Gagea Salisb. conmprizes about 275 species. In the World Checklist for Gagea (Govaerts 2006) 594 names were listed. It is a genus of small bulbous plants in the family Liliaceae, endemic to Eurasia and North Africa. A single circumpolar species, a former Lloydia is now included in Gagea (Peruzzi 2012). The greatest number of species can be found in Kazakhstan in the Tien Shan and Pamir-Alai. This coincides with the greatest richness of Tulipa (Zonneveld 2010). In Flora Neerlandica (van Oostrom and Reichgelt 1964) four species are recorded for The Netherlands and in Heukels Flora of The Netherlands (van der Meijden 2005) a fifth is added.
To elucidate the relationships between Gagea species, the classical taxonomic traits based on morphological characters, chromosome numbers (Peruzzi 2003(Peruzzi , 2012 and sequencing data (Peterson et al. 2008;Zarrei et al. 2009) are here supplemented with data on nuclear DNA content. From only five species genome size was determined earlier (Greilhuber et al. 2000;Vesely et al. 2011;Leitch et al. 2007). Taxonomy of Gagea is rather difficult and the main useful characters so far are: the chromosome numbers, the number and type of bulbils, the number and width of the leaves, the presence of red coloration at the base of the leaf, the hairiness of the flower stalk, the shape of the petals and the number of flowers on a scape. Newer investigations are also based on morpho-anatomical data (Peruzzi 2012).

Open Access
*Correspondence: ben.zonneveld@naturalis.nl 1 NBC Naturalis, Herbarium Section, P.O. Box 9517, 2300 RA Leiden, The Netherlands Full list of author information is available at the end of the article 186 different accessions from The Netherlands were measured in an attempt to understand the relationships within the Dutch gageas. These values were compared with an additional 41 taxa from Germany. Nuclear DNA content can conveniently be measured by flow cytometry using propidium iodide, a stoichiometric DNA stain that intercalates in the double helix. Where many species in a genus have the same chromosome number, differences in DNA 2C-value have proven to be very effective in delimiting infrageneric divisions in a number of taxa (Ohri 1998). The evolution of genome size (Greilhuber 1979) has received increased attention during recent years (Greilhuber 2005). The smallest angiosperm genome size reported so far is for Genlisia margarethae Hutch. with 2C = 0.13 pg (Greilhuber et al. 2006). The record holders for maximum genome size were for eudicots Viscum album L. with 2C = 205.8 pg and for monocots Paris japonica with 2C = 304.5 pg (Pellicer et al. 2010). Flow cytometry was successfully used to measure the 2C-value for the genera Hosta Tratt., Helleborus L., Clivia Lindl., Nerine Herb., Agapanthus L'Hér., Galanthus L., Narcissus L., Gasteria Duval. Tulipa L. etc. by Zonneveld (2001Zonneveld ( , 2003Zonneveld ( , 2008Zonneveld ( , 2009Zonneveld ( , 2010, Zonneveld and Van Iren (2001), Duncan (2003, 2006), and Zonneveld et al. ( , 2012. In this paper it is shown that genome size is helpful to discriminate between the species of Gagea from The Netherlands (Fig. 1).
Nuclear DNA content as measured by using flow cytometry may conveniently be used to produce systematic data. It is applicable even in dormant bulbs or sterile plants for the monitoring of the trade in bulbous species. In the case of Gagea, it is difficult to ascribe a plant to a taxon in the often non-flowering state. Genome size is a good way to determine the species and their ploidy. A different genome size infers usually a different ploidy or a different species. However, the reverse is not true: if plants have the same genome size it does not automatically mean that they are the same species, it might be a coincidence.
Based on van den Berg and te Linde (2003) and new observations, morphological descriptions were given for the species, correlating it with the measured genome weights. New ploidies were inferred for Gagea villosa which, apart from 14 diploids, had six plants with an inferred tetraploid amount of DNA and for G. pratensis that was found to have, apart from the tetraploid, the hexaploid and the very common pentaploid accessions, also a genome size inferring decaploidy.

