Evolutionary Relationships in Tribe Lycieae (Solanaceae)

We examine evolutionary relationships among the three genera in tribe Lycieae using DNA sequence data from the nuclear granule-bound starch synthase gene (GBSSI, waxy). Tribe Lycieae is comprised of the large cosmopolitan genus Lycium and the predominately South American genera Grabowskia and Phrodus. Phylogenetic results strongly suggest that Lycium contains Grabowskia and may also include Phrodus. Further, we examine relationships among several clades of American Lycium and within the monophyletic Old World lineage. This study has the largest taxon sampling to date for tribe Lycieae, with 85% of the named species. Increased sampling within eastern Asia and South America, with the world’s highest species-richness of Lycium, as well as the addition of more rapidly evolving genetic markers, are the areas on which to focus future work. INTRODUCTION Tribe Lycieae Hunz. is one of the largest tribes within Solanaceae (ca. 87 species) and includes ca. 83 species in the large genus Lycium, Grabowskia (3–4 species), and monotypic Phrodus microphyllus (Hunziker, 1977; D’Arcy, 1991; Hunziker, 2001). Phrodus microphyllus and the Grabowskia species have limited geographic distributions. Phrodus microphyllus is endemic to the Atacama and Coquimbo regions of Chile, and Grabowskia species are restricted to South America including the Galapagos Islands, although one wide-ranging species (G. boerhaviaefolia) occurs in both South America and Mexico. In contrast, Lycium is one of only three cosmopolitan genera in Solanaceae, along with Solanum and Physalis (Hunziker, 2001). Lycium is most species-rich in southern South America, southern Africa and southwestern North America. In addition, a number of species are distributed in the Mediterranean and across Asia, and one species is apparently native to Australia. Sister to tribe Lycieae is tribe Nolaneae (Olmstead et al., 1999; Levin and Miller, 2005), which includes only the genus Nolana (ca. 83 species; D’Arcy, 1991; Tago-Nakazawa and Dillon, 1999), endemic to Chile and Peru, with one species on the Galapagos Islands. Species in tribe Lycieae are uniformly woody shrubs that generally inhabit arid to semi-arid environments. Hunziker (2001) characterized members of Lycieae by developing buds with imbricate corolla aestivation, and fruits that are usually berries or, alternatively, drupaceous having 2–4 seeds. Fruit type has served as the distinguishing character among the three genera, varying from fleshy fruits with two “pyrenes” of 1–2 seeds each (i.e., each seed or pair of seeds is surrounded by a stony layer) in Grabowskia to mucilaginous, multi-seeded berries with two apical sclerifications in Phrodus microphyllus (Bernardello and Hunziker, 1987; Hunziker and Bernardello, 1995; Bernardello and Chiang-Cabrera, 1998; Hunziker, 2001) to true berries in most Lycium. 225 VI International Solanaceae Conference Eds.: D.M. Spooner et al. Acta Hort. 745, ISHS 2007 The majority of Lycium tend to produce red, yellow, or black, fleshy, multi-seeded (>10 seeds) berries, although four American species (L. athium, L. minimum, L. ameghinoi and L. californicum) have drupaceous fruits containing two seeds with each seed surrounded by a stony layer (Bernardello, 1986; Miller, 2002). Five other North American Lycium species (L. cooperi, L. macrodon, L. puberulum, L. shockleyi and L. schaffneri) have modified berries that have partial sclerification and a reduced seed number (Chiang-Cabrera, 1981; Miller, 2002). The worldwide distribution of Lycium poses questions regarding its biogeographical history. Recently, Levin and Miller (2005) have shown that the Old World Lycium species are monophyletic and nested within an American group of Lycium species. Among the New World Lycium, the North and South American species are not in distinct clades; thus, geography is not a good predictor of phylogenetic relationships. Given that Grabowskia, Phrodus and Nolana are all endemic to the Americas, it is likely that Lycium arose in the Americas and then dispersed to the Old World. Further, Lycium probably arose in South America, given that its closest relatives, Nolana, Sclerophylax and Jaborosa, are all endemic to South America (Di Fulvio, 1961; Tago-Nakazawa and Dillon, 1999; Hunziker, 2001). Within Lycieae, previous work (Miller, 2002; Levin and Miller, 2005) suggests that generic definitions require reconsideration. There is clear evidence that a small group of North American Lycium is more closely related to a monophyletic Grabowskia than to the other species of Lycium. Interestingly, most of the species in this small group of Lycium species share a hardened fruit and glaucous leaves, characteristics that are more similar to Grabowskia species than to other species of Lycium. Further, the placement of Phrodus is equivocal in Levin and Miller (2005); it may be nested within Lycium or sister to the rest of the tribe. In the present study our goals center around the addition of sequence data for the nuclear GBSSI region and increased taxon sampling, mainly from South America and southern Africa, in order to 1) determine the placement of Phrodus microphyllus relative to other members of Lycieae, 2) evaluate species circumscriptions within Grabowskia, 3) better understand relationships among the various clades of New World Lycium species, and 4) examine relationships within Old World Lycium, especially the placement of L. australe, the only species of Lycium native to Australia. MATERIALS AND METHODS Taxon Sampling Included in this study are 68 species of Lycium (85% of the genus), including 16 North American species (one species ranges into the Pacific islands), 20 South American species, all 27 African species (including a species from the Canary Islands), four Asian and one Australian species. Multiple accessions including named varieties of the closely related South American L. chilense and L. ciliatum were included, as were several accessions of the North American species L. californicum, which has both monomorphic and dimorphic populations (Yeung et al., 2005; Miller and Levin, unpubl. data). We have also sampled the other genera in tribe Lycieae, including three species of Grabowskia (six total accessions sampled across the wide geographic range of G. boerhaviaefolia) and monotypic Phrodus microphyllus. Also included are two Nolana species, the sister group to Lycieae (Levin and Miller, 2005), as well as representatives of other genera closely related to Lycieae, including Sclerophylax and Jaborosa (Olmstead et al., 1999; R. Olmstead and L. Bohs, pers. commun.). All 89 taxa with voucher information and GenBank accession numbers are in Table 1. DNA Extraction, Amplification and Sequencing Total genomic DNA was extracted from fresh or silica gel-dried leaf material following Miller (2002) and Levin et al. (2004). We amplified exons 2 through 10 (ca. 1800 bp) of the nuclear GBSSI (waxy) gene