Open Access

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

SpringerPlus20154:395

https://doi.org/10.1186/s40064-015-1167-4

Received: 21 January 2015

Accepted: 17 July 2015

Published: 5 August 2015

Abstract

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.

Keywords

Gagea The NetherlandsDNA 2C-valueGenome sizePloidy levelNew decaploid G. pratensis

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.

Declarations

Authors’ contributions

BtL and LJB collected the plants, provided morphological and biogeographical data and corrected the manuscript. BZ did the flow cytometry and drafted the manuscript. BtL provided and made all figures. All authors read and approved the final manuscript.

Acknowledgements

I like to thank Tanja Pfeiffer for providing most of the German material.

Compliance with ethical guidelines

Competing interests The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
NBC Naturalis, Herbarium Section
(2)
Berglinde BV

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