An explosive innovation: Phylogenetic relationships of Solanum section Gonatotrichum (Solanaceae)

Abstract Solanum is one of the largest plant genera and exhibits a wide range of morphological diversity. Solanum section Gonatotrichum, the focus of this study, is unique within the genus because of its fruits that swell with turgor pressure and explosively dehisce to disperse the seeds. We infer phylogenetic relationships within section Gonatotrichum 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 resulting phylogenetic trees support the monophyly of the section with the inclusion of Solanum lignescens, a species not previously thought to belong to the group due to the presence of stellate hairs. This inclusion of this species in section Gonatotrichum suggests that the simple, often geniculate hairs of species in the group may represent reduced stellate hairs. The presence of heterantherous flowers appears to be derived in the section, but this character is largely lost in Solanum parcistrigosum.


Introduction
Solanum (Solanaceae), with approximately 1500 species, is one of the 10 largest fl owering plant genera (Frodin 2004;Bohs 2005). Recent species-level taxonomy (Knapp et al. 2004; http://www.solanaceaesource.org) and numerous molecular phylogenetic studies (e.g., Bohs 2005;Weese and Bohs 2007) have helped to defi ne infrageneric groups within the genus, some of which correspond to formally named subgenera or sections. Weese and Bohs (2007) recognized 12 to 15 major clades in Solanum, one of which they called the Brevantherum clade. Th is large clade includes species with short, broad anthers that lack spines but commonly have stellate hairs or lepidote scales. It encompasses members of the formally named sections Brevantherum Seithe, Extensum D'Arcy, Lepidotum Seithe, Stellatigeminatum A. Child, and Gonatotrichum Bitter. Solanum section Cernuum Carvalho & G. J. Sheph. belongs to the clade on the basis of morphological data, but no sequence data have been available to place it in a molecular phylogeny.
Solanum section Gonatotrichum is morphologically unusual within the Brevantherum clade because the species traditionally placed within it have simple rather than stellate hairs and unique fruits with explosive dehiscence (see below ;Bitter 1912;Nee 1989Nee , 1999Weese and Bohs 2007). Prior to molecular studies, its affi nities were thought to be with the Morelloid clade of the non-spiny Solanums, not with the Brevantherum clade (D'Arcy 1972(D'Arcy , 1991Nee 1999;Child and Lester 2001). Th e taxonomy of section Gonatotrichum has been poorly understood and its sectional limits have been unclear. Until recently, this section was thought to contain two species (Nee 1989). A forthcoming revision (Stern et al. in review) and this contribution recognize eight species within it: S. adscendens Sendtn., S. defl exum Greenm., S. evolvuloides Giacomin & Stehmann (recently described, see Giacomin and Stehmann 2011), S. hoffmanseggii Sendtn., S. lignescens Fernald, S. manabiense S.Stern, S. parcistrigosum Bitter, and S. turneroides Chodat. Th e purpose of this study is to clarify the circumscription of section Gonatotrichum and to investigate the phylogenetic relationships of the species of the section as well as its placement within the larger Brevantherum clade.
Species in section Gonatotrichum are native to North, Central, and South America. Th ey are herbs or small, woody shrubs with short infl orescences and simple hairs (except S. lignescens, which has stellate pubescence; see below). Flowers in the section have corollas ranging from 1-2.5 cm in diameter. Th e largest fl owers are those of S. turneroides and S. evolvuloides, which also exhibit marked heteranthery, in which one fi lament is nearly double the length of the other four. Th e fruits of species in section Gonatotrichum are unique within the genus. Th ey have a thin pericarp with a watery mesocarp held under pressure until they explosively dehisce. Th e fruits are white, yellow, or green, nearly transparent, and turgid before explosive dehiscence and defl ated and shriveled after dehiscence.
Some species within section Gonatotrichum are relatively widespread (S. defl exum, S. parcistrigosum, and S. turneroides), whereas others are narrowly distributed and relatively inconspicuous, making them among the least collected species of Solanum. As Bitter (1912) noted in the original description of the section, the group has a large geographic disjunction, with species found in the southwestern USA, Mexico, and Central America as far south as Costa Rica and then again in southern South America (Bolivia, Paraguay, northwestern Argentina, and southern Brazil). Th e recent description of S. manabiense from coastal Ecuador (Stern and Bohs 2009) and identifi cation of previously undetermined specimens, particularly those of S. hoff manseggii from Pará and Tocantins states in Brazil, has lessened the area of this disjunction; however, the section is absent from large areas of South America. Th is is likely due to the preference of the species for lower-altitude, dry habitats that are widely spaced on the continent.
Species placed in section Gonatotrichum have been subjected to two previous phylogenetic studies. Bohs (2005) and Weese and Bohs (2007) obtained sequence data for three species of the section (S. defl exum, S. turneroides, and "S. adscendens," later reidentifi ed as S. parcistrigosum). In both studies they formed a strongly supported monophyletic group sister to the remaining sampled members of the Brevantherum clade. Concurrently, morphological and fi eld studies of the group identifi ed two new species, S. manabiense (Stern and Bohs 2009) and S. evolvuloides (Giacomin and Stehmann 2011), and clarifi ed species limits and nomenclatural problems in the section. Th e presence of explosively dehiscent fruits in the Mesoamerican S. lignescens suggested that it also belongs to the group despite its stellate rather than simple pubescence (Stern et al. in review).
In this paper we use molecular phylogenetic methods to 1) examine the phylogenetic relationships of section Gonatotrichum with other members of the genus, 2) test the monophyly of section Gonatotrichum, 3) test the monophyly of species within the section and examine selected species-level relationships, and 4) examine geographical distributions and morphological patterns within the section.

