A Chloroplast DNA Phylogeny of Solanum Section Lasiocarpa

Solanum section Lasiocarpa includes about a dozen species with a center of diversity in theNewWorld tropics. Solanum lasiocarpum and S. repandum (sometimes considered to be conspeciŽc as S. ferox) have an Old World distribution in Asia and the PaciŽc Islands. Several species in this section produce edible fruits, and two, the lulo or naranjilla (S. quitoense) and the cocona (S. sessiliorum) are cultivated commercially. Phylogenetic relationships in Solanum section Lasiocarpa were investigated using sequence data from the chloroplast trnT-trnL spacer, the trnL-trnF spacer, and the trnL gene, including the trnL intron. Sampling included 24 accessions from section Lasiocarpa and 14 accesssions of other Solanum species as outgroups. All species considered to belong to section Lasiocarpa by previous authors were examined with the exception of the recently described S. atheniae. Solanum robustum and S. stagnale, sometimes considered to belong to section Lasiocarpa, are excluded from the group on the basis of the trn data. The remaining species in the section form a monophyletic group, with three well-supported clades within it: S. hirtum, S. pectinatum-sessiliorum-stramonifolium, and the remainder of the species in the section. Sequences of S. lasiocarpum and S. repandum are extremely similar, and these two Asian taxa cluster with the New World S. candidum and S. pseudolulo on the trn trees. Solanum section Lasiocarpa (Dunal) D’Arcy comprises approximately a dozen species of perennial shrubs or small trees with a center of distribution in northwestern South America. Morphological characters that deŽne the section include difoliate sympodial units, large repand leaves, unbranched inorescences, stellate corollas, and fruits covered with stellate hairs with reduced lateral rays (Whalen et al. 1981). The section was monographed by Whalen et al. (1981), who recognized 13 species. Eleven are native to the northern Andes of Venezuela, Colombia, Ecuador, and Peru, and three have ranges that extend into Central America (S. candidum, S. hirtum) or northeastern South America through the Guianas into northern Brazil (S. stramonifolium). Several species in the section produce edible fruits and two, S. quitoense (the lulo or naranjilla) and S. sessiliorum (the cocona) are economically important fruit crops in Latin America (Heiser 1969, 1985a). Solanum quitoense has been introduced to Panama, Costa Rica, and Guatemala and is now naturalized in Central America. Solanum lasiocarpum and S. repandum are found in Asia and the PaciŽc Islands. Although treated as separate taxa by Whalen et al. (1981), Heiser (1996) considered them conspeciŽc under the name S. ferox. Dunal (1852), Morton (1976), and Hunziker (2001) included the South American S. robustum in section Lasiocarpa, but Whalen et al. (1981) excluded it from the section due to differences in branching pattern, leaf shape, and fruit trichomes. Subsequent to Whalen et al.’s (1981) treatment, S. stagnalewas removed from the section and placed in the S. polytrichum group within Solanum subgenus Leptostemonum (Dunal) Bitter (Whalen 1984; Child 1998; Nee 1999). Symon (1985) described S. atheniae from New Guinea and postulated that it belonged to section Lasiocarpa. Solanum section Lasiocarpa belongs to the spiny subgroup of the genus Solanum, usually recognized as Solanum subgenus Leptostemonum. Previous authors such as Dunal (1852), Seithe (1962), Danert (1970), D’Arcy (1972), and Whalen (1984) have regarded subgenus Leptostemonum as a natural group based on the shared presence in most species of spines, stellate hairs, and tapered anthers. Molecular phylogenetic studies based on chloroplast DNA restriction sites (Olmstead and Palmer, 1997) and nuclear and chloroplast sequence data (Bohs and Olmstead 1997, 1999, 2001; Bohs, in press) indicate that Solanum species that bear spines as well as stellate hairs comprise a monophyletic group, termed the Leptostemonum clade by Bohs (in press). Solanum wendlandii, a representative of Solanum section Aculeigerum Seithe, falls outside the clade comprised of the other spiny Solanum taxa (Bohs andOlmstead 1997, 1999, 2001; Bohs, in press). Solanum sectionAculeigerum includes six species that bear spines but lack stellate hairs. In this paper, Solanum subgenus Leptostemonum is used in the traditional sense to refer to all taxa of the genus that bear spines. The term Leptostemonum clade is used in accordance with Bohs (in press) to refer to the monophyletic group of spiny Solanum taxa exclusive of Solanum section Aculeigerum. Molecular studies based on chloroplast DNArestriction sites and chloroplast ndhF sequence data using a broad range of sampling from Solanum indicate that