Phylogenetic Relationships in Solanum Section Androceras (Solanaceae)

The Leptostemonum clade of Solanum contains approximately 350–450 species, including the cultivated eggplant, S. melongena . This clade is characterized by the presence of prickles and apically attenuate anthers. Solanum section Androceras , the focus of this study, is a group of ca. 12 species belonging to the Leptostemonum clade. This section is unusual in the genus because of its mostly north temperate distribution and distinctive zygomorphic, heterantherous, and enantiostylous flowers. We infer phylogenetic relationships among 43 Solanum taxa, including 11 species and all varieties of sect. Androceras , using DNA sequence data from two nuclear regions (ITS and the granule-bound starch synthase gene [GBSSI or waxy ]) and the chloroplast region trnT-F . The combined phylogenetic tree supports sect. Androceras as a monophyletic group sister to Solanum sect. Crinitum . Only one of the three series proposed by previous taxonomists, ser. Pacificum , is supported as monophyletic. Solanum tenuipes from the northern Chihuahua Desert is sister to the remaining species in sect. Androceras . Species-level relationships were also examined and it was found that two species, S. heterodoxum and S. citrullifolium, are not monophyletic. The ancestral flower color in sect. Androceras appears to be violet, with white and yellow flowers restricted to more derived clades. Characters formerly used to diagnose ser. Androceras , such as exclusively branched hairs and lack of complex foliar flavonoids, appear to have evolved more than once in the section. Keywords— enantiostyly , heteranthery , ITS , Mexico , trnT-F, waxy. 886 SYSTEMATIC BOTANY [Volume 35 by Whalen (1979a) , such as the presence of methoxylated aglycones, 8-hydroxyflavonoids and various flavones in sers. Violaceiflorum and Pacificum that are absent in ser. Androceras. The major chemical differences between sers. Violaceiflorum and Pacificum are flavones with chrysoeriol type B-rings in ser. Pacificum and the presence of 8-oxygenated flavonols in ser. Violaceiflorum ( Whalen 1978a ). Although Whalen (1979a) revised sect. Androceras and included a cladistic analysis based on 14 morphological and chemical traits, to date there have been limited molecular phylogenetic studies of this section. Two species of sect. Androceras , S. rostratum and S. citrullifolium , were included in molecular phylogenies of the entire Leptostemonum clade and were strongly supported as sister taxa ( Levin et al. 2006 ; Bohs et al. 2007 ). These studies place sect. Androceras sister to sect. Crinitum Child with moderate support (84% bootstrap and 1.0 posterior probability in Levin et al. 2006 ). This relationship had not previously been proposed due to the fact that sect. Crinitum is a South American group of large shrubs and trees with fruits that may reach 10 cm in diameter and large flowers that are not heterantherous. A close relationship between sect. Androceras and S. sisymbriifolium of sect. Cryptocarpum Dunal has been proposed in the past due to their similar leaves, inflorescences, and accrescent calyces ( Dunal 1813 , 1852 ; Walpers 1844 ; Danert 1970 ; Whalen 1979a ; Lester et al. 1999 ). Both Weese and Bohs (2007) and Bohs et al. (2007) have found that S. sisymbriifolium is sister to a clade composed of sect. Androceras and sect. Crinitum . Whalen (1979a) favored sect. Nycterium (Venten.) Walp. as the sister group to sect. Androceras based on morphological similarities, but molecular studies unequivocally place the members of sect. Nycterium quite distant from sect. Androceras ( Levin et al. 2006 ; Bohs et al. 2007 ; Weese and Bohs 2007 ). While these studies provide hypotheses about relationships between sect. Androceras and other Solanum sections, they did not extensively sample from within the section. In this paper we use molecular phylogenetic methods to 1) test the monophyly of sect. Androceras as currently circumscribed, 2) examine the phylogenetic relationships of sect. Androceras with closely related members of the Leptostemonum clade, 3) test the monophyly of Whalen’s (1979a) series and species within sect. Androceras , and 4) examine selected species-level relationships to test hypotheses of character evolution and speciation proposed by Whalen (1979a) . Materials and Methods Taxon Sampling— Eleven of the 12 species and all 10 varieties in sect. Androceras sensu Whalen (1979a) were sampled for this study ( Table 1 ). We were unable to obtain high quality genomic DNA for Solanum leucandrum , which is known only from the type locality in Puebla, Mexico, due to a lack of available herbarium material. Specimens were determined using keys found in Whalen (1979a) , with almost half of the specimens determined by the late Michael D. Whalen himself (indicated with asterisks in Appendix 1). We also included six members of sect. Crinitum as well as S. sisymbriifolium , both shown by previous molecular studies to be closely related to sect. Androceras ( Levin et al. 2006 ; Bohs et al. 2007 ). Five other more distantly related species from the Acanthophora and Bahamense clades of the Leptostemonum clade were included to ensure sufficient outgroup sampling, and the tree was rooted using S. betaceum, an even more distantly related Solanum from outside the Leptostemonum clade. The final data set included 43 accessions, representing 11 named species of sect. Androceras as well as 12 outgroup species. All taxa, along with voucher information and GenBank accession numbers, are listed in Appendix 1. DNA Extraction, Amplification, and Sequencing— Total genomic DNA was extracted from fresh, silica gel-dried, or herbarium material using the DNeasy plant mini extraction kit (Qiagen, Inc., Valencia, California). Amplification for each gene region followed standard procedures described in Taberlet et al. (1991) , Bohs and Olmstead (2001) , and Bohs (2004) for the trnT-L and trnL-F intergeneric spacer regions; Levin et al. (2005) for waxy ; and Levin et al. (2006) for ITS. The ITS region was amplified as a single fragment using primers ITSleu1 ( Bohs and Olmstead 2001 ) and ITS4 ( White et al. 1990 ) using PCR conditions described in Bohs and Olmstead (2001) . When possible, trnT-F and waxy were amplified as single fragments using primers a and f for trnT-F ( Taberlet et al. 1991 ) and primers waxyF and waxy2R for waxy ( Levin et al. 2005 ). Amplification conditions for trnT-F followed Bohs and Olmstead (2001) ; conditions for waxy followed Levin et al. (2005) . When necessary, overlapping fragments were amplified and assembled, using primers a with d, and c with f to amplify trnT-F , and primers waxyF with 1171R, and 1058F with 2R to amplify waxy . Specimens not amplifying for waxy were amplified in Table 1. Species of Solanum sect. Androceras , including the series and their distributions according to Whalen (1979a) . All taxa except S. leucandrum were sampled in this study. Solanum section Androceras (Nutt.) Marzell Geographic Distributions