Plant material
The plant material used in this study was collected from locations across The Netherlands and Germany as described in Table 1. It was mainly obtained from B. te Linde, Stichting Berglinde, Babberich and a few from L. Duistermaat from NCB Naturalis, Leiden, The Netherlands. Further material came from T. Pfeiffer from the Ernst-Moritz-Arndt-University of Greifswald, Germany. The German plants, supplied with chromosomes counts, were used to infer the ploidy of the Dutch plants. Material of known origin was used. Vouchers will be lodged in the Herbarium of Naturalis Leiden (L). Figures 2,3,4,5,6,7,8 show the spread of the taxa in the Netherlands.

Flow cytometric measurement of DNA 2C-value
For the isolation of nuclei, a few cm of leaf or a single bulbil was chopped together with a piece of Agave americana L. ' Aureomarginata' or Agave attenuata L. as an internal standard (see below). The chopping was done with a new razor blade in a Petri dish in 0.25 ml nucleiisolation buffer to which 0.25 mg RNase/ml was added (Zonneveld and van Iren 2001). After adding 1.75 ml propidium iodide solution (50 mg PI/l in isolation buffer) the suspension with nuclei was filtered through a 20 μm nylon filter. The fluorescence of the nuclei at 585 nm was measured half an hour and 1 h after addition of propidium iodide excitation, using a BD Accuri C6 flow cytometer equiped with a 488 nm laser suitable for propidium iodide. Data were analyzed by means of BD Accuri Cflow Plus software provided by the supplier. Plots were first gated to exclude debris on a scatter diagram (Fl2-A vs FL1-A) and counted against FL2-A on a logarithmic scale. The 2C DNA content of the sample was calculated as the sample peak mean, divided by the Agave peak mean, and multiplied with the amount of DNA of the Agave standard. Two different samples, with each at least 5,000 nuclei, were measured twice for each clone. Most histograms revealed a Coefficient of Variation of less than 5%. The standard deviation was calculated for the DNA content of each species, using all relevant measurements.

Internal standard and absolute DNA content values
When measuring nuclear DNA content by means of flow cytometry, it is necessary to chop tissue from the plant of interest together with an internal standard. This standard must be as close as possible to the plants of interest and not overlap with the ploidy area of interest. If they are too close together the peak values interfere with each other. Linearity is checked by comparing the different ploidies as found within leaves and roots of many plants. In this way, variation in signal intensities due to staining kinetics, to light absorption and quenching by sample components, as well as to instrument and other variables, is reduced to a minimum. Agave americana was chosen as internal standard for Gagea. For Gagea minima and G. villosa, with 2C-values that more or less coincided with Agave americana, Agave attenuata was used. Agave is available yearround, does not mind several weeks without water and, being a large plant, a single specimen can serve a lifetime, thereby further reducing variation in readings. It also has a low background in propidium iodide measurements, and show a single G 0 peak, almost lacking G 2 arrest.
Fresh male human leucocytes [2C = 7.0 pg; 1 pg = 10 −12 g = 0.978 × 10 9 base pairs (Doležel et al. 2003)] were chosen as primary standard (Tiersch et al. 1989). This yields 2C = 15.9 pg for nuclei of Agave americana L. and 8.0 pg for A. attenuata. Based on a published male human genome size of 6.294 × 10 9 base pairs the nucleus was calculated as containing 6.436 pg (Doležel et al. 2003). However this is based on a human sequence where the size of the very large repeat sequences could not accurately be determined. So in the end the genome size could be closer to 7 pg than now envisioned.

General
Morphologically the species of Gagea are rather difficult to differentiate. They are all small bulbous plants with grass like leaves and mostly yellow flowers. Moreover they are visible above the soil surface only about 2 months a year in early spring. The Dutch Gagea can be divided over four out of 12-14 different sections. G. lutea, G. × pomeranica and G. pratensis belong to section Gagea whereas G. minima, G. villosa and G. spathacea each belong to a separate section. Gageas have been measured from 186 localities in The Netherlands (Tables 1, 2) and they are compared with 41 accessions from Germany. They are shown to comprise six taxa with several inferred ploidies.

