PHYLOGENETICS OF THE FLORALLY DIVERSE ANDEAN CLADE
STACEY DEWITT SMITH
AND DAVID A. BAUM
Department of Botany, University of Wisconsin, Madison, Wisconsin 53706 USA
Recent molecular phylogenetic studies of Solanaceae have identified many well-supported clades within the family and have
permitted the creation of a phylogenetic system of classification. Here we estimate the phylogeny for Iochrominae, a clade of
Physaleae sensu Olmstead et al. (1999), which contains 34 Andean species encompassing an immense diversity of floral forms
and colors. Using three nuclear regions, ITS, the second intron of LEAFY, and exons 2 to 9 of the granule-bound starch synthase
gene (waxy), we evaluated the monophyly of the traditional genera comprising Iochrominae and assessed the extent of
interspecific hybridization within the clade. Only one of the six traditionally recognized genera of Iochrominae was supported as
monophyletic. Further, comparison of the individual nuclear data sets revealed two interspecific hybrid taxa and a third possible
case. These hybrid taxa occur in the Amotape–Huancabamba zone, a region between the northern and central Andes that has the
greatest diversity of Iochroma species and offers frequent opportunities for hybridization in areas of sympatry. We postulate that
periodic hybridization events in this area coupled with pollinator-mediated selection and the potential for microallopatry may have
acted together to promote diversification in montane Andean taxa, such as Iochrominae.
Key words: floral evolution; granule-bound starch synthase; interspecific hybridization; LEAFY; phylogeny; pollination;
reticulate evolution; speciation.
The tropical Andes comprise the pre-eminent hotspot of
plant biodiversity, with approximately 15% of all plant species
native to that region (Myers et al., 2000). Many plant families,
though cosmopolitan, have centers of diversity in western
South America, for example, Ericaceae, Orchidaceae, and
Solanaceae (Dressler, 1981; D’Arcy, 1991; Luteyn, 2002). An
important contributor to the origin of this diversity is the
topological and environmental variation resulting from the
uplift of the Andes (Gentry, 1982; Hooghiemstra et al., 2002).
Phylogenetic studies support an association between the
diversification of Andean plants (von Hagen and Kadereit,
2003; Kay et al., 2005) and animals (Patton and Smith, 1992;
Bates and Zink, 1994; Brower, 1994) and the major episodes of
Andean uplift, beginning in the early Miocene (ca. 20 mya) and
ending in the Pliocene (ca. 3 mya) (Hoorn et al., 1995;
Hooghiemstra and van der Hammen, 1998). Indeed, the
parallel invasions of higher elevations by numerous plant
groups and the coincident radiations of pollinating animals,
e.g., hummingbirds (Bleiweiss, 1998), may explain the
‘‘explosive’’ speciation seen in some Andean groups (Gentry,
1982; Luteyn, 2002). Here we investigate the phylogenetic
history of Iochrominae, a group of Andean Solanaceae, which
have radiated in floral morphology and pollination system
(Cocucci, 1999) and which may serve as a model system for
other Andean radiations.
Recent phylogenetic analyses using plastid genes have
greatly clarified relationships within Solanaceae and allowed
for the creation of a phylogenetic system of classification
(Olmstead and Sweere, 1994; Olmstead et al., 1999; Martins
and Barkman, 2005; Olmstead et al., University of Wash-
ington, personal communication). Iochrominae sensu Olmstead
et al. (1999) is a clade of Physaleae comprising around 34
mainly Andean species traditionally assigned to six genera:
Acnistus Schott, Dunalia H.B.K, Eriolarynx (Hunz.) Hunz.,
Iochroma Benth., Saracha R. and P., and Vassobia Rusby
(Table 1). In the Olmstead et al. (1999; R. G. Olmstead,
University of Washington, unpublished manuscript) scheme,
Iochrominae together with Physalinae and Withaninae form the
large clade Physaleae, which is sister to Capsiceae. Although
the phylogenetic classification was not accompanied by
a morphological reassessment, Iochrominae can be distin-
guished from other subtribes in Physaleae by the fact that they
are all woody shrubs or small trees and often have showy
tubular flowers. In a recent morphological phylogenetic
analysis (Sawyer, 2005), all Iochrominae genera except one,
Acnistus, were found to be monophyletic, united most notably
by the rounded-mucronate shape of the fruiting calyx margin
and the presence of sclerosomes in the fruit wall.
