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, 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).

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 (2001, 2003, 2008, 2009, 2010), Zonneveld and Van Iren (2001), Zonneveld and Duncan (2003, 2006), and Zonneveld et al. (2003, 2012). In this paper it is shown that genome size is helpful to discriminate between the species of Gagea from The Netherlands (Fig. 1).

Fig. 1

Scans of petals of Gagea pratensis in the Netherlands. In the left column petals from a fresh inflorescence. On the left the inner petals (left the upper side and on the right the bottom side). On the right the outer petals (left the upper side and on the right the bottom side). In the right column petals from an inflorescence after bloom. On the left the inner petals (left the upper side and on the right the bottom side). On the right the outer petals (left the upper side and on the right the bottom side). The tetraploid gagea is collected in a park near the river Berkel in Almen. The pentaploid (a) gagea is collected in a churchyard in Wassenaar. (All gageas in the western part of the Netherlands are of this type). The pentaploid (b) is collected in a road verge near Fromberg. The pentaploid (c) is collected in a park in Zutphen. (These large gageas resemble Gagea megapolitana). The hexaploid gagea is collected in a park near the river IJssel in Deventer.

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.

Methods

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.

Table 1

Accessions of Gagea from The Netherlands and Germany (in italics), with their nuclear DNA content, inferred ploidy, average, standard deviations and localities

