Abstract
Genome size estimations are reported for nine Gagea species, belonging to two different sections. The lowest value was found in Gagea cf. peduncularis from Turkey (sect. Didymobulbos), 2C = 12.06 pg, likely diploid, the highest in a pentaploid G. pratensis from S France (sect. Gagea), 2C = 39.45 pg. Monoploid genome size or 1Cx values range from 5.81 pg in G. foliosa (sect. Didymobulbos) to 9.16 pg in G. sicula, from the same section.
Introduction
The genus Gagea Salisb. (Liliaceae) was the object of several recent cytotaxonomic studies, concerning chromosome number and karyotype asymmetry analysis (e.g. Peruzzi Citation2003, Citation2008; Peruzzi and Aquaro Citation2005; Peruzzi Citation2012a). Although Gagea is the largest genus amongst Liliaceae, with about 300 taxa (Tison et al. Citation2013), genome size is currently known only for 10 species ranging from 2C = 12.48 pg in Gagea pusilla (F.W. Schmidt) Sweet to 2C = 75.80 pg in G. pratensis (Pers.) Dumort., both from sect. Gagea (Greilhuber et al. Citation2000; Leitch et al. Citation2007; Veselý et al. Citation2012; Zonneveld et al. Citation2015). Moreover, these values were mostly not linked to ploidy levels verified by chromosome counts. A further study was devoted to relative genome size variation (with inference on ploidy levels) in the Gagea reticulata (Pall.) Schult. & Schult.f. species complex (sect. Platyspermum) in Iran (Zarrei et al. Citation2012).
This paper begins a series of contributions aimed to fill this gap in Gagea karyological knowledge.
Material and methods
Plant material
Plant material (leaves) was collected from plants cultivated in the Botanic Garden of Pisa and originally collected across the Mediterranean area, for a total of 13 accessions belonging to nine species and two sections (Table ). In all accessions beyond one of G. cf. peduncularis, chromosome numbers were known from our previous studies (Peruzzi Citation2003, 2008 2012b; Peruzzi & Aquaro Citation2005; Peruzzi, Astuti, et al. Citation2009; Table ).
Table 1. Source of studied material, with previously published information on chromosome number, ploidy level and chromosome size range of the same individuals.
Flow cytometry
Nuclear DNA content was assessed by flow cytometry according to the protocol of Marie and Brown (Citation1993). Measurements were taken on nuclei extracted from fresh leaves. Conditions recommended by Doležel and Bartoš (Citation2005) to estimate genome size were fulfilled. For larger genomes, the propidium iodide concentration was raised to 100 μg ml−1. Two calibration standards, differing in DNA amount, were used: Artemisia arborescens L. (origin Crete, 2C = 11.43 pg, Garcia et al. Citation2006) for the majority of samples and Pisum sativum L. ‘Long Express’ (2C = 8.37 pg, Marie and Brown Citation1993) for just one accession. The nuclei were isolated by chopping together leaf of the sample and the internal standard using a razor blade in plastic Petri dishes containing 800 ml of Galbraith’s nuclear isolation buffer (Galbraith et al. Citation1983) which was supplemented with 10 mM sodium metabisulphite and 1% polyvinylpyrrolidone 10,000 Mr.
The suspension of nuclei was filtered through nylon mesh (pore size 50 μm). After RNase treatment (2.5 U ml−1; Roche, Meylan, France), the nuclei were stained with 75–100 μg ml−1 propidium iodide (Sigma-Aldrich, Lyon, France), a DNA intercalating dye without base-specificity. DNA content of at least 5000 nuclei was determined for each sample using a PartecCyFlow SL3 532 nm laser cytometer (Munster, Germany). The cytometer linearity was supervised by checking the relative positions of 2C and 4C nuclei found in leaf tissue. All measurements were repeated twice. The 2C DNA content was calculated multiplying the ratio between the peak (modal) positions in the histogram of fluorescence intensities of the Gagea species and the internal standard by known DNA content of the internal standard.
Results
The results obtained are shown in Table . In the panel of investigated species three ploidy levels occurred (2x, 3x and 5x). The minimum 2C value was found in Gagea cf. peduncularis from Turkey (sect. Didymobulbos), 2C = 12.06 pg, the highest in a pentaploid G. pratensis from S France (sect. Gagea), 2C = 39.45 pg. Monoploid genome size (1Cx) values ranged from 5.81 pg in triploid G. foliosa (sect. Didymobulbos) to 9.16 pg in diploid G. sicula, from the same section. Almost the same 2C values were obtained for two G. lojaconoi populations despite the use of two different standards (Artemisia and Pisum).