Although it contains only one-third the number of species in
its probable sister group Physalinae, Iochrominae boasts
a greater floral diversity, spanning all major flower colors
and forms found in the entire Solanaceae. Iochrominae flowers
may be red, orange, yellow, green, blue, purple, or white, and
the corolla varies from rotate to tubular, with over eight-fold
variation in tube length across species (Shaw, 1998; Hunziker,
2001; Table 1). In contrast, a vast majority of taxa within
Manuscript received 7 December 2005; revision accepted 1 May 2006.
The authors thank S. Hall, O. Jadan, D. Neill, C. Padilla, W. Quizhpe,
A. Rodriguez, H. Vargas, V. Zak and in particular, S. Leiva G., for
assistance during fieldwork and plant collection; N. W. Sawyer for
determinations of Cuatresia and Larnax; M. Nee for determinations of
Lycianthes and Capsicum; S. Keel for determination of Salpichroa;D.G.
Howarth for advice with LFY; R. G. Olmstead for providing DNA of
Leucophysalis grandiflora and Tubocapsicum anomalum; L. Bohs for
material of Dunalia brachyacantha,Saracha punctata, and Acnistus
arborescens; G. van der Weerden for material of E. lorentzii; A. Tye for
help in obtaining samples of I. ellipticum; P. E. Berry, L. Bohs, J. W.
Boughman, N. I. Cacho, M. Nee, R. G. Olmstead, D. M. Spooner, K. J.
Sytsma, and T. J. Theim for helpful discussion; two anonymous reviewers
for useful comments; B. Larget for advice regarding Bayesian analyses; K.
Elliot for assistance with illustrations; and R. A. Smith for editing. Finally,
the authors acknowledge financial support from the National Science
Foundation grant DEB-0309310, the University of Wisconsin chapter of
Graduate Women in Science, the Marie Christine Kohler Fellowship, the
American Society of Plant Taxonomists, the University of Wisconsin
Tinker-Nave Fund, and the O. N. Allen Memorial Fund.
Author for correspondence (e-mail: firstname.lastname@example.org)
American Journal of Botany 93(8): 1140–1153. 2006.
Physalinae, Withaninae, and Capsiceae have small, white or
yellow, rotate flowers, and there are no instances of long,
tubular, red or purple corollas in these three clades. Thus, the
brightly colored tubular flowers likely represent a derived
feature that arose within or at the base of Iochrominae.
The great floral diversity of Iochrominae sensu Olmstead et
al. (1999) has misled classifications based on morphology. For
example, Hunziker’s (2001) morphologically delimited Iochro-
minae included Oryctes S. Watson, a monotypic tubular-
flowered genus native to California and Nevada. Oryctes has
since been shown to be nested within Physalinae, probably
sister to Leucophysalis (Whitson and Manos, 2005; Olmstead
et al., University of Washington, personal communication).
Similarly, Sawyer’s (2005) morphological cladistic analysis of
Physaleae identified an Iochrominae clade that included all
genera except Acnistus, which appeared with Tubocapsicum in
a distant clade. Although Acnistus and the monotypic Japanese
Tubocapsicum share small campanulate-infundibuliform flow-
ers with valvate bud aestivation, molecular studies strongly
suggest that Tubocapsicum is more closely related to other
Physaleae (e.g., Nothocestrum and Withania) than to Acnistus
and other Iochrominae (Olmstead et al., 1999; Olmstead et al.,
University of Washington, personal communication).
Another challenge in the systematics of Iochrominae is the
potential for hybridization among species and across generic
boundaries. Horticulturists have generated several hybrids
(e.g., I. australe 3I. cyaneum), and botanists have occasionally
encountered hybrid populations in nature (Shaw, 1998; S. D.