Species	pg/2C	Average	stdev	Locality	coll. nr	Locality (counties NL)
Gagea minima diploid
Gagea minima	14.8	14.9	0.6	Leyduin, Bloemendaal	BtL11-0092	N Holland
Gagea minima	14.9			Leyduin, Bloemendaal	L Duistermaat	N Holland
Gagea villosa diploid
Gagea villosa	16.5	17.0	0.4	Doddendaal	BtL11-053	Gelderland
Gagea villosa	16.7			Deventer	BtL11-085	Overijsel
Gagea villosa	16.7			Zutphen, Hanzehof	BtL11-003	Gelderland
Gagea villosa	16.8			Elten	BtL11-001	Germany
Gagea villosa	16.8			Eys	BtL11-002	Limburg
Gagea villosa	16.8			Zevenaar	BtL11-084	Gelderland
Gagea villosa	16.9			Zeist	BtL11-037	Utrecht
Gagea villosa	17.0			Lent	BtL11-055	Gelderland
Gagea villosa	17.0			Valkenburg	BtL11-077	Limburg
Gagea villosa	17.2			Zevenaar	BtL11-078	Gelderland
Gagea villosa	17.3			Zalk	BtL11-017	Overijsel
Gagea villosa	17.4			Mijnsherenland	BtL14-002	Gelderland
Gagea villosa	17.3			Spijk	BtL14-003	Gelderland
Gagea villosa	17.2			Amerongen	BtL14-004	Utrecht
Gagea villosa tetraploid
Gagea villosa	31.5	32.3	0.7	Heelsum	BtL11-052	Gelderland
Gagea villosa	31.7			Zwolle, Nahuysplein 1	BtL11-016	Overijsel
Gagea villosa	31.8			Zwolle, Zandhove	BtL13-079	Overijsel
Gagea villosa	32.6			Bussloo, begraafplaats t.o Zutphenboer	BtL13-088	Gelderland
Gagea villosa	32.8			Zwolle, Nahuysplein	BtL13-080	Overijsel
Gagea villosa	33.2			Azewewijn, kerkhof	BtL13-102	Gelderland
Gagea pratensis tetraploid
Gagea pratensis	32.1	32.8	0.4	Deventer	BtL11-085	Overijsel
Gagea pratensis	32.1			Kootwijk	BtL11-073	Gelderland
Gagea pratensis	32.2			Almen 2	BtL11-066	Gelderland
Gagea pratensis	32.2			Legden	BtL13-028	Germany
Gagea pratensis	32.3			Almen 1	BtL11-040	Gelderland
Gagea pratensis	32.3			Rhienderen 1	BtL11-067	Gelderland
Gagea pratensis	32.4			Suderas, Wichmond	BtL13-053	Gelderland
Gagea pratensis	32.4			Bussloo, begraafplaats t.o Zutphenboer	BtL13-087	Gelderland
Gagea pratensis	32.5			Babberich	BtL11-014	Gelderland
Gagea pratensis	32.6			Voorst	BtL11-062	Gelderland
Gagea pratensis	32.7			Gietelo	BtL13-002	Gelderland
Gagea pratensis	32.7			Ravenswaarden	BtL13-009	Gelderland
Gagea pratensis	32.7			Keppel, klein	BtL13-050	Gelderland
Gagea pratensis	32.8			Almen	BtL11-012	Gelderland
Gagea pratensis	32.9			Trent 2	Tre11-xx	Germany
Gagea pratensis	32.9			Gingst	Gin11-xx	Germany
Gagea pratensis	32.9			Meppen, stadswal	BtL13-077	Germany
Gagea pratensis	33.0			Subzow	Sub11-18	Germany
Gagea pratensis	33.0			Elbe Jasebeck	BtL13-043	Germany
Gagea pratensis	33.0			Deventer, Brinkgrave	BtL13-084	Overijsel
Gagea pratensis	33.1			Almen 3	BtL11-074	Gelderland
Gagea pratensis	33.2			Zirchow	Zir11-xx	Germany
Gagea pratensis	33.2			Deventer, Brinkgrave	BtL13-015	Overijsel
Gagea pratensis	33.3			Beek	BtL11-045	Gelderland
Gagea pratensis	33.3			Klarenbeek	BtL11-061	Gelderland
Gagea pratensis	33.3			Trent	Tre11-xx	Germany
Gagea pratensis	33.3			Kampens dl	BtL13-035	Overijsel
Gagea pratensis	33.4			Gorssel, oud kerkhof	BtL13-083	Gelderland
Gagea pratensis	33.7			Bingerden 1a	BtL11-075	Gelderland
Gagea pratensis	33.7			Gietelo	BtL13-002	Gelderland
Gagea pratensis pentaploid
Gagea pratensis	37.9	39.9	0.5	Cortenoever 1	BtL11-087	Gelderland
Gagea pratensis	37.9			Leyduin	BtL11-041	N. Holland
Gagea pratensis	38.3			Ressen	BtL11-056	Gelderland
Gagea pratensis	38.5			Bingerden 2	BtL11-094	Gelderland
Gagea pratensis	38.5			Bemmel 1	BtL11-033	Gelderland
