First record of a natural hexaploid population for Valeriana officinalis: genome size is confirmed to be a suitable indicator of ploidy level in the species

Abstract

A recent study of genome size in Valeriana officinalis has established for a Pyrenean population a C-value divergent from those assessed in the currently known diploid, tetraploid and octoploid cytotypes, which proportionally indicates a hexaploid cytotype. If confirmed, this would constitute the first record of a natural hexaploid population for Valeriana officinalis. If not, these data would diminish the present strong support for accurate deduction of ploidy levels from genome sizes within the species. In order to discern between these two alternative interpretations, chromosome counts were carried out in the population of interest. Counting reveals 2n = 42, that is to say, the hexaploid cytotype. This result completes the polyploid series of V. officinalis and confirms the suitability of nuclear DNA amount for inferring ploidy level in the species. Such a finding raises the question of the origin and the extreme rarity of the hexaploid cytotype in V. officinalis. Furthermore, given that ploidy level impacts upon many plant features, including the chemical compound profile, the hexaploid population might constitute a new chemotype for this medicinal species.

Keywords:

• C-value
• chemotype
• chromosome
• cytotype
• polyploid
• valerian
• Valerianaceae

Introduction

Valerian (Valeriana officinalis L. s.l.) is an important medicinal plant whose use dates back to antiquity, and is believed to have sedative, nervine, antispasmodic and relaxant properties (reviewed by Upton and Petrone 1999; Patočka and Jakl 2010). The species is naturally distributed across Eurasia and is cultivated in many other regions. Valeriana officinalis s.l. constitutes an aggregate of taxa of controversial taxonomic levels, circumscriptions and phylogenetic histories, showing a complexity to which the polyploid series (of 2x, 4x and 8x) displayed by the taxon greatly participates (Skalińska 1947, 1951; Titz 1969; Keller 1973; Evstatieva et al. 1993). The species exhibits a constant chromosome base number, x = 7, the smallest in Valerianaceae (other base numbers are x = 8, 11, 13, 15). Genome sizes increase with polyploidy, from 2C = 2.97 pg up to the highest value in the family, 2C = 8.32 pg, found in the octoploid form of V. officinalis (Hidalgo et al. 2010). Recent studies revealed a strong correlation between ploidy levels and 2C-values, and affirmed the suitability of genome size for inferring ploidy level within the species (Hidalgo et al. 2010; Klatte-Asselmeyer et al. 2011). However, a population from the Pyrenees presented an intermediate genome size between the values obtained for tetraploid and octoploid plants, which would correspond proportionally to the hexaploid cytotype (Hidalgo et al. 2010). This being the case, this would constitute the first record of a hexaploid population in V. officinalis. However, it cannot be ruled out that the population might correspond to a tetraploid or an octoploid cytotype. Indeed, gain or loss of DNA coupled with a polyploidy event, a phenomenon documented in many plant groups (Yang et al. 2011, and references therein), would explain the divergence in genome size between this Pyrenean population and other populations at same ploidy level. If this assumption is verified, genome size would not be as reliable a ploidy level indicator in the species as previously thought.

In order to discern between these two alternative interpretations, the present study aimed to establish the chromosome number of this Pyrenean population of Valeriana officinalis.

Materials and methods

The studied population of Valeriana officinalis comes from Catalonia, Spain: Lleida, Vielha, 1600 m, 8 August 2011, Joan Vallès (BCN 30336). This population exhibits intermediate morphological features, which impede its assignment to a given infraspecific taxon currently recognized in Catalonia [subsp. officinalis, subsp. tenuifolia (Vahl) Schübl. & G. Martens, subsp. sambucifolia (J.C. Mikan ex Pohl) Čelak. and var. vayredana Sennen; Bolòs and Vigo 1983, 1995; Vázquez Pardo et al. 2007].

Adult plants were taken from the wild and further grown in pots in the greenhouse. Root tip meristems were collected, pretreated with 0.002 M 8-hydroxyquinoline for 3 h at 16°C, fixed in absolute ethanol, trichloromethane, and glacial acetic acid (6:3:1) and stored in the fixative at 4°C for at least two days before processing. Samples were hydrolysed in 1 N HCl for 5 min at 60°C, stained with 1% aqueous aceto-orcein for 1 h minimum, and squashed into a drop of 45% acetic acid-glycerol (9:1) on slides. Pictures of metaphase plates were taken with a digital camera (Zeiss AxioCam HRm) mounted on a Zeiss Axioplan microscope, and images were analysed with Axio Vision Ac version 4.2.

Correlation between genome sizes and ploidy levels was established with Microsoft Excel 2010 (Microsoft Corp., Redmond, WA, USA) using the data from the present study and from Hidalgo et al. (2010), and tested for significance with the statistical software STATA 10.0 (Stata Corporation, College Station, TX, USA).

Results

Chromosome counting in the population of interest establishes 2n = 6x = 42 (Figure 1A).

Figure 1 (A) Metaphase plate of Valeriana officinalis showing 2n = 6x = 42 chromosomes. Scale bar: 10 μm. (B) Scatter plot showing correlation between the genome sizes and ploidy levels in Valeriana officinalis, using data from Hidalgo et al. (2010) and the present study.

Discussion

This result constitutes the first record of a hexaploid cytotype in Valeriana officinalis. Ploidy levels have been substantially implicated in the circumscription of subspecific entities within this taxon, which comprises a multitude of chromosome numbers and cytogeographic data throughout the time (Goldblatt and Lowry 2011, and references therein). However, despite this considerable amount of data, the hexaploid cytotype has remained to date unknown in the species, even in the case of V. officinalis populations presenting mixed ploidy levels (Klatte-Asselmeyer et al. 2011). It is noted that hexaploids have been found in a species closely related to V. officinalis, namely V. coreana Briq. (with 6x as the unique ploidy level; Probatova and Sokolovskaya 1981), and in V. transjenisensis Kreyer (with 2x, 6x and 8x represented; Panarctic Flora, http://www.nhm.uio.no/english/research/infrastructure/paf/900105, accessed 2 February 2012, and references therein).

This study, which provides a fourth ploidy level with which to determine the association between genome size and ploidy level in V. officinalis, establishes a strong positive linear correlation between these two parameters (p < 0.0001; Figure 1B). Consequently, genome size is confirmed as a suitable tool for inferring the ploidy level within the species, which is crucial to survey at large scale the karyological and cytogenetic diversity of this medicinal plant. Further studies aiming to address the questions of the origin and the extreme rarity of the hexaploid cytotype in V. officinalis are being carried out. The results would recommend a more in-depth survey of the species in the Catalan Pyrenees, a region showing a great diversity of genomes issued from polyploid-driven evolution for this medicinal plant (this study, Vázquez Pardo 2007; Hidalgo et al. 2010).

Although it seems that valepotriates or their metabolites play a role in the therapeutic properties of V. officinalis, these have not been definitely attributed to a particular compound, and they are thought to be explained by the synergistic effects of multiple active constituents (Upton and Petrone 1999; Andreatini et al. 2002; Vanaclocha and Cañigueral 2003, and references therein). The use of valerian thus exclusively relies on cultivation. The composition of the essential oil of V. officinalis has been found to depend on the cultivar, plant age, harvesting time, and geographical origin (Letchamo et al. 2004, and references therein). Taking into consideration that polyploidy is another important factor that shapes the chemical profile of plants (e.g. Kokate et al. 2008; in Lippia, Pierre et al. 2011; in Atriplex, Sanderson 2011), further research is needed to investigate whether the chemotype – and putatively associated medicinal properties – corresponding to the 6x cytotype of Valeriana officinalis differs from those of other ploidy levels.

Acknowledgements

This work was subsidized by the Ministerio de Educación y Ciencia, Spain (Project CGL2010-22234-C02/BOS) and Generalitat de Catalunya (“Ajuts a Grups de Recerca Consolidats” 2009/SGR/00439). OH benefited from a Juan de la Cierva postdoctoral contract from the Ministerio de Ciencia e Innovación, Spain.