Smith, personal observation). The ease of crossing, the
overlapping species ranges of many Iochrominae, and the
observation of natural hybrids suggest that hybridization may
have been important in the evolutionary history of Iochromi-
nae. Combined with external sources of information such as
morphology, biogeography, and cytology, phylogenetic esti-
mation using multiple genetic markers can help identify
instances of hybridization.
In this study, we used three nuclear regions, the internal
transcribed spacer (ITS), exons 2 through 9 of the nuclear
granule-bound starch synthase gene (GBSSI or waxy), and the
second intron of LEAFY (LFY) to estimate the phylogeny of
Iochrominae. Both ITS and waxy have been useful in clarifying
specific and generic relationships in Solanaceae (e.g., Marshall
et al., 2001; Peralta and Spooner, 2001; Whitson and Manos,
2005). LFY introns are increasingly utilized for resolving
interspecific relationships and identifying hybrid taxa (e.g., Oh
and Potter, 2003; Howarth and Baum, 2005), although this is
the first study to use LFY in Solanaceae systematics. Our
specific objectives were to evaluate the monophyly of the six
traditional genera of Iochrominae and to assess the extent of
interspecific hybridization. We close by considering our results
in a biogeographical context.
MATERIALS AND METHODS
Taxon sampling—This study includes a nearly complete sampling of
Iochrominae (Table 1) and a broad sampling of related lineages in the Solanoid
radiation. Thirty-three of the 34 commonly recognized species of Iochrominae
(all but Eriolarnyx iochromoides) were sampled in this study, as well as three
as yet undescribed taxa (Appendix 1). The status of these unnamed taxa is
under review by S. Leiva G., and for the purposes of this study, we will use
temporary names, indicated by quote marks, based on their likely species
epithets (S. Leiva G., Herbario Antenor Orrego, personal communication). For
Iochroma peruvianum, a species known only from the type collection, our
determination remains tentative because we were unable to find the species in
its type locality and have here sampled individuals from another locality that
closely resemble the type but have some small differences. For one ingroup
species, A. arborescens, multiple individuals were included because the species
is extremely widespread and variable.
Three ingroup taxa were suspected to have recent hybrid ancestry: Iochroma
‘‘sagasteguii,’’ I. ayabacense, and I. stenanthum. These species are endemic to
northern Peru and are often found in sympatry with other species of Iochroma
and Acnistus. They share some characteristics of Iochroma (e.g., tubular
flowers, purple coloration in the latter two) and some of Acnistus (e.g., yellow-
green markings inside the corolla lobes), making their taxonomic affinity
unclear. Preliminary chromosome counts for one of these three species, I.
ayabacense suggest that it is n¼12 (S. D. Smith and V. Kolberg, University of
Wisconsin, unpublished data) as are other species and genera of Iochrominae
(Hunziker, 2001, and references therein). We, therefore, considered these taxa
to be possible homoploid hybrids.
The 10 outgroup taxa were selected by reference to the plastid phylogeny of
Solanaceae (Olmstead et al., 1999) and included Nicandreae (Nicandra),
Solaneae (Solanum), Capsiceae (Capsicum and Lycianthes) and other members
of Physaleae (Leucophysalis,Physalis, Salpichroa, Tubocapsium, and With-
eringia) (Appendix 1). Also, included were the Andean genera Cuatresia and
Larnax, which have not yet been incorporated into the phylogenetic
TABLE 1. Summary information for genera of Iochrominae. Number of species is from recent treatments and descriptions (Acnistus [Hunziker, 1982],
Eriolarynx [Hunziker, 2000, 2001], Dunalia [Hunziker, 1960, 2001], Iochroma [Leiva, 1995; Leiva et al., 1998, 2003; Shaw, 1998], Saracha
[Alvarez, 1996], Vassobia [Hunziker, 1984, 2001]).
