The Case of Artemisia Crithmifolia L. (Asteraceae, Anthemideae)
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CARYOLOGIA Vol. 62, no. 2: 152-160, 2009 Changes in genome size in a fragmented distribution area: the case of Artemisia crithmifolia L. (Asteraceae, Anthemideae). Pellicer Jaume1, Sònia Garcia2, Teresa Garnatje2 and Joan Vallès1* 1 Laboratori de Botànica, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII, s.n., 08028 Barcelona, Catalonia, Spain. 2 Institut Botànic de Barcelona (CSIC-ICUB), Passeig del Migdia s.n., Parc de Montjuïc, 08038 Barcelona, Cata- lonia, Spain. Abstract — Artemisia crithmifolia is a hexaploid shrub which inhabits the coastal Atlantic sand dunes of Western Europe, from the Southern Iberian Peninsula to the Netherlands, reaching the British Isles. Genome size data of 45 populations of A. crithmifolia, covering its entire distribution area, were obtained using the flow cytometry method. The 2C nuclear DNA content in this species ranged from 14.27 to 15.72 pg, the mean value being 14.98 pg. A negative correlation between nuclear DNA amount and latitude has been found, and statistically significant differences between two groups resulting from the fragmentation of the distribution area were evidenced. Key words: 2C value, Compositae, dunes, flow cytometry, nuclear DNA amount. INTRODUCTION species occupies the maritime sands, principally at the back of the dunes in process of stabilization The genus Artemisia L. is one of the largest on the Northern Atlantic beaches, being part of of the Asteraceae, with more than 500 species two associations, Corynephoretum atlanticum and (OBERPRIELER et al. 2007). Different taxonomic Roseto-Ephedretum (KUHNZH O LTZ -LO RDAT 1927), rearrangements, based on morphological traits, which are closely related (VANDEN 1958). have been carried out, and five large subgenera Artemisia crithmifolia, a species from subgenus are considered at present (Absinthium DC., Ar- Dracunculus, presents capitula with glabrous re- temisia, Dracunculus Besser, Seriphidium Besser ceptacles, the outer florets female and the remain- and Tridentatae (Rydb.) McArthur). The genus ing ones functionally male, as commonly reported is widely dispersed across the Northern Hemi- in the subgenus. The panicle branches are not sphere although a few species are distributed in sticky and the leaf lobes are short, fleshy, convex the Southern Hemisphere (LING 1982; VALLÈS but not keeled beneath, according to TUTIN et al. and GARNATJE 2005). Artemisia crithmifolia L. is a hexaploid shrub (1976). This species is closely related to A. camp- distributed over the Western coast of Europe, estris L. at morphological and molecular levels from the Southern Iberian Peninsula to the North- (TO RRELL et al. 1999). Its taxonomical considera- ern Netherlands, reaching the British Isles. Two tion has been variable, as morphological features important disjunctions have been detected along have been used to describe not only the species its range, which are coincidental with the North- as an independent one, but also some subspecific ern coasts of both the Iberian Peninsula [where entities subordinated to A. campestris. For this it seems to be extinct with the exception of one reason, different synonyms related to A. crithmi- population (AED O et al. 1990)] and France. This folia can be found in the literature [e.g. A. camp- estris L. subsp. maritima Arcangeli; A. campestris L. subsp. lloydii (Rouy) Cout., A. gayana Besser., A. lloydii (Rouy) A.W. Hill]. All these taxonomic * Corresponding author: phone: +34-934024490; fax: considerations could be related to the fact that the +34-9340235879; e-mail: [email protected] species presents a certain degree of morphological genome size in artemisia crithmifolia (asteraceae) 153 variability, maybe due to different environmental parameter along the geographical distribution conditions where it grows. This taxon shows mor- range in a given species; iii) to evaluate whether phological and ecological affinities with another some morphological differences observed be- member of the A. campestris complex, A. camp- tween populations are also reflected at genome estris subsp. sericea (Fr.) Lemke et Roth., growing size level; and iv) to study possible changes of ge- in Baltic coastal sand dunes, and also with some nome size related to insularity. related Asian taxa, such as A. jordanica Danin and A. monosperma Delile. BA kk ER (1976), in his study on phytogeo- MATERIAL AND METHODS graphical aspects of the vegetation in the Atlantic province of Europe, postulated the relationship Table 1 shows the provenance of all the popu- between the presence of A. crithmifolia (cited as lations investigated with collectors, dates and A. lloydii) and other chamaephytes and the main- voucher information. In order to confirm the tenance of a warm refuge during the glacial pe- presence or not of the studied species, different riods in the Southern part of this province. This transects covering the Atlantic coast along its author rules out that