Table 2. Genome size estimations in Gagea: present and previous data.
Discussion
According to the classification of Leitch et al. (Citation1998), all the taxa studied can be considered as having genomes of mostly intermediate (7 < 2C ≤ 28 pg) size, except the large genome (2C = 39.45 pg) of pentaploid G. pratensis.
It is interesting to note that, within sect. Gagea, the diploid G. tisoniana (endemic to Central Italy; Peruzzi et al. Citation2007; Falcinelli et al. Citation2012), has a slightly variable genome size (2C = 14.07–14.25 pg from three different accessions), seemingly higher than that of the taxonomically very close G. pusilla (likely diploid as well) (2C = 12.48 pg; Veselý et al. Citation2012). The pentaploid G. pratensis, on the other hand, shows a genome size (2C = 39.45 pg) very similar to that reported for – only putatively pentaploid plants – by Zonneveld et al. (2015: 2C = 39.90 ± 0.50 pg), and to the hexaploid G. lutea (2C ranging from 39.50 to 42.70 pg and 2n = 6x = 72; Greilhuber et al. Citation2000; Veselý et al. Citation2012). This could be explained by 1Cx genome size reduction in polyploids, as already pointed out by Peruzzi, Leitch, et al. (Citation2009) in Liliaceae.
Concerning sect. Didymobulbos, with the exception of diploid narrow endemic G. sicula (showing also the highest 1Cx value) and G. cf. peduncularis (unknown ploidy level), the studied species were all triploid, with a 2C values ranging from 17.42 to 23.54 pg. The value reported by Veselý et al. (Citation2012) for “Gagea chrysantha” (2C = 18.17 pg) is certain to refer to another taxon (G. chrysantha s.s. is a narrow endemic to Sicily; Tison et al. Citation2013), and likely to refer to G. lojaconoi (as many others in the Mediterranean: Tison et al. Citation2013; Kayikçi et al. Citation2014). This is also supported by the data in our study, where G. lojaconoi resulted with 2C = 18.94–18.96 pg and G. chrysantha with higher values (2C = 20.21). Tison et al. (Citation2013), in spite of a clear morpho-anatomical differentiation, found complete molecular identity within the so-called G. chrysantha aggregate (G. amblyopetala, G. chrysantha, G. lojaconoi), with only G. sicula somehow distinct on molecular grounds. Our results highlight also differences in genome size among these closely related taxa. Concerning G. peduncularis, despite the only chromosome count available in literature being 2n = 3x = 36 (Peruzzi and Caparelli Citation2007, made on Italian material), the very low genome size observed suggests instead a diploid level (but karyological verification is required).
Our results enlarge considerably the genome size knowledge in the genus Gagea, which now covers 18 taxa (about 6% of the total estimated number), including all the three Italian endemics (G. chrysantha, G. sicula, G. tisoniana; Peruzzi et al. Citation2014).
It will be of particular interest to add higher ploidy level taxa to the picture, such as for instance wide-range species as G. fragifera (Vill.) Erh.Bayer & G.López (7x), as well as diploids from other sections (Peruzzi Citation2012a).
Given the relevant within-ploidy level genome size variation evidenced in this study (about 1.5-fold in diploids, 1.4-fold in triploids), we recommend chromosome count checking to be coupled in future genome size studies in this genus, to infer ploidy levels. This was not done, for instance, by Zarrei et al. (Citation2012) and by Zonneveld et al. (Citation2015), whose conclusions on ploidy might be questionable. Indeed, for instance, the latter authors inferred nearly the same genome size in G. × pomeranica R.Ruthe 2C = 46.80 ± 0.10 pg, and G. pratensis 2C = 45.60 pg ± 1.10, and G. spathacea, 2C = 46.70 ± 0.80 pg. They assigned different ploidy levels to these accessions (6x the first two, 9x the third) arbitrarily, without any chromosome count checking, only based on previous karyological literature knowledge.
Acknowledgements
Many thanks are due to Janis Ruksans and Dörte Harpke for having provided bulbs of Gagea cf. peduncularis from Turkey and to Mickael Bourge and Spencer C. Brown for their assistance in cytometry at the Imagif Platform IbiSA, Gif-sur-Yvette, France. Flavia Domizia Nardi’s stay in Paris benefited from an ERASMUS grant provided by the University of Pisa from December 2013 to March 2014.
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