Genus No. species No. sampled Distribution Elevation (m a.s.l.) Distinctive features
Acnistus 1 1 Southern Mexico, Central America, the
Caribbean, northern South America
and eastern Brazil
300–2000 Campanulate or funnel-shaped fragrant flowers;
valvate bud aestivation; triangular corolla lobes;
green markings inside corolla lobes; edible fruit
Dunalia 5 5 Colombia to Argentina 1600–3700 Often spiny; a few dioecious or gynodioecious;
tubular flowers with wing-like appendages of stapet
Eriolarynx 3 2 Bolivia and Argentina 1000–3000 Rotate or campanulate flowers with a dense ring
of trichomes at base of corolla tube; stapet with
small projections (‘‘auricles’’)
Iochroma 21 þ3
Colombia to Peru, with one species
in the Galapagos*
1100–3500 Tubular, often colorful flowers, with inflated calyces in
Saracha 2 2 Venezuela to Bolivia 2700–4500 Occasionally spiny; coriaceous to subcoriaceous leaves;
campanulate or funnel-shaped flowers; pyrenes in fruit
Vassobia 2 2 Bolivia, Argentina, Paraguay,
Uruguay and Brazil
300–2700 One spiny; campanulate flowers mostly glabrous;
stapet with auricles
Note: *The range for Iochroma excludes the two southern Andean species shown in this study not to belong in Iochroma.
August 2006] SMITH AND BAUM—PHYLOGENETICS OF IOCHROMINAE 1141
classification scheme for the family, but appear to belong in Physaleae (R. G.
Olmstead et al., University of Washington, unpublished manuscript).
Data collection—Total genomic DNA was extracted from silica-dried leaf
material (Chase and Hills, 1991) using a modified 23CTAB protocol (Doyle
and Doyle, 1987). ITS was amplified as described in Baum et al. (1998) with
primers ITS leu.1 (Andreasen et al., 1999) and ITS4 (White et al., 1990) and
sequenced with these two primers plus ITS2 (White et al., 1990) and ITS3B
(Baum et al., 1994).
The waxy region was amplified using primers 50and 3 0and sequenced
using primers GBSSI-A, -B, -C
, and -D
designed by Peralta and Spooner
(2001). For difficult taxa, four Iochrominae specific primers were designed:
F41, F420, R991, and R1235 (Appendix 2). Each 25 lLwaxy PCR reaction
contained 2.5 lL103PCR Buffer (Qiagen, Valencia, California, USA), 2.5 lL
of 25 mM MgCl
, 1.0 lL of 10 mM dNTPs, 1.0 lL of each primer (10 lM
solutions), 0.125 lLTaq polymerase (5 units/lL), and approximately 100 ng
of template DNA. The PCR program was 958C for 2 min, then 35 cycles of
958C for 45 s, 568C for 30 s, 728C for 3 min, followed by a final extension of
728C for 5 min.
The second intron of LFY was initially amplified and cloned from a subset
of taxa using degenerate primers F2 and R1 (Howarth and Baum, 2005). These
sequences were used to create Solanoid specific primers (LFYSOL-F7, -F68,
-R700, -R1000 in Appendix 2). The PCR reactions for LFY differed from the
waxy reactions in that they contained 2.0 lL MgCl
and 1.0 lL Q-solution
(Qiagen). The PCR program for LFY amplification was 958C for 4 min, then 35
cycles of 958C for 1 min, 488C for 1 min, 728C for 3 min, followed by a final
extension of 728C for 5 min. All PCR products were purified with AMPure
using manufacturer’s protocols (Agencourt Bioscience, Beverly, Massachu-
Copy number and allelic variants are of concern when using nuclear genes
for phylogenetics. The ITS region, as part of the repeating units of rDNA in the
nuclear genome, undergoes concerted evolution, potentially homogenizing the
many copies (Hamby and Zimmer, 1992). This may explain why direct
sequencing was possible for ITS for all taxa. With the single or low copy
nuclear loci, LFY and waxy, direct sequencing often failed to yield a single
sequence. In these cases, PCR products were gel-purified with the QIAquick
gel extraction kit (Qiagen) and cloned using the pGEM-T easy vector system
(Promega, Madison, Wisconsin, USA) following the manufacturer’s protocol.
