Analysis of the taxonomic subdivision within the genus Helleborus by nuclear DNA content and genome-wide DNA markers
Research highlights
▶ One common chromosome number for all Helleborus species. ▶ Nuclear DNA contents of Helleborus species varied between 18 and 33 pg/2C. ▶ Genome-wide DNA markers support the genus classification based on morphological characteristics. ▶ Two newly described species were validated in its section.
Introduction
Helleborus are rhizomatous, herbaceous perennials that flower from late winter until early spring. As members of the Ranunculaceae, they bear a characteristic fruit consisting of a cluster of follicles and multiovular carpels (McLewin and Mathew, 1995). Different approaches have been undertaken to better understand the classification and phylogeny of this plant family. This group has been divided into subfamilies and tribes either by morphological or anatomical characteristics (Prantl, 1888, Hutchinson, 1923, Hoot, 1991, Wang and Ren, 2008), cytological (Langlet, 1932, Gregory, 1941) or serological studies (Jensen, 1968) and recent molecular data (Hoot, 1995, Ro et al., 1997, Wang et al., 2005). As a result, within the Ranunculaceae, Helleborus belongs to the subfamily Ranunculoideae, which comprises all genera with Ranunculus-type chromosomes (Ro et al., 1997), but their relationship to other genera in that subfamily is still topic of debate.
Helleborus species are distributed over different parts of Europe and West Asia, with the exception of one species, H. thibetanus, which is native to East Asia. Since the descriptions of Braun and Bouché (1861), two morphological groups have been distinguished in the genus according to caulogenesis; the Caulescentes and Acaules. Caulescent Helleborus, including H. argutifolius, H. foetidus and H. lividus, have tough stems supporting the leaves and flowers and a less developed rhizome. They grow a large terminal inflorescence with many flowers. Species belonging to the acaulescent group are characterised by underground rhizomes that produce shoots with basal leaves, leafless flower stems with leaf-like bracts and rather few flowers per stem (McLewin and Mathew, 1995). H. thibetanus can be considered as acaulescent by its basic appearance, but the rhizome and the roots are atypical for this group because the inflorescence appears before the true leaves start to expand (McLewin and Mathew, 1995), and it is the only hellebore with hypogeal germination (McLewin and Mathew, 1999). One characteristic shared by H. thibetanus and H. vesicarius is that, in the early summer months, the plant goes dormant and disappears during summer (McLewin and Mathew, 1999). H. niger represents an intermediary between the caulescent and acaulescent Helleborus (Fig. 1). Its acaulescent traits include basal leaves and leafless flower stems, but it possesses undivided bracts and often produces only one flower per stalk and hybridises easily with the caulescent species H. argutifolius and H. lividus but not with the acaulescent (McLewin and Mathew, 1995). The species H. vesicarius does not fit into any of these groups. Although its leaf and stem structures are typical for the acaulescent Helleborus (Fig. 1) (McLewin and Mathew, 1999), its thick inflorescence stalks and pollen morphology show great similarity to the caulescent group (Nowicke and Skvarla, 1983).
The classification of the Helleborus species into two groups seemed useful for horticultural purposes (McLewin and Mathew, 1995), but phylogenetically insufficient for the abovementioned reasons. Therefore, Mathew (1989) suggested a classification dividing the genus into six sections (Table 1) according to plant structure, the ability to hybridise, pollen morphology and seed characteristics. Werner and Ebel (1994) suggested the division of Helleborus into two subgenera, Helleborus and Helleborastrum, according to the species hypsophylls, with sections Griphopus, Chenopus and Helleborus belonging to the subgenus Helleborus and Syncarpus, Dicarpon and Helleborastrum to the subgenus Helleborastrum. Furthermore, two new Italian Helleborus species, H. abruzzicus and H. liguricus, have been described recently (Thomsen, 2008), and no molecular genetic data describing their relationship to other species has been reported thus far, so that to date, 22 species can be listed (Table 1). Out of these, H. niger and H. × hybridus, a group of hybrids that have H. orientalis as one parent in common, have attained the highest commercial interest as demonstrated by increasing market shares. Some traits of other species, for example flower colour, growth type, scent and disease tolerance, are valuable for the horticultural improvement of hellebores and could be introduced by interspecific hybridisation. To utilize this breeding tool, it is necessary to obtain additional information on genetic relationships within this genus.
To clarify relationships within the Ranunculaceae and within the genus itself, cytological studies of some representatives within the genus were carried out (Gregory, 1941, D’Amato and Bianchi, 1989, Yuan and Yang, 2006). These investigations revealed that all studied species have the same chromosome number of 2n = 32 (Gregory, 1941, D’Amato and Bianchi, 1989, Yuan and Yang, 2006, Bennett and Smith, 1976, Dobes et al., 1997, Castro and Rosselló, 2007). In another attempt to evaluate Helleborus taxonomy, the nuclear DNA contents of the Helleborus species were estimated (Zonneveld, 2001). The genome size (C-value) is known to correlate with the geographical distribution of plants (Bennett, 1976) and can be useful for the examination of phylogenetic dimensions (Bennett et al., 2000) and the taxonomic classification of species or the relationships between those in narrow taxonomic groups (Ohri, 1998). Therefore, it is a valuable method for the analysis or verification of plant taxonomy. Zonneveld's results were in agreement with the genus division recommended by Mathew (1989) and Werner and Ebel (1994).
The first phylogenetic evaluation of Helleborus on the basis of the chloroplast (trnL-F and matK) and ribosomal ITS DNA sequence data was done by Sun et al. (2001), which supported the genus’ division into sections, but did not resolve the problematic nature of the section Helleborastrum. Close relationships between species, and intraspecific morphological variation (Servettaz et al., 1988), make it difficult to differentiate and estimate the relationship between them. An initial taxonomic differentiation of four species and eight populations from Italy was done by Fico et al. (2005) based on RAPD markers (randomly amplified polymorphic DNA), which revealed a clear discrimination between the species and populations of H. bocconei, H. niger, H. odorus and H. viridis.
Due to the contradictory results regarding the taxonomic classification of the genus Helleborus and the discovery of new species, in this study, we set out to gather novel information on genetic relationships within the genus by combining different analyses with molecular marker data. The objectives of this study were (i) to confirm the chromosome numbers of representative species of all sections, (ii) to confirm the nuclear DNA content of 18 species and three hybrids and determine this nuclear DNA content for three new species and one hybrid and (iii) to evaluate genetic relationships within the genus Helleborus by the use of the multilocus AFLP technique (Vos et al., 1995). The integrative approach of this study is the basis for the establishment of future breeding strategies.
Section snippets
Plant materials
In total, 21 out of 22 described Helleborus species, 11 genotypes of H. niger, 11 genotypes of H. orientalis/H. × hybridus and four Helleborus hybrids were used in this study (Table 1). At the beginning of the study, no plant material of H. occidentalis, formerly regarded as subspecies of H. viridis, was available; therefore, it was not integrated. Plants of most of these species were obtained by the company Heuger, Glandorf, Germany. In addition, leaf material of six species was kindly provided
Cytological analysis
Chromosomes were counted for the representative species of all six Helleborus sections, except for the section Syncarpus, because no mitotically active root tips were available for H. vesicarius (Fig. 2). All other species of the monospecific sections, namely H. foetidus, H. niger and H. thibetanus, and H. argutifolius and H. lividus in the section Chenopus, were analysed. For the Helleborastrum section, chromosomes were counted for H. atrorubens, H. cyclophyllus, H. dumetorum, H. × hybridus, H.
One common chromosome number for all Helleborus species
Chromosome numbers are a dynamic characteristic in eukaryotic evolution. Therefore, the analysis of chromosomes by counting and karyotyping offer the opportunity to investigate plant families, genera and species for phylogenetic and breeding purposes. All species examined in this study had a chromosome number of 2n = 32, which was in agreement with data previously described in the literature (Gregory, 1941, D’Amato and Bianchi, 1989, Yuan and Yang, 2006, Bennett and Smith, 1976, Dobes et al., 1997
Acknowledgements
The authors thank Gartenbaubetrieb Josef Heuger, Glandorf, Germany and Will McLewin for supporting this work with plant and leaf material of certain Helleborus species and for useful discussion. We thank Dr. Joerg Fuchs for providing the internal reference standards for flow cytometric analysis. This work was supported by the German Federal Ministry of Food, Agriculture and Consumer Protection administrated by the Federal Agency for Agriculture and Food [grant number 28-1-41.035-06].
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