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Chemical and Morphological Study of a Putative Hybrid Between Luzuriaga Radicans and L. Polyphylla (Monocotyledoneae: Luzuriagaceae)

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Chemical and Morphological Study of a Putative Hybrid Between Luzuriaga Radicans and L. Polyphylla (Monocotyledoneae: Luzuriagaceae) TeillierNew Zealand et al.—Putative Journal of hybrid Botany, between 2008, Vol.Luzuriaga 46: 321–326 radicans and L. polyphylla 321 0028–825X/08/4603–0321 © The Royal Society of New Zealand 2008 Chemical and morphological study of a putative hybrid between Luzuriaga radicans and L. polyphylla (Monocotyledoneae: Luzuriagaceae) SEBASTIÁN TEILLIER is endemic to New Zealand; L. polyphylla (Hook.) Escuela de Arquitectura del Paisaje J.F.Macbr., which is endemic to Chile; and L. radi- Universidad Central de Chile cans Ruiz et Pav., which grows in southern Chile Santa Isabel 1186, Santiago, Chile and Argentina (Rodríguez & Marticorena 1987). Thus, this genus is a clear example of the floristic ALEJANDRO URZÚA links between distant landmasses of the Southern Facultad de Química y Biología Hemisphere. Luzuriaga polyphylla grows between Universidad de Santiago de Chile c. 37 and 45°S and L. radicans between 35 and 45°S Casilla 40, Santiago-33, Chile (Rodríguez & Marticorena 1987). Preliminary obser- HERMANN M. NIEMEYER* vations of these two species occurring sympatrically Departamento de Ciencias Ecológicas at the Valdivian temperate rainforests of Puyehue Facultad de Ciencias National Park revealed individuals that possessed Universidad de Chile some characters of one of the accepted species and Casilla 653, Santiago, Chile other characters of the other species; those individu- *Author for correspondence: niemeyer@abulafia. als also presented flowers with a scent similar to one ciencias.uchile.cl of the species (the scent of the other species is unap- parent) and anthers coloured as one of the species but not as the other. These observations suggested Abstract Patterns of headspace volatiles and the occurrence of a putative hybrid between the two flavonoids extracts from flowers of sympatric described species. populations of Luzuriaga radicans Ruiz et Pav. Flower headspace volatiles have been used to (Monocotyledoneae: Luzuriagaceae), L. polyphylla distinguish hybrids from parental species (Ishizaka (Hook.) J.F.Macbr., and a Luzuriaga taxon which et al. 2002). Flavonoid patterns have also been used presented some morphological characters of one of to compare hybrids with their parents (Bhargava et the species and other characters of the other species, al. 2005), and to distinguish between species and suggest that this latter taxon is a hybrid between the putative hybrids (Freeman et al. 1991; Wyatt & two accepted species. Hunt 1991; Heimler et al. 1993; Voirin et al. 1999; Menadue & Crowden 2000; Bayly et al. 2001), and Keywords flower headspace volatiles; flower fla- between closely related taxa (Kellow et al. 2005). vonoid patterns; hybridisation Given the differences detected in flower scent and colour of anthers, we analysed the volatiles in the headspace and the patterns of flavonoids in extracts of flowers of the sympatric Luzuriaga taxa men- INTRODUCTION tioned. The genus Luzuriaga (Monocotyledoneae: Luzur- iagaceae) is composed of four species: L. marginata Benth. & Hook.f., which grows in Chile, Argentina, and the Falkland Islands; L. parvifloraKunth, which MATERIAL AND METHODS Study area The study was carried out in the Puyehue National Park, Anticura sector, in southern Chile (40°40′S; B07056; Online publication date 29 July 2008 72°10′W; 364 m above sea level). The main forest Received 20 December 2007; accepted 2 July 2008 type is the Valdivian temperate rainforest dominated 322 New Zealand Journal of Botany, 2008, Vol. 46 by evergreen species such as Eucryphia cordifolia 25 × 0.46 cm) column. Gradient elution was done (Cunoniaceae), Aetoxicon punctatum (Aetoxicace- with a mobile phase consisting of water (solution A) ae), Nothofagus dombeyii (Nothofagaceae), and and MeOH (solution B) as follows: 0–5 min, isocrat- Laurelia philippiana (Atherospermataceae) (Muñoz ic elution 60%A/40%B; 5–30 min, linear gradient 1980). The material collected was identified by from 60%A/40%B to 40%A/60%B; and 30–45 min, Sebastián Teillier, Universidad Central de Chile. isocratic elution 40%A/60%B. Detection was ac- Voucher specimens were stored at the Herbarium complished with a Waters 2996 diode-array-detector of Universidad de Concepción (CONC). (DAD), and spectra were recorded at wavelengths between 200 and 800 nm. All compounds showed Analysis of floral scents one band between 264 and 269 nm corresponding Three flower samples of each taxon (between 40 to band I of ring A of a flavonoid, and another band and 70 g fresh weight per sample) were gathered between 341 and 362 nm corresponding to band II from many individual plants in the same locality of ring B of a