Five to eight clones from each product were sequenced.
Sequencing used ABI sequencing reagents (Applied Biosystems, Foster
City, California, USA). Each 10-lL cycle sequencing reaction contained 1.0 lL
of purified PCR product, 2 pM of primer, 2 lL Big Dye, and 2 lLof
sequencing buffer and was cycled through a program of 948C for 2 min, then 30
cycles of 948C for 20 s, 478C for 20 s, and 608C for 3 min. Reactions were
cleaned using CleanSEQ (Agencourt) and run on ABI PRISM 3700 DNA
analyzer at the University of Wisconsin Sequencing Facility. Sequences were
edited in Sequencher (Gene Codes Corp., Ann Arbor, Michigan, USA) and
aligned manually in MacClade 4.0 (Maddison and Maddison, 2000). Unique
clones were maintained in the alignment until data collection was complete.
Minor allelic variants (five or fewer substitutions per kilobase) were combined
to create a consensus sequence for the species with differences coded as
ambiguities. When alleles from a single species differed markedly in sequence
or in length (due to indels) but still formed a clade in phylogenetic analyses, the
allele giving the shortest branch in a parsimony tree was kept for analysis. In
cases where the two alleles of a given species did not consistently form as
a clade, both were kept for analysis. We use the term ‘‘divergent alleles’’ for
such sequences for the remainder of the paper.
Sequences were examined for evidence of intragenic recombination by
visual examination of the spatial distribution of different site patterns.
Additionally, the sequences were analyzed for evidence of recombination
using the MaxChi (Maynard Smith, 1992) and GENECONV (Padidam et al.,
1999) methods (with default settings) in the program RDP (Martin and Rybicki,
2000). These methods have a limited ability to detect recombination, but are
still potentially informative (Posada and Crandall, 2001; Posada, 2002). Final
sequence alignments were deposited in TreeBASE (study accession number
Phylogenetic reconstruction—For parsimony analyses, all characters were
equally weighted, and gaps were treated as missing characters. Heuristic
searches were conducted in PAUP*, version 4.0b10 (Swofford, 2002), using
1000 random taxon addition sequences (holding two trees at each step) with
tree-bisection-reconnection (TBR) branch swapping and keeping up to 100
most parsimonious trees (MPTs) per random addition replicate. Similar to
Catala´n et al. (1997), we next completed a heuristic search using the same
settings but with 5000 random taxon additions and retaining only trees not
compatible with the strict consensus of the first parsimony search (by enforcing
the strict consensus as a reverse constraint). If the second search returned only
trees longer than first search, then we considered the MPTs from the first search
an adequate sample of parsimony tree space. If we found shorter trees, we
repeated the process until no additional MPTs were recovered. To estimate
clade support, heuristic searches were completed for 1000 bootstrap replicates
with 10 random sequence additions (holding one tree at each step), TBR branch
swapping, and maxtrees set to 100.
For likelihood analyses, the best fitting model was chosen by hierarchical
likelihood ratio tests. Likelihood scores were calculated in PAUP* (Swofford,
2002) for the following models (in order of increasing complexity): JC, K2P,
HKY, HKYþC, HKYþCþI, GTRþC, and GTRþCþI (Swofford et al., 1996,
and references therein). The most-parsimonious tree (MPT) with the highest
likelihood under the JC model was used for calculating likelihoods under more
complex models. Likelihood searches were carried out in PAUP* (Swofford,
2002) using the best fitting model with all the MPTs used as starting trees, TBR
branch swapping, and model parameters estimated during the hierarchical
likelihood ratio tests.
Bayesian analyses were performed with MrBayes, version 3.1.1 (Ronquist
and Huelsenbeck, 2003). The ITS and LFY intron data sets were each treated as
a single data partition, whereas the waxy data set was divided into three
partitions: first and second codon positions, third codon positions, and introns.