this presence can be due to range of distribution were explored. In all cases, lime content in the soils. In their work on general fresh young leaves were collected for flow cyto- threats to plant species growing in the coastal hab- metric measurements. Herbarium vouchers have itats, VAN DER MAAREL & VAN DER MAAREL -VERS - been deposited in the Herbarium BCN (Centre LUYS (1996) present A. crithmifolia in an uncertain de Documentació de Biodiversitat Vegetal, Uni- status of conservation but needing attention. We versitat de Barcelona). The internal standard agree with these authors that the development of used, Petunia hybrida Vilm. ‘PxPc6’ (2C = 2.85 urban-industrial-recreational facilities, especially pg; MARIE and BR O WN 1993), was cultivated in the in dunes, has caused the loss of the extensive areas greenhouses of the Facultat de Farmàcia (Uni- where these plants used to grow. We have already versitat de Barcelona) and the Institut Botànic de experienced this fact during the sampling for the Barcelona (CSIC-ICUB). It was provided by the present work. Institut des Sciences du Végétal, Gif-sur-Yvette The DNA C-value for a species is the amount (France). of nuclear DNA in the unreplicated haploid ge- DNA content assessment - Fresh young leaves from nome of a gamete (SWIFT 1950). It seems that C- the plants studied were co-chopped using a razor value tends to be characteristic of a taxon, and blade in a plastic Petri dish with an internal stand- quite constant within a species, the C accounting ard in 600 μl of Galbraith’s isolation buffer (GAL - for constant. Since the C-value term was coined BRAITH et al. 1983) and supplemented with 100 μl/ to date, many studies concerning the relationships ml ribonuclease A (RNase A, Boehringer, Meylan, between genome size and biological and ecologi- France). A sample containing only the standard cal traits, involving aspects like polyploidy, cell was first prepared and analysed to determine its physiology, biogeographic distribution, genetic peak position. Nuclei were filtered through a 30- plasticity or breeding system, have been carried mm nylon filter in order to eliminate cell debris out (e.g., BENNETT 1972; JASIENS K I and BAZZAZ before adding 36 μl of propidium iodide (Sigma- 1995; MAC GILLI V RAY and GRI M E 1995; GRI M E Aldrich Química, Alcobendas, Madrid, Spain) in 1996; VIN O GRAD ov 2003; LEITCH and BENNETT a final concentration of 60 μg/ml. Samples were 2004; CHASE et al. 2005; PRICE et al. 2005; BEAU - kept on ice for 20 min before measurement. Five LIEU et al. 2007; GARCIA et al. 2008), suggesting individuals per population were analysed. Two that genome size does in fact have an evolutionary samples from each individual were extracted and effect or actually its size is a consequence of evolu- measured independently. Fluorescence analysis tion. Reports on infraspecific variation of genome was carried out using an Epics XL flow cytometer size have been conducted, and the idea of a rela- (Coulter Corporation, Hialeah, Florida). The total tive constancy of genome size within a species has nuclear DNA content was calculated by multiply- been evidenced (BARANYI and GREILHUBER 1996; ing the known DNA content of the standard by BENNETT et al. 2000). the quotient between the 2C peak positions of the The aims of the present work are: i) to contrib- target species and the standard in the histogram ute to the enlargement of genome size knowledge of fluorescence intensities, under the assumption in plants, especially in the Asteraceae and in the that there is a linear correlation between the fluo- genus Artemisia, on which many of our works are rescent signals from stained nuclei of the unknown focused; ii) to study the constancy of the C-value specimen, the known internal standard, and DNA 154 pellicer, garcia, garnatje and vallès content. We also calculated the mean half peak than the one calculated in the precedent work. coefficient of variation (HPCV) corresponding to In any case, the 2C values reflect the constancy ten samples. of the hexaploid level, which is the only one re- Statistical analyses - One-way ANOVA has been ported in the taxon studied (2n = 6x = 54; KA- carried out to test differences between the data- WATANI and OHN O 1964; PAST O R 1992; OLI V A and sets. We have indeed performed analyses either VALLÈS 1994; TO RRELL et al. 2001; and references considering two groups (populations growing therein). These values indicate that a certain inter- in the Northern part of the distribution area, in- populational variability exists in this species along cluding France, Belgium and the Netherlands, its distribution area, even if this intraspecific vari- and those at the Spanish-Portuguese coasts at the ation can be considered as not very high, accord- South) or three groups (those of the Iberian Pe- ing to the results reported for several plant groups ninsula, the French group and the Belgian-Dutch (DO L E ž E L and BART O š 2005; GARCIA et al. 2006; one). We have done it both ways because, al- 2008 and references therein).