flavonoid. The UV spectra and reten- and placed inside a glass jar provided with an inlet tion times of all flavonoids detected suggest they and an outlet. At the inlet, a compressed air cylin- correspond to flavonol glycosides (Mabry et al. der containing synthetic air made from extra pure 1970; Martson & Hostettmann 2006). Quantitation oxygen and nitrogen, with no detectable organic was based on peak areas in chromatograms taken at impurities, was attached through a regulator which 360 nm. The three samples collected were analysed controlled the air flow. At the outlet, a column was independently. attached which contained Porapak Q (100 mg). Vola- tile entrainment (c. 4 min per gram of sample, with Data analysis an air flow of 0.5 L/min) commenced immediately Similarity in the composition of flower headspace after the flowers had been cut from the plants. The volatiles and flower flavonoid extracts was compared columns were taken back to the laboratory and the with the Jaccard similarity coefficient, which uses volatiles adsorbed on the Porapak Q eluted with 1 ml presence-absence data, and with the Renkonen index, of dichloromethane. These extracts were analysed which uses quantitative composition data (Krebs in a GC-MS (gas chromatograph: Hewlett-Packard 1989). In the latter, mean values of composition were model HP5891; mass spectrometric detector with used. Comparisons between the same taxon should integrated data system: Hewlett-Packard model give values of 1 and 100, respectively (complete HP5972) with a SPB-5 (25 m × 0.2 mm id) capillary similarity), and comparisons between different taxa column. Ionisation by electron impact (70 eV) was should give values between 0 and 1, and between 0 carried out at 280°C. The GC oven was programmed and 100, respectively (partial similarity). to remain at 50°C for 10 min, to increase up to 280°C at a rate of 5°C/min, and then to remain at 280°C for 45 min. The identification of compounds in the RESULTS AND DISCUSSION chromatographic profiles was achieved by compari- son of their mass spectra with a library database, and Table 1 summarises and Fig. 1 illustrates some was confirmed by comparison of retention indices morphological features which differ between L. with those of authentic standards or with values polyphylla and L. radicans. The putative hybrid from the literature. shares characteristics with both accepted species, and closely resembles L. polyphylla except for the Analysis of flavonoids shape and dimensions of tepals. After we collected headspace volatiles, flower Table 2 lists the compounds identified in the samples were dried in the shade, taken back to the flower headspace volatiles. A total of 25 compounds laboratory and milled. The flower powder (between were identified, six of which occurred only in L. 4 and 10 g per sample) was extracted with 250 ml of radicans, nine only in L. polyphylla, and two only EtOH with agitation at room temperature for 24 h. in the putative hybrid. These latter might have been The extracts obtained were concentrated to dry- produced as a result of new gene combinations in ness under reduced pressure at 30°C. The residues the putative hybrid. Of the nine compounds identi- were solubilised in 10 ml MeOH, of which 20 μl fied in the putative hybrid, three were shared with were used for each HPLC analysis. Separation was L. radicans and four with L. polyphylla. The two achieved with an analytical HPLC (Waters 600), major compounds in the hybrid were also the major using a reverse-phase Symmetry (5 μm particle size; compounds in L. polyphylla. Teillier et al.—Putative hybrid between Luzuriaga radicans and L. polyphylla 323 Fig. 1 Morphological charac- teristics of leaves, flowers, and reproductive organs of Luzuriaga radicans (Lr), L. polyphylla (Lp), and their putative hybrid (hyb). Table 1 Some morphological characters of Luzuriaga radicans, L. polyphylla, and their putative hybrid, based on measurements of 50 individuals of each taxon collected at the Anticura sector of the Puyehue National Park, Chile. Character L. radicans Putative hybrid L. polyphylla Leaf shape falcate straight straight Leaf length (mm) 20–40 8–25 8–25 Leaf width (mm) 4–10 3–14 3–14 Nerves in leaf (No.) 9–13 5–7 5–7 Grouping of flowers cymes with 2–4 flowers solitary flowers solitary flowers (commonly 2) Tepal shape convex convex concave Tepal length (mm) 16–18 19–21 12–13 Tepal width (mm) 5–7 6–8 9–11 Tepal width (internal/external) >1 >1 <1 Ratio between length of tepals and stamens 3:1 2:1 2:1 Anthers attached at base versatile versatile Ratio between length of anthers and filament >1 <1 <1 Connivent anthers yes no no Colour of anthers orange creamy creamy Flower aroma strong weak unapparent 324 New Zealand Journal of Botany, 2008, Vol. 46 Table 2 Volatile compounds entrained from the headspace of flowers ofLuzuriaga radicans, L. polyphylla, and their putative hybrid. GC area (%)2 Compound Retention index1 L. radicans
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