Thus, the combined data set had five total data partitions. Each partition was
assigned the best fitting model as suggested by likelihood ratio tests using
MPTs from each partition as described previously. Transition/transversion
ratio, substitution rates, state frequencies, gamma shape parameters, and
proportion of invariant sites were unlinked across partitions and estimated
during the Markov Chain Monte Carlo (MCMC) runs. For the individual and
combined data sets, we conducted four independent MCMC runs, each with
two internal runs (nruns ¼2), to give eight tree files for each data set. Each run
was initiated with a different starting seed and comprised four linked chains
with temperature of 0.2. The chains were run for 5 000 000 generations,
sampling every 100 generations, except for ITS, for which we used 15 000 000
generations, sampling every 150 generations. Adequate mixing (sampling of
tree and parameter space) was judged by movement among chains and
acceptance rates, which should be between 10 and 70%, and, most importantly,
by convergence among independent runs with different starting points
(Huelsenbeck et al., 2002). Inadequate mixing in some initial runs was
corrected by adjusting the temperature and re-running the analysis. We
considered that the runs had converged when the convergence diagnostics
provided in sump output approached 1 and when clade credibilities (post burn-
in), branch lengths, and topologies were similar across the four independent
runs. We discarded 10% of trees as our burn-in period, which appeared to be
very conservative given visual inspection of likelihood-by-generation plots.
Posterior probabilities (PP) were averaged across runs.
Statistical tests—We estimated the g1 statistic, a measure of phylogenetic
signal, for each data set in PAUP* using 10 000 random trees. Significance of
the statistic was assessed following Hillis and Huelsenbeck (1992).
Incongruence between the three data sets was estimated with the
incongruence length difference (ILD) test (Farris et al., 1994), implemented
as the partition homogeneity test in PAUP*. The test was conducted with 1000
replicate partitions, each subjected to heuristic parsimony searches, comprising
10 random taxon addition replicates with TBR branch swapping and keeping
no more than 100 trees per random addition replicate. The difference in
phylogenetic signal from the three data sets as manifested in differing tree
topologies was further examined using Wilcoxon signed-ranks (WSR) tests,
also known as Templeton tests (Templeton, 1983), implemented in PAUP*. A
detailed description of the use of WSR tests to compare phylogenetic
hypotheses is given in Larson (1994). Constrained searches completed in
conjunction with WSR tests were carried out with the same settings as
unconstrained parsimony searches (described previously).
We also examined incongruence between data sets in a Bayesian framework
as described in Buckley et al. (2002). We determined whether the combined
topology existed within the 95% credible set of trees from each gene. If not, we
assumed that the gene in question evolved under a different topology or that the
model of evolution was inappropriate. In these cases, we attempted to localize
areas of discordance by comparing individual clade credibilities between the
individual and combined analyses.
1142 AMERICAN JOURNAL OF BOTANY [Vol. 93
Phylogenetic analyses of individual data sets—ITS—
Sequences were completed for all taxa (Appendix 1) and
easily aligned to provide a matrix of 803 characters (described
in Table 2). We found no evidence of intragenic recombination,
either by visual inspection or by use of MaxChi and
GENECONV methods in the program RDP (P.0.05).
Relative to LFY and waxy, ITS had low consistency and low
phylogenetic signal (Table 2), resulting in many more MPTs.
Our initial Bayesian analyses showed variation in clade
credibilities across runs, but lengthening the runs to
15 000 000 generations (sampling every 150 generations)
produced identical majority-rule consensus trees with less than
5% difference in PP across independent runs for clades with
over 50% PP. Also, the convergence diagnostic was 1.0 for
each run and acceptance rates were between 10 and 70%,
indicating good mixing. Despite thorough exploration of tree
space, ITS provided little resolution at any level and showed
only a few strongly supported clades, which appeared
consistently in the MP, ML, and Bayesian analyses. At the
broader level, ITS data suggested that the mostly closely
related taxa to Iochrominae are other Physaleae (bootstrap
support [BS] 59%, PP ¼1.0; Fig. 1), but it did not provide
support for any specific Physaleae lineage being sister to
Iochrominae. Iochrominae appeared monophyletic (BS ¼86%,
PