Evolution of alkaloid biosynthesis in the genus Narcissus Strahil Berkov a,b,⇑ , Vanessa Martínez-Francés c , Jaume Bastida d , Carles Codina d , Segundo Ríos c a Agrobioinstitute, 8 Dragan Tzankov, Sofia 1164, Bulgaria b Institute of Biodiversity and Ecosystem Research at the Bulgarian Academy of Sciences, 23 Acad. G. Bonchev str, Sofia 1113, Bulgaria c Estación Biológica-Jardín Botánico Torretes, Instituto Universitario de Biodiversidad CIBIO, Universidad de Alicante, Alicante, Spain d Departament de Productes Naturals, Facultat de Farmàcia, Universitat de Barcelona, Avda. Juan XXIII s/n, 08028 Barcelona, Spain article info Article history: Received 1 June 2013 Received in revised form 30 October 2013 Available online xxxx Keywords: Endemic Narcissus plants Amaryllidaceae alkaloids Biogeography Evolution Phylogeny abstract In an attempt to reveal the relationships between alkaloid biosynthesis and phylogeny, we investigated by GC–MS the alkaloid patterns of 22 species and 3 hybrids (from 45 locations) from seven main sections of the genus Narcissus (Amaryllidaceae). The results indicate that the first alkaloids to evolve in the genus Narcissus were of the lycorine- and homolycorine-type. The alkaloid pattern of the Nevadensis section supports its recent separation from the Pseudonarcissus section. The plants of Narcissus pallidulus (Ganymedes section) show a predominance of Sceletium-type compounds, which are quite rare in the Amaryllidaceae family. Two successful evolutionary strategies involving alkaloid biosynthesis and leading to an expansion in taxa and occupied area were determined. Firstly, a diversification of alkaloid patterns and a high alkaloid concentration in the organs of the large Narcissus species (in the Pseudonarcissus section) resulted in an improved chemical defence in diverse habitats. Secondly, both plant size and alkaloid biosynthesis were reduced (in the Bulbocodium and Apodanthi sections) relegated to dry pastures and rocky places. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The Amaryllidaceae constitute a cosmopolitan, mainly pantrop- ical family (Meerow et al., 1999), which has received different systematic treatments throughout botanical history. According to Fay and Chase (1996), the Amaryllidaceae form a monophyletic group (based on molecular data) with the Agapanthaceae and Alliaceae. Meerow et al. (1999) and Meerow and Snijman (2006), however, maintain the independent status of Amaryllidaceae based on morphological features and also due to their exclusive alkaloid biosynthesis. The early diversification of the main Eurasian Clade of the Amaryllidaceae (including the genus Narcissus) was most likely centered in North Africa and the Iberian peninsula (Meerow et al., 2006; Gage et al., 2011), but the greatest diversity of Narcis- sus species is found in the Iberian peninsula (Fernandes, 1968a), where 90% of all species are present. Allopatric derivation from North African species and diversification at the specific and sub- specific level most probably occurred during the last full glacial period (Meerow et al., 2006). The genus Narcissus comprises about 50–180 species according to different authors (Fernandes, 1968a; Web, 1978; Blanchard, 1996; Rivera et al., 2006), with many interspecific hybrids, poly- ploid forms (Fernandes, 1951, 1967, 1968b), and wide geographic dispersion in various microhabitats, which explains its enormous morphological variability. The habitats of the Narcissus species range from lowlands to mountain sites, mainly in restricted fragile areas, including grass- land, shrubland, woods, river banks, and rocky crevices (Rivera et al., 2006; Martínez-Francés et al., 2009; Ríos et al., 2010). The phylogeny of Narcissus has not been well established, although some approaches, like those of Graham and Barrett (2004) and RØnsted et al. (2008), combining analysis of trnL-F and ndhF DNA sequence data, and Zonneveld (2008), using the genome size, have shown that the basal clade corresponds to the subgenus Hermione (Haw.) Spach. This subgenus includes the sections Serotini, Aurelia and Tazettae with a basal chromosome number of n = 5. In the opposite clade, the most heterogeneous subgenus Narcissus appears with a basal chromosome number of n =7(Graham and Barrett, 2004; RØnsted et al., 2008; Zonneveld, 2008; Santos-Gally et al., 2011). The classic section Pseudonarcissus appears to consist of two separate groups. The first group consists of Narcissus segurensis, Narcissus yepesii, Narcissus alcaracensis, Narcissus nevadensis and Narcissus longispathus. Since the description of the three species in the Segura–Alcaraz Mountain (N. segurensis, N. yepesii, N. alca- racensis) by Ríos et al. (1999), the group has been a subject of controversy. Medrano and Herrera (2008) regard it as a synonym 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.11.002 ⇑ Corresponding author at: Institute of Biodiversity and Ecosystem Research at the Bulgarian Academy of Sciences, 23 Acad. G. Bonchev str, Sofia 1113, Bulgaria. Tel.: +359 89 7063311; fax: +359 2 9635408. E-mail address: berkov_str@yahoo.com (S. Berkov). Phytochemistry xxx (2014) xxx–xxx Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Please cite this article in press as: Berkov, S., et al. Evolution of alkaloid biosynthesis in the genus Narcissus. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2013.11.002