Gagea pratensis	38.6			Zwolle 2	BtL11-015	Overijsel
Gagea pratensis	38.6			Hattem, Heezenberg	BtL13-089	Gelderland
Gagea pratensis	38.7			Angerlo	BtL11-027	Gelderland
Gagea pratensis	38.7			Ravenswaarden	BtL11-058	Gelderland
Gagea pratensis	38.7			Den Haag, Westerduinpark	BtL13-031	Z. Holland
Gagea pratensis	38.8			Castricum, kerkhof	BtL13-085	N. Holland
Gagea pratensis	38.9			Didam	BtL13-048	Gelderland
Gagea pratensis	38.9			Zwolle, Vecht	BtL13-081	Overijsel
Gagea pratensis	39.0			Loil	BtL11-024	Gelderland
Gagea pratensis	39.0			Angerlo	BtL11-026	Gelderland
Gagea pratensis	39.0			Babberich 2	BtL11-072	Gelderland
Gagea pratensis	39.0			Den Haag, Marlot	BtL13-032	Z. Holland
Gagea pratensis	39.0			Lisse, Huis te Spekke	BtL13-024	Z. Holland
Gagea pratensis	39.1			Haarlem 1a	BtL11-039	N. Holland
Gagea pratensis	39.1			Leiden, Rhijnhof	BtL13-042	Z. Holland
Gagea pratensis	39.1			Keppel, groot	BtL13-051	Gelderland
Gagea pratensis	39.2			Bemmel	BtL11-032	Gelderland
Gagea pratensis	39.2			Velp 2	BtL11-029	Gelderland
Gagea pratensis	39.2			Bemmel 2	BtL11-032	Gelderland
Gagea pratensis	39.2			Noordwijk, Gooweg	BZ13-023	Z. Holland
Gagea pratensis	39.2			Haarlem, Schootersingel	BtL13-036	N. Holland
Gagea pratensis	39.2			Driehuis begraafplaats	BtL13-037	N. Holland
Gagea pratensis	39.2			Huize Baak	BtL13-052	Gelderland
Gagea pratensis	39.2			Deventer, Drouwelerkolk	BtL13-013	Overijsel
Gagea pratensis	39.2			Hattemerwaard	BtL13-093	Gelderland
Gagea pratensis	39.2			Velzen, Beeckenstein	BtL13-101	N. Holland
Gagea pratensis	39.3			Huissen, pastorie	BtL13-040	Gelderland
Gagea pratensis	39.3			Voorhout	BtL13-022	Z. Holland
Gagea pratensis	39.3			Eldrik	BtL13-049	Gelderland
Gagea pratensis	39.3			Kampen, stadswal	BtL13-033	Overijsel
Gagea pratensis	39.3			Mehr	BtL13-027	Germany
Gagea pratensis	39.4			Bingerden 1b	BtL11-076	Gelderland
Gagea pratensis	39.4			Kampen 1a	BtL11-019	Overijsel
Gagea pratensis	39.4			Brummen 1	BtL11-068	Gelderland
Gagea pratensis	39.4			Warmond	BtL13-021	Z. Holland
Gagea pratensis	39.5			Fromberg	BtL11-093	Limburg
Gagea pratensis	39.5			Oud-Zevenaar	BtL11-023	Gelderland
Gagea pratensis	39.5			Elten	Elt11-xx	Germany
Gagea pratensis	39.5			Drempt	BtL11-042	Gelderland
Gagea pratensis	39.5			Zutphen, Hanzehof	BtL13-001	Gelderland
Gagea pratensis	39.5			Zutphen, kanaal 16 jan 2013	BtL13-003	Gelderland
Gagea pratensis	39.5			Epse	BtL13-005	Gelderland
Gagea pratensis	39.5			Wassenaar	BtL13-006	Z. Holland
Gagea pratensis	39.5			Sassenheim	BtL13-010	Z. Holland
Gagea pratensis	39.5			Zutphen, De Hoven	BtL13-011	Gelderland
Gagea pratensis	39.5			OegstgeesT	BtL13-012	Z. Holland
Gagea pratensis	39.5			IJmuiden berm	BtL13-038	N. Holland
Gagea pratensis	39.5			Velzen, Kanaalweg	BtL13-086	N. Holland
Gagea pratensis	39.5			Achthoven	BtL14-005	Gelderland
Gagea pratensis	39.6			Haarlem	BtLs.n.	N. Holland
Gagea pratensis	39.6			Hoog-Keppel	BtL11-043	Gelderland
Gagea pratensis	39.6			Zwolle, Zandhove	BtL13-082	Overijsel
Gagea pratensis	39.6			Velzen, kerkhof	BtL13-100	N. Holland
Gagea pratensis	39.6			sdl Zutphen, Hanzehof	BtL14-08	Gelderland
Gagea pratensis	39.6			sdl Zutphen, Hanzehof	BtL14-008	Gelderland
Gagea pratensis	39.7			Zutphen	Btl11-022	Gelderland
Gagea pratensis	39.7			Cortenoever 2	Btl11-020	Gelderland
Gagea pratensis	39.8			Beverwijk, Scheybeek	BtL13-039	N. Holland
Gagea pratensis	39.8			Zwolle, Engelse werk	BtL13-078	Overijsel
Gagea pratensis	39.9			Hummelo	BtL11-044	Gelderland
Gagea pratensis	39.9			Leiden, Rhynhof	BZ12-01	Z. Holland
Gagea pratensis	39.9			Den Haag 2	BtL11-070	Z. Holland
Gagea pratensis	40.0			sdl Zutphen, Hanzehof	BtL14-008	Gelderland
Gagea pratensis	40.0			sdl Zutphen, Hanzehof	BtL14-008	Gelderland
Gagea pratensis	40.1			Rhienderen 2	BtL11-067	Gelderland
Gagea pratensis	40.1			Zutphen, Hanzehof	BtLs.n.	Gelderland
Gagea pratensis	40.2			Cortenoever 3	BtL11-021	Gelderland
Gagea pratensis	40.2			Weurt	BtL11-054	Gelderland
Gagea pratensis	40.2			Heelsum, kerk	BtL14-011	Gelderland
Gagea pratensis	40.3			Brummen 2	BtL11-071	Gelderland
Gagea pratensis	40.3			sdl Zutphen, zwembad	BtL14-009	Gelderland
Gagea pratensis	40.4			Doesburg	BtL11-028	Gelderland
Gagea pratensis	40.4			Middachten	BtL11-046	Gelderland
Gagea pratensis	40.4			parent Zutphen, zwembad	BtL14-010	Gelderland
Gagea pratensis	40.5			Spankeren	Btl11-060	Gelderland
Gagea pratensis	40.6			Olburgen	Btl11-047	Gelderland
Gagea pratensis	40.6			Steenderen	Btl11-048	Gelderland
Gagea pratensis	40.7			Zutphen, begraaf plaats	BtL13-054	Gelderland
Gagea pratensis	40.9			Groessen	BtL11-035	Gelderland
Gagea pratensis	40.9			Oud-Zevenaar	BtL11-057	Gelderland
Gagea pratensis hexaploid
Gagea pratensis	43.8	45.6	1.1	Zirchow/U 2	ZiU11-xx	Germany
Gagea pratensis	44.0			Trent 2	Tre11-xx	Germany
Gagea pratensis	44.3			Vaassen, Canneburg 194/478	BtL11-095	Limburg
Gagea pratensis	45.0			Bronkhorst, slotheuvel	BtL11-049	Gelderland
Gagea pratensis	45.1			Deventer, De Worp	BtL13-004	Overijsel
Gagea pratensis	45.1			Culemborg, Stroomrug	BtL13-008	Gelderland
Gagea pratensis	45.2			Brummen, Engelenburg	BtL13-017	Gelderland
Gagea pratensis	45.3			Oosterbeek, gazon	BtL11-051	Gelderland
Gagea pratensis	45.4			Meppen begraafplaats	BtL13-072	Germany
Gagea pratensis	45.4			Deventer, Blauwijk	BtL13-014	Overijsel
Gagea pratensis	45.4			Marle, uiterwaardgrasland	BtL13-090	Overijsel
Gagea pratensis	45.7			Brummen, Ganzenei	BtL13-091	Gelderland
Gagea pratensis	45.8			Meppen stadswal	BtL13-071	Germany
Gagea pratensis	46.0			Elst, Johan de Wittstraat	BtL11-030	Gelderland
Gagea pratensis	46.0			Altenkirchen	Alt11-19	Germany
Gagea pratensis	46.1			Olst, gementehuis	BtL13-016	Overijsel
Gagea pratensis	46.6			Ravenswaarden	BtL13-099	Gelderland
Gagea pratensis	47.2			Empe	BtL11-063	Gelderland
Gagea pratensis	47.4			Brummen 3	BtL11-064	Gelderland
Gagea pratensis	47.9			Brummen 4	BtL11-069	Gelderland
Gagea pratensis decaploid
G. pratensis	75.0	75.8	1.1	Kampen 1b	BtL11-018	Overijsel
G. pratensis	76.6			Kampen 1c	BtL11-060	Overijsel
Gagea × megapolitana hexaploid
Gagea × megapolitana	46.7	46.8	0.1	Gingst 2	Gin11-xx	Germany
Gagea × megapolitana	46.8			Meppen	BtL13-071	Germany
Gagea lutea hexaploid
Gagea lutea	41.7	42.7	0.6	Trent 1	DEs.n.	Germany
Gagea lutea	41.9			Haarlem, Spaem en Hout	BtL11-039	N. Holland
Gagea lutea	42.1			Epe, Dinkel	BtL13-030	Germany
Gagea lutea	42.1			Vreden, Berkel	BtL13-029	Germany
Gagea lutea	42.1			Miste	BtL13-103	Gelderland
Gagea lutea	42.3			Roden	BtL11-081	Drenthe
Gagea lutea	42.3			Kelmis, Hohntal	BtL11-079	Belgie
Gagea lutea	42.4			Zuidlaren	BtL11-089	Drenthe
Gagea lutea	42.4			Griebenow	DE-s.n.	Germany
Gagea lutea	42.6			Meppen, stadswal	BtL13-070	Germany
Gagea lutea	42.6			Bron Berkle	Bbe11-xx	Germany
Gagea lutea	42.6			Bredevoort	BtL11-011	Gelderland
Gagea lutea	42.8			Haarlem 1c	BtL11-038	N. Holland
Gagea lutea	42.8			D Meppen	BtL13-026	Germany
Gagea lutea	42.8			Millingerwaard	BtL14-001	Z. Holland
Gagea lutea	42.9			Gesher	Ges11-xx	Germany
Gagea lutea	42.9			Haarlem, Spaem en Hout	BtL11-38	N. Holland
Gagea lutea	42.9			Elbe, Jasebeck	BtL13-044	Germany
Gagea lutea	43.2			Aerdenhout	BtL13-066	N. Holland
Gagea lutea	43.4			Winterswijk, Vreehorst weg	BtL13-055	Gelderland
Gagea lutea	43.8			Bronkhorst	BtL11-005	Gelderland
Gagea lutea	43.9			Schoorl	BtL13-094	N. Holland
Gagea lutea var. glauca	41.9	42.3	0.3	Anloo, grasland	BtL13-064	Drenthe
Gagea lutea var. glauca	41.9			Veenhof, Berm	BtL13-057	Drenthe
Gagea lutea var. glauca	42.0			Groningen, Noorderplantsoen	BtL13-045	Groningen
Gagea lutea var. glauca	42.1			Zeegse	BtL11-090	Drenthe
Gagea lutea var. glauca	42.1			Bellingwolde, berm	BtL13-067	Groningen
Gagea lutea var. glauca	42.2			Midwolda, Ennemaborg	BtL13-047	Groningen
Gagea lutea var. glauca	42.2			Eext Berm	BtL13-058	Drenthe
Gagea lutea var. glauca	42.2			Westeresch	BtL11-080	Drenthe
Gagea lutea var. glauca	42.3			Sterrenbospark	BtL11-088	Groningen
Gagea lutea var. glauca	42.3			Gieten	BtL11-009	Drenthe
Gagea lutea var. glauca	42.3			Doetinchem, Zumpe 16 jan 2013	BtL13-007	Gelderland
Gagea lutea var. glauca	42.4			Midlaren	BtL13-059	Drenthe
Gagea lutea var. glauca	42.8			Wedde, Huis Te Wedde, onder linde	BtL13-069	Groningen
Gagea lutea var. glauca	42.8			Appingedam, Ekenstein	BtL13-046	Groningen
Gagea lutea var. glauca	43.0			Eext, Brink	BtL13-056	Drenthe
Gagea lutea var. glauca	42.9			Naumburg	BtL14-012	Germany
Gagea × pomeranica pentaploid
Gagea × pomeranica	34.2	34.9	0.6	Vitense	Vit11-11	Germany
Gagea × pomeranica	34.3			Zirchow/U	ZIU11-xx	Germany
Gagea × pomeranica	34.3			Zirchow/U 2	ZIU11-xx	Germany
Gagea × pomeranica	34.4			W. Baggendorf	WBa11-15	Germany
Gagea × pomeranica	34.8			Zirchow/U 2	ZiU11-xx	Germany
Gagea × pomeranica	35.1			Semlow 2	Sem11-xx	Germany
Gagea × pomeranica	35.2			Semlow	Sem11-xx	Germany
Gagea × pomeranica	35.2			Poseritz	Pos11-12	Germany
Gagea × pomeranica	35.3			Semlow 2	Sem11-xx	Germany
Gagea × pomeranica	35.5			Zirkow/R	ZiR11-12	Germany
Gagea × pomeranica	36.0			Semlow	Sem11-xx	Germany
Gagea spathacea nonaploid
Gagea spathacea	45.4	46.7	0.8	Ootmarsum, de Voort	BtL13-063	Z. Holland
Gagea spathacea	45.4			Zeegse	BtL11-091	Drenthe
Gagea spathacea	45.5			Losser	BtL11-082	Overijsel
Gagea spathacea	45.9			Samerot, eiken-haagbeukenbos	BtL13-074	Germany
Gagea spathacea	46.1			Vasse, beekoever	BtL13-076	Overijsel
Gagea spathacea	46.1			Brummen, Ganzenei, stroomrug	BtL13-092	Gelderland
Gagea spathacea	46.3			Amen	BtL14-006	Drenthe
Gagea spathacea	46.4			Bentheim, langs pad	BtL13-073	Germany
Gagea spathacea	47.0			Roden, Havezate	BtL11-081	Drenthe
Gagea spathacea	47.0			Peizermade, bosrand	BtL13-096	Drenthe
Gagea spathacea	47.2			Roden, Havezate	BtL13-097	Drenthe
Gagea spathacea	47.3			Wüllen, eiken-haagbeukenbos	BtL13-068	Germany
Gagea spathacea	47.5			Nietap	BtL13-098	Drenthe
Gagea spathacea	47.6			Bentheim, gazon	BtL13-041	Germany
Gagea spathacea	47.7			Ootmarsum, weilandrand	BtL13-075	Overijsel
Gagea spathacea	47.9			Varik, eikenbos	BtL13-065	Drenthe
Gagea spathacea	48.1			Peize	BtL13-095	Drenthe

All were measured against Agave americana, but for G. minima and G. villosa A. attenuta was used.

BtL B. te Linde, stdev standard deviation, coll.nr collection number.

Fig. 2

The distribution of Gagea minima and Gagea spathacea in the Netherlands.

Fig. 3

The distribution of Gagea villosa in the Netherlands.

Fig. 4

The distribution of Gagea lutea and var. glauca in the Netherlands.

Fig. 5

The distribution of tetraploid Gagea pratensis in the Netherlands.

Fig. 6

The distribution of pentaploid Gagea pratensis in the Netherlands.

Fig. 7

The distribution of hexaploid Gagea pratensis in the Netherlands.

Fig. 8

The distribution of decaploid Gagea pratensis 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 nuclei-isolation 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 year-round, 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 G0 peak, almost lacking G2 arrest.

Fresh male human leucocytes [2C = 7.0 pg; 1 pg = 10−12 g = 0.978 × 109 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 × 109 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.

Results

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.

Table 2

Summary of genome sizes in pg (2C), number of accessions and inferred chromosome numbers of species of Dutch and German gageas

Species	Average pg/2C	Inferred ploidy	Chromosome number	Number of accessions
Gagea minima (L.) Ker Gawl.	14.9	Diploid	2x = 24	2
Gagea villosa (M.Bieb.) Sweet	17.0	Diploid	2x = 24	14
Gagea villosa (M.Bieb.) Sweet	32.3	Tetraploid	4x = 48	6
Gagea lutea (L.) Ker Gawl.	42.7	Hexaploid	6x = 72	22
Gagea lutea var. glauca L.Klein	42.3	Hexaploid	6x = 72	16
Gagea spathacea (Hayne) Salisb.	46.7	Nonaploid	9x = 108	17
Gagea pratensis (Pers.) Dumet.	32.8	Tetraploid	4x = 48	30
Gagea pratensis (Pers.) Dumet.	39.9	Pentaploid	5x = 60	85
Gagea pratensis (Pers.) Dumet.	45.6	Hexaploid	6x = 72	20
Gagea pratensis (Pers.) Dumet.	75.8	Decaploid	10x = 120	2
G. × pomeranica R.Ruthe	34.9	Hexaploid	5x = 60	11
G. × megapolitana Henker	46.8	Hexaploid	6x = 72	2

The term ‘inferred ploidy’ indicates that the ploidy is derived from the genome size and not based on chromosome counts. It is preferred to the proposed term ‘DNA ploidy’ (Suda et al. 2006) as this seems more ambiguous. Inferred decaploidy is found for the first time in G. pratensis. The hybrid G. × megapolitana, is only collected in Germany so far. The largest genome contains roughly 60 × 109 more base pairs than the smallest. A difference of 1 pg amounts to a difference of nearly 1 × 109 base pairs, so far exceeds a single taxonomic character.

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.

Fig. 9

Gagea pratensis tetraploid April 6, 2015 in a churchyard in Beek.

Fig. 10

Gagea pratensis pentaploid (a) March 17, 2015 in a churchyard in Wassenaar.

Fig. 11

Gagea pratensis pentaploid (b) April 3, 2011 in Bingerden.

Fig. 12

Gagea pratensis pentaploid (c) March 23, 2014 in a park in Zutphen.

Fig. 13

Gagea pratensis hexaploid April 22, 2011 in a road verge in Brummen.

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.

Conclusions

Five species and different inferred ploidies are recorded for The Netherlands, as summarized in Table 2, some of the latter for the first time. Inferred decaploidy in G. pratensis was not demonstrated earlier. G. minima has an inferred diploid size. G. minima was only recently (1994) recognized as a new species for the Netherlands (Diemeer 2005). It is not clear whether it reached Haarlem by itself or was imported with lime trees from abroad. After all, Linnaeus lived there for 3 years only a kilometer away. The nearest known locality is 300 km away in Germany. G. pratensis is inferred to have four cytotypes: tetraploid, pentaploid, hexaploid and decaploid. Remarkable is the high number, 85 out of 186 accessions, of the pentaploid cytotype. Although it is largely sterile, bulbs seem to be a very effective way for vegetative multiplication, just as found for G. spathacea (Pfeiffer et al. 2012). Gagea lutea is only found in an inferred hexaploid form. The nonaploidy reported for G. spathacea would suggests a low basic genome size. This is corroborated by the fact that G. spathacea belongs to a section different from the others. Flow cytometry could provide the correct identification in most cases. It is a taxonomic and diagnostic tool that is applicable even in the case of dormant bulbs or sterile plants, and therefore has applications for conservation monitoring. Future research of the Dutch gageas could focus on combining chromosome counts and flow cytometry of the same samples, especially in the case of G. villosa. The fertility of the pentaploid G. pratensis needs further investigation. Sequencing of the forma glauca of G. lutea could reveal if it is a separate species or not.