Taxon sampling
Seven of the eight recognized species of section Gonatotrichum were sampled. We were unable to obtain high quality genomic DNA for S. hoff manseggii, an undercollected species from Amazonian Brazil. Multiple accessions were sampled for four species, with four accessions sampled for S. parcistrigosum, and two each for S. evolvuloides, S. manabiense, and S. defl exum. In addition to the species of section Gonatotrichum, we included members of Solanum as outgroups guided by results from previous studies showing other members of the Brevantherum clade to be sister to the section (Bohs 2005;Weese and Bohs 2007). Members of section Geminata (G. Don) Walp. were included as a more distant outgroup and the tree was rooted with S. betaceum, a member of section Pachyphylla (Dunal) Dunal. 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). PCR amplifi cation 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. Th e ITS region was amplifi ed 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 amplifi ed 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). PCR conditions for trnT-F followed Bohs and Olmstead (2001); conditions for waxy followed Levin et al. (2005). When necessary, overlapping fragments were amplifi ed 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.
PCR products were cleaned using the Promega Wizard SV PCR Clean-Up System (Promega Corporation, Madison, Wisconsin). Th e University of Utah DNA Sequencing Core Facility performed sequencing on an ABI automated sequencer. Sequences were edited in Sequencher (Gene Codes Corp., Ann Arbor, Michigan) and all new sequences were submitted to GenBank; accession numbers are listed in Appendix 1.

Sequence alignment and analysis
Sequence alignment for all of the gene regions was straightforward and performed visually using Se-Al (Rambaut 1996). Th e aligned dataset is available as Appendix 2 (see Appendix 2: Aligned Dataset).

Parsimony analyses
Parsimony analyses were performed on each dataset separately and on the combined dataset using PAUP*4.0b10 (Swoff ord 2002). All characters were weighted equally in analyses that implemented TBR branch swapping with 1,000 heuristic random addition replicates, each limited to 1,000,000 swaps per replicate. Gaps were treated as missing data. Bootstrapping (BS; Felsenstein 1985) was used to evaluate branch support with 1,000 random addition replicates and TBR branch swapping limited to 1,000,000 swaps per replicate.

Bayesian analyses
Prior to Bayesian analyses, a general model of nucleotide evolution was selected for the separate and the combined datasets using the AIC criterion identifi ed in Modeltest 3.7 (Posada and Crandall 1998). MrBayes 3.1 (Hulsenbeck and Ronquist 2001) was used to analyze each of the separate and combined datasets. For each analysis, fi ve million generations were run using eight Markov chains, each initiated from a random tree and sampled every 1,000 generations. Each of the analyses reached a standard deviation below 0.01 between the chains and all parameters from each analysis were visualized graphically to determine the trees discarded as burn-in prior to achieving stationarity.

Phylogenetic analyses
Th e parsimony strict consensus and Bayesian majority rule consensus trees of all datasets diff ered only in the degree of resolution, with Bayesian tree topologies more resolved than parsimony trees (Table 1). Clades with low posterior probabilities, typically those below 0.90 PP but occasionally those with up to 1.0 PP in Bayesian analyses were often collapsed in parsimony strict consensus trees. Descriptive statistics for individual and combined genes are provided (Table 1). More nodes were strongly supported by combining the data than were obtained in any of the separate analyses.

Topological conflicts
Our discussion will largely be based on the parsimony strict consensus tree of the combined data set, which is a conservative hypothesis of phylogenetic relationships, but areas of the tree that receive strong support in the Bayesian analysis that are less strongly supported in the parsimony analysis will be noted (Fig. 1). Th e parsimony strict consensus trees for the individual markers are also presented ( Fig. 2-4). In parsimony analyses, each DNA sequence region consistently identifi ed the same major, well-supported groups corresponding to the Brevantherum clade and section Gonatotrichum comprising identical species, but relationships within these major clades were often not strongly supported (BS values < 90 %), or were unresolved, and thus cannot be considered confl icting under Wiens' (1998) criteria. Within section Gonatotrichum, one incongruence of note among the various datasets is the placement of S. adscendens, S. lignescens, and S. defl exum. In the plastid (Fig. 3) and combined trees ( Fig. 1), S. adscendens is sister to the remainder of the species in the section (98% BS, 1.0 PP in the trnT-F tree, 52% BS, 0.78 PP in the combined tree). In the ITS tree, S. lignescens is sister to the remaining species in section Gonatotrichum, but this relationship is unsupported (Fig. 2). In the waxy tree, S. adscendens, S. lignescens, and S.    defl exum form a well-supported clade (91% BS, 1.0 PP) sister to the remaining species of section Gonatotrichum (Fig. 4).

Phylogenetic relationships
Solanum section Gonatotrichum emerges as monophyletic in all analyses and strongly supported in all except the ITS-only tree. Th e section is strongly supported (99% BS, 1.0 PP in the combined tree) as sister to the remainder of the species sampled from the Brevantherum clade, which form a monophyletic group in all analyses (100% BS, 1.0 PP in the combined tree; for sampling of Brevantherum clade see Fig. 1). Species with duplicate accessions sequenced (S. parcistrigosum, S. evolvuloides, S. manabiense, and S. defl exum) were all monophyletic in the combined tree. Within section Gonatotrichum, S. turneroides is strongly supported as sister to S. parcistrigosum in the combined tree (100% BS, 1.0 PP), and S. evolvuloides is sister to this clade (82% BS, 0.98 PP). Solanum manabiense from coastal Ecuador is resolved as sister to this clade (89% BS, 1.0 PP). Solanum defl exum and S. lignescens form a clade (89% BS, 1.0 PP). Th e fi nal species, S. adscendens, is sister to the remainder of the species of section Gonatotrichum in the combined tree, although this relationship is poorly supported (52% BS, 0.78 PP) and, as noted above, is not recovered in the ITS and waxy trees.