Gagea minima (L.) Ker Gawl.-section Minimae
Gagea minima is a small plant with 1 (or 2) narrow 2-3(5) mm wide leaves and 1-3 flowers per scape. G. minima with 2C = 14.9 pg from two localities, together with G. villosa, are the only two inferred diploid species found in The Netherlands.

Gagea villosa (M.Bieb.) Sweet-section Didymobolbos
Gagea villosa is a hairy, largely sterile plant with numerous bulbils. Fourteen accessions of G. villosa from the Dutch provinces of Gelderland, Overijsel and Zuid-Holland are inferred to be diploid with 16.9 pg. Six accessions of G. villosa are inferred to be tetraploid with on average 32.3 pg. This is based on the basic value of 7-8 pg as in the other species (except G. spathacea) and the published counts of 24 and 48 chromosomes (http://www. tropicos.org/gagea).

Gagea pratensis (Pers.)Dumort.-section Gagea
Gagea pratensis is a glabrous plant with up to four flowers per scape. Characteristic are the two nude egglike, horizontal bulbils. Gagea pratensis can be found in The Netherlands with four different inferred ploidies. They can be recognized in that the tetraploid has the leaf sheath circling the stem halfway, the pentaploid three-quarter and the hexaploid and the decaploid completely. They are shown in Figs. 9, 10, 11, 12 and 13. The tetraploids (30 accessions) have a DNA 2C-value (nuclear DNA content) of on average 32.8 pg, the pentaploids (85 accessions) have on average 39.9 pg and the hexaploids (20 accessions) have on average 45.6 pg. The pentaploids could be hybrids between the tetraploid and the hexaploid cytotypes. Even a decaploid with 75.8 pg has been found. The pentaploid form of G. pratensis is by far the most common Gagea in The Netherlands with 39.5 pg from 85 out of 186 localities. The same ploidy is counted in all 7 populations of G. pratensis from Mecklenburg (Germany) (Henker 2005). Therefore it seems most likely that the decaploid plant is derived from the frequently found pentaploid G. pratensis that has in this case doubled its genome. As often in polyploids, DNA might have been lost and a similar loss is found in the hexaploid G. pratensis but not in the lower ploidies. The inferred decaploid plants have not been reported before for G. pratensis. Being pentaploid in most cases, it comes as no surprise that G. pratensis is considered to be sterile (van der Meijden 2005). Taxa with anorthoploid chromosome sets often show a highly irregular meiosis. An exception are large plants from Zutphen, NL that are fully fertile and differ morphologically with a large basal leaf and 4-8 flowers to a stem. They have a genome size similar to pentaploid G. pratensis, but look more like G. × megapolitana Henker (Henker 2005). Out of 50 germinated seeds, five seedlings measured from the Zutphen locality had the same genome size as their parents. This is peculiar for a pentaploid. Earlier analysis of seedlings of the triploid Hosta 'Sum and Substance' show different, but lower genome sizes in the seedlings (Zonneveld and Pollock 2012). Pfeiffer et al. (2013) report also that some pentaploid populations of G. pratensis are partially fertile. None of the calculated genome sizes of the possible hybrids between G. lutea and G. pratensis would fit the plants from Zutphen. Hence more research is required to explain these results.

Gagea lutea (L.) Ker Gawl.-section Gagea
Gagea lutea is a glabrous plant with leaves of more than 1 cm wide and up to seven flowers per stem. The bulbs have a diameter of 0.75-1.5 cm and form numerous bulbils. Apart from hexaploid G. pratensis also G. lutea is inferred to be hexaploid with 2C = 42.7 pg, collected in 22 localities. Gagea lutea var. glauca (a synonym of G. lutea (L.)Ker Gawl.) differs in its glaucous leaves, slightly larger petals, lower fertility and the anthropogenic habitats it grows in. The flowering time of the glaucous forms starts about 2 weeks later when transplanted in the garden. Gagea var. glauca is restricted to the northern part of the Netherlands and is found in localities separate from the green-leaved form. However, with 42.3 pg for 16 different accessions there is no significant difference in genome size.