section Lasiocarpa may be a relatively basal lineage within the Leptostemonum clade and that it may be sister to Solanum sectionAcanthophoraDunal (Olmstead and Palmer 1997; Bohs, in press). Whereas these broad scale studies sampled only one to two species from the section, species-level relationships in section Lasiocarpa have been the subject of numerous investigations using morphological data, crossing studies, isozyme electrophoresis, karyotype analyses, and cpDNA restriction 178 [Volume 29 SYSTEMATIC BOTANY TABLE 1. Sources of Solanum DNA accessions used in this study. Seeds, leaves, or DNA extracts provided by 1 L. Bohs, University of Utah, Salt Lake City, UT. 2 R. G. Olmstead, University of Washington, Seattle, WA. 3 A. Bruneau, McGill University, Montreal, Canada. 4 C. B. Heiser, Jr., Indiana University, Bloomington, IN. 5 J. Miller, Amherst College, Amherst, MA. a For further collection and voucher data see Appendix in Whalen et al. (1981). BIRM samples bear the seed accession number of the University of BirminghamSolanaceae collection. Nijmegen accession numbers refer to the Solanaceae collection at the University of Nijmegen, The Netherlands. Solanum section Lasiocarpa: S. candidum Lindl.3—Stoutamire s.n. (IND) from Heiser S249a, Mexico: Veracruz (AY266250). S. candidum Lindl.1—Bohs 2898 (UT), Costa Rica: La Cangreja (AY266237). S. felinum Whalen4—Benitez de Rojas 8915 (IND), Venezuela: Colonia Tovar (AY266252). S. hirtum Vahl3—Whalen 730 (QCA), Ecuador (AY266254). S. hirtum Vahl3—Jones s.n. (IND) from Heiser S404a, Costa Rica: Guanacaste (AY266253). S. hyporhodium A. Braun & Bouché3—Whalen 717 (BH), Venezuela: Sucre (AY266238). S. hyporhodiumA. Braun & Bouché4—Carreno Espinosa 8214 (IND), Venezuela: Sucre (AY266255). S. lasiocarpum Dunal4—Ansyar 9605 (IND), Indonesia: Pandang (AY266256). S. pectinatum Dunal4—Peeke 8512 (IND), Ecuador: Limoncocha (AY266227). S. pectinatum Dunal1—Bohs 2899 (UT), Bolivia: Santa Cruz (AY266230). S. pseudolulo Heiser3—Plowman et al. 4276 (GH)a, Colombia: Meta, Sierra de la Macarena (AY266258). S. pseudolulo Heiser5—Bohs DNA extract 995, Nijmegen #824750021 (AY266242). S. quitoense Lam.1—Bohs 2873 (UT), Costa Rica (AY266228). S. quitoense Lam.4—Heiser s.n. Bohs DNA extract 996, Ecuador: Quito market (AY266243). S. repandum G. Forst.3—Heiser 8215 (IND), Fiji (AY266229). S. repandum G. Forst.4—Ashley 8627 (IND), Solomon Islands: Malaita (AY266234). S. sessiliorum Dunal3— Dickson 458 (BH) from Whalen 859 (HUT), Peru (AY266261). S. sessiliorum Dunal var. sessiliorum4—Heiser 8255 (IND), Ecuador: Yanzatza (AY266260). S. stramonifolium Jacq. var. inerme (Dunal) Whalen4—Pickersgill 154 (IND), Peru: Iquitos (AY266244). S. stramonifolium Jacq. var. inerme (Dunal) Whalen3—Whalen & Salick 860 (BH), Peru: Pasco, Iscozacin (AY266263). S. vestissimum Dunal3—Dickson 456 (BH) from Plowman 13431 (F), Venezuela (AY266264). S. vestissimum Dunal4—Movilla s.n. (IND) from Heiser S432a, Colombia: Santa Marta (AY266247). Outgroups: S. abutiloides (Griseb.) Bitter & Lillo2—RGO S-73 (WTU), BIRM S.0655 (AY266236). S. acerifolium Dunal1—Bohs 2714 (UT), Costa Rica (AY266249). S. capsicoides All.1—Bohs 2451 (UT), Peru (AY266251). S. dulcamara L.2—no voucher, USA (AY266231). S. jamaicense Mill.2—RGO S-85 (WTU), BIRM S.1209 (AY266239). S. luteoalbum Pers.1—Bohs 2337 (UT), BIRM S.0042 (AY266257). S. mammosum L.2—RGO S-89 (WTU), BIRM S.0983 (AY266232). S. melongena L.2—RGO S-91 (WTU), BIRM S.0657 (AY266240). S. palinacanthum Dunal1—Bohs 3151 (UT), Bolivia (AY266233). S. pseudocapsicum L.2— no voucher, BIRM S.0870 (AY266241). S. robustum Wendl.4—Bohs 3084 (UT), Argentina: Corrientes, Perichón (AY266259). S. sisymbriifolium Lam.1—Bohs 2533 (UT), Argentina (AY266235). S. stagnale Moric.4—Carvalho 3213 (IND), Brazil: Bahia, Valença (AY266262). S. tenuispinum Rusby1—Bohs 2475 (UT), Bolivia (AY266245). S. torvum Sw.1—RGO S-101 (WTU), BIRM S.0839 (AY266246). S. wendlandii Hook. f.1—no voucher, BIRM S.0488 (AY266248). site data (Heiser 1972, 1985b, 1987, 1989; Whalen et al. 1981;Whalen and Caruso 1983; Bernardello et al. 1994; Bruneau et al. 1995).Many of these studies were aimed at examining the evolutionary history of the Asian disjuncts and the origin and evolution of S. quitoense. Despite the accumulation of an impressive amount of data, a consensus has not been reached regarding the phylogenetic relationships of the taxa of this group due to conicting topologies from different data sets and to low resolution in some parts of the trees. Evidence suggests that the Asian species S. repandum and S. lasiocarpum are sister taxa (Heiser 1986, 1987; Bernardello et al. 1994; Bruneau et al. 1995) or even conspeciŽc (as S. ferox; Heiser 1996), but the closest relatives of this clade are debated. The inclusion of