Discussion
Our data, like those of previous studies, show that Solanum section Gonatotrichum belongs to the Brevantherum clade (sensu Bohs 2005; Weese and Bohs 2007). Th is clade consists of approximately 60 species of herbs, shrubs and trees found in tropical and subtropical regions of the New World (Bohs 2005). Centers of diversity occur in the Andes and in eastern Brazil, where many species are common in secondary vegetation and disturbed habitats. All members of the clade lack prickles and have relatively short, broad anthers. However, members of section Gonatotrichum are strikingly diff erent from the rest of the species of the Brevantherum clade. Traditionally, the section included small annuals or perennials with reduced infl orescences and simple hairs, whereas the remainder of the Brevatherum clade includes shrubs to sizable woody trees with stellate or lepidote pubescence and often large, branched infl orescences. Th e explosively dehiscent fruits of section Gonatotrichum diff er from those of the rest of the Brevantherum clade, which are variously colored, fl eshy in texture, and not dehiscent at maturity. Th ese results indicate that section Gonatotrichum forms a monophyletic group including the Mesoamerican S. lignescens. Th is species had not been considered to be a member of the section Gonatotrichum in previous taxonomic treatments (Bitter 1912(Bitter , 1913Nee 1989). Nee (1999) placed S. lignescens in section Brevantherum due to its shrubby habit and stellate hairs, although he indicated that it had no obvious close relatives within the section and suggested that perhaps it might belong in section Gonatotrichum. Our molecular data place S. lignescens within section Gonatotrichum and, in the combined and waxy trees, as sister to S. defl exum, a species with exclusively simple hairs found from the southwestern USA to Mexico and Central America. Th e fruits of S. lignescens are explosive berries like those of the rest of the section, indicating that fruit morphology may be a synapomorphy for section Gonatotrichum, but that habit and hair morphology may be somewhat variable within it.
Hairs have been used extensively for identifi cation of species and sections in Solanum and have their own standardized terminology in the genus (see Roe 1971 for a overview). While the presence of specifi c hair types can be diagnostic, many groups or even species can have multiple hair types. Section Brevantherum sensu Roe (1972), placed in the Brevantherum clade by Bohs (2005) and Weese and Bohs (2007), exemplifi es this complexity, with some species having up to six diff erent hair types (see Table 1, Roe 1972). Section Gonatotrichum was previously distinguished within the genus due to the presence of simple, geniculate hairs with a short basal cell and a 90° bend between this and the second cell in at least some of its members (Bitter 1912(Bitter , 1913Nee 1989). Geniculate hairs are present in some species in the section (S. hoff manseggii and S. parcistrigosum) but absent in other species (S. adscendens, S. defl exum, S. evolvuloides, and S. manabiense) with one species (S. turneroides) having intermediate hairs that are fl attened along the stem but lack a 90° bend between the basal and second cells. Our results show that a species with stellate hairs, S. lignescens, also belongs in section Gonatotrichum, which previously was thought to contain only simple-haired species. Th e placement of S. lignescens as sister to the simple-haired S. defl exum in some analyses suggests that simple hairs may have evolved from branched hairs in the latter species and perhaps in the four species sister to the S. lignescens/S. defl exum clade in the combined and waxy trees. Further morphological, developmental, and phylogenetic study of various species of the Brevantherum clade may clarify the patterns of hair evolution throughout the group and distinguish between simple hairs that are pleisiomorphic versus those derived via ray reduction from branched-haired ancestors.
We sampled multiple accessions per taxon in four of the seven species of section Gonatotrichum included in the phylogeny (S. evolvuloides, S. defl exum, S. manabiense, and S. parcistrigosum). In the combined and waxy trees, all four species were monophyletic. Th e four accessions of S. parcistrigosum were not monophyletic in the trnT-F tree, and the two accessions of S. defl exum did not form a clade in the ITS tree. However, neither of these cases of species non-monophyly were strongly supported. It appears that species limits, at least within these four taxa, are fairly distinct.
Within section Gonatotrichum, the phylogeny from the combined data exhibits clear geographic patterns. Th e species ranging from North to Central America (S. lignescens and S. defl exum) form a strongly supported clade, as do the southern South American species (S. evolvuloides, S. parcistrigosum, and S. turneroides) with the Ecuadorian S. manabiense sister to the southern South American species. Th e position of the Brazilian S. adscendens as sister to the rest of the clade in the combined and trnT-F trees does not fi t this biogeographic pattern and is confounded by the incongruence between the trnT-F and waxy datasets indicated above, although neither of these datasets place S. adscendens with the remainder of the southern South American species. Future molecular studies should attempt to include S. hoff manseggii, a poorly known species from Amazonian Brazil and the only member of section Gonatotrichum for which sequence data are not available. Based on morphology, particularly the geniculate hairs that lay fl at along the stem, the similar sized fl owers and fruits, and the overall distribution, we speculate that S. hoff manseggii will likely be closely related to the southern South American species S. parcistrigosum. Sequence data is needed to confi rm this relationship and determine its phylogenetic affi nities to the rest of the South American taxa of the clade.
Although buzz pollination is virtually universal in Solanum, heteranthery is an unusual trait in the genus and has been shown to have evolved multiple times independently within it ). It has been particularly well studied in the temperate spiny weed, S. rostratum Dunal, a member of Solanum section Androceras (Nutt.) Marzell in the subgenus Leptostemonum (Dunal) Bitter (Bowers 1975;Vallejo-Marín et al. 2009). Th is species has four yellow stamens that serve a "feeding" function, providing pollen that is used as food for the bee pollinator. Pollination is achieved by an elongate, brown lower stamen that is specialized for placing pollen on areas of the insect where it cannot be easily removed and used for food (Vallejo-Marín et al. 2009). In section Gonatotrichum, S. evolvuloides and S. turneroides are both strongly heterantherous, with the lowermost fi lament extending to 2-5 mm, thereby reaching double the length of the other stamens. Th ese two species also have fl owers that open during the morning hours and close by midday (Nee 1989;S. Stern and L. Giacomin pers. obs.) with the fl owers of S. turneroides also strongly fragrant, an unusual trait in the genus. Th e pollinators of these species are unknown and it is also unknown whether the upper stamens are modifi ed for "feeding" and the lower stamen modifi ed for "pollinating" as described for S. rostratum above. Th e third member of this clade, S. parcistrigosum, has fl owers that are only weakly heterantherous with the fi lament of the lowermost stamen only ca. 1 mm longer than the other stamens.
Future work on section Gonatotrichum should include sequencing of more gene regions and species accessions, especially targeting S. hoff manseggii and S. adscendens, to clarify biogeographic patterns in the group. Nothing is known about chromosome numbers or potential hybridization among taxa, and the function of heteranthery in pollination of several species within the group is unclear. Detailed studies of the development and morphology of hairs in Solanum and in section Gonatotrichum in particular may reveal that simple hairs may have arisen by two diff erent evolutionary pathways, either from plesiomorphically simple hairs or by reduction from branched hairs. Finally, more in-depth studies of the entire Brevantherum clade are needed to clarify its species limits, phylogenetic relationships, and morphological and biogeographical patterns.