Gagea spathacea(Hayne) Salisb.-section Spathaceae
Gagea spathacea is a glabrous plant with 1-3 flowers per stem and is usually found in fairly moist places. Gagea spathacea is only present as a nonaploid plant across (Eastern) Europe (Westergaard 1936;Henker 2005;Pfeiffer et al. 2012). It is observed in about 70 localities in The Netherlands, of which material was collected at 12 localities with an average of 2C = 46.5 pg. This implies a low basic (Cx) value of 5.2, instead of 7.4-8.4 for the other three species. The virtually sterile G. spathacea (Pfeiffer et al. 2012) seems to be a nearly monoclonal plant able to occupy a significant range by dispersal of bulbils (Pfeiffer et al. 2011;2013).

Hybrid species
Hybridization and polyploidy are amongst the most important evolutionary mechanism in plants. The parents can be deduced by comparing the genome size of possible parents and their offspring. If parents have say 20 and 30 pg then their offspring will mostly have 25 pg. In more complicated allopolyploids the contribution of each parent can often be calculated. In Gagea inferred polyploids run from triploid to decaploid (Peruzzi 2003) whereby several species show different ploidies (Henker 2005). Three different hybrids have been described that have the same parents, G. lutea and G. pratensis but combining different ploidies. These three hybrids are here discussed under the names as found in the literature. They are not found in The Netherlands, but two of the hybrids were obtained from Germany (Table 1). The hybrids mostly occur in anthropogenically disturbed sites like churchyards, parks and marginally used meadows. Their parent species are found in forests (G. lutea) and forest edges (G. pratensis). Based on maternal inheritance of the plastids G. pratensis provide the female gametes for G. × pomeranica and G. megapolitana (Peterson et al. 2009).

Gagea × pomeranica R.Ruthe
The pentaploid G. × pomeranica (R.Ruthe) Henker with two genomes of the tetraploid G. pratensis and three genomes of the hexaploid G. lutea (Peterson et al. 2009). However, in the case of G. × pomeranica, 11 accessions were obtained from Germany that had on average a nuclear DNA content of 34.9 pg. This differs considerably (2.2 pg) from the calculated genome size of 37.1 pg, based on the basic values for G. lutea and G. pratensis. One explanation could be that this hybrid is an old one and has lost DNA. Another possibility is that other species are involved. Pfeiffer et al. (2013) have shown that backcrosses of the hybrid, mostly with the fully fertile hexaploid G. lutea as pollen parent are possible. However, backcrosses of G. × pomeranica (34.9 pg) with G. lutea (42.6 pg) might give higher 2C-values not lower, but these were not observed.

Gagea × marchica Henker. Kiesew., U.Raabe, Rätzel
Recently another sterile pentaploid hybrid was described as G. marchica Henker et al. (2012) It is described as falling morphologically between the pentaploid G. (×) pomeranica and the hexaploid G. (×) megapolitana with 57, 59 but probably 60 chromosomes. If it is supposed to be the reversed hybrid (compared to the parents of G × pomeranica) between hexaploid G. pratensis and tetraploid G. lutea the problem arises that a tetraploid G. lutea has not been reported so far (Pfeiffer et al. 2013).

Gagea × megapolitana Henker
A third hybrid with the same parents is the hexaploid G. × megapolitana Henker with three genomes of the hexaploid G. pratensis and three genomes of the hexaploid G. lutea (Peterson et al. 2009). It was obtained from two localities in Germany with on average 2C = 46.8 pg.
In the world checklist for monocots (Govaerts 2006) G. megapolitana is accepted as a species. However Peterson et al. (2009) have clearly shown that it is a hybrid between the hexaploids G. pratensis and G. lutea. The genome size provides a firm argument for this hybridity and confirm the suggestion of Peterson et al. (2009) for the parents and the ploidy of G. × megapolitana.