S. stagnale, S. robustum, and S. atheniae in section Lasiocarpa has not been critically examined and the data have been inconclusive with respect to the wild relatives of the putative domesticates S. quitoense and S. sessiliorum. The present study examines species-level phylogenetic relationships in Solanum section Lasiocarpa using chloroplast trn sequence data. These data shed light on the circumscription of the section, the relationships of the Asian taxa, and the wild relatives of the lulo and cocona, and demonstrate the utility of trn sequence data for examining species-level phylogeny within Solanum. MATERIALS AND METHODS All species placed in section Lasiocarpa by Whalen et al. (1981) were sampled, including S. stagnale and S. robustum. Solanum atheniae is known only from the type (Symon 1985) and no material was available for sampling. In most cases, two accessions were sampled from each species of section Lasiocarpa. Outgroup taxa included ten species from Solanum subgenus Leptostemonum and four species representing taxa from various non-spiny Solanum clades. Outgroups were chosen to represent a variety of diverse Solanum clades based on previous molecular studies. In addition, sampling included Žve representatives fromSolanum sectionAcanthophora, which was identiŽed as the sister group to section Lasiocarpa in previous analyses based on chloroplast DNA data (Olmstead and Palmer 1997; Bohs, in press). Collection, voucher, and GenBank information is given in Table 1. DNAwas extracted from fresh or silica dried leaf material using protocols described in Bohs and Olmstead (1997, 2001) and Bohs (in press). AmpliŽcation of the entire trnT (UGU)—trnF (GAA) region used primers a and f of Taberlet et al. (1991) in 25 ml reactions as described in Bohs and Olmstead (2001) with a PCR program of 928 C for 7 min followed by 30 cycles of 928 C for 1 min, 458 C for 1 min, 728 C for 5 min, and a single cycle of 728 C for 7 min. PCR products were cleaned using QiaQuick spin columns (Qiagen, Inc., Valencia, CA) and sequenced on an ABI automated sequencer using primers a through f of Taberlet et al. (1991). Sequence data were edited and contigs constructed using Sequencher (Gene Codes Corp.) and sequences were aligned by eye using Se-Al (Rambaut 1996). Indel alignments took into account the mechanisms and patterns of evolution in non-coding sequences outlined in Kelchner (2000). All sequences were submitted to GenBank (Table 1) and the data sets and representative trees are 2004] 179 BOHS: PHYLOGENY OF SOLANUM SECTION LASIOCARPA deposited in TreeBASE [accession numbers S907 (study) and M1490 (matrix)]. The trn region sampled here includes two coding regions (trnL 59 and 39 exons), two intergenic spacers (trnT–trnL and trnL–trnF spacers), and the trnL intron (for diagrams and sequences of this region in tobacco, see Yamada et al. 1986). To explore the informativeness of each of these regions in the context of Lasiocarpa phylogeny, each of the non-coding regions was analyzed separately using parsimony and the results were compared with those from the complete data sets. To explore the effects of indels and indel coding on the phylogenetic results, several analyses were performed on the aligned data set. The Žrst used the complete aligned nucleotide sequence data set, with gaps treated as missing data. The second excluded indels from the sequence data matrix. For subsequent analyses, 32 phylogenetically informative gap characters (i.e., those shared by two or more taxa) whose homology could be conŽdently assessed were coded as separate presence/absence characters according to the simple indel coding scheme of Simmons and Ochoterena (2000). The third analysis used the nucleotide sequence data with indels excluded and the 32 presence/absence gap characters added. The fourth analysis used the complete aligned sequence data, including indel regions, with the addition of the 32 presence/absence gap characters. Parsimony analyses were conducted with PAUP* 4.0b10 (Swofford 2002) using the heuristic search algorithm with the TBR, MulTrees, and Steepest Descent options, equal weights for all characters and character state changes, and 500 random-order entry replicates. Bootstrap analyses were performed with 500 replicates using the heuristic search option, TBR and MulTrees, Maxtrees set to 1,000, and rearrangements limited to 1,000,000 per replicate. Sequence data from the ITS region were obtained froma subset of the Lasiocarpa species used in the trn study using protocols described in Bohs and Olmstead (2001). ITS sequence divergencewas extremely low among Lasiocarpa taxa and provided little phylogenetic information. These ITS sequences were deposited in GenBank (numbers AY263455—AY263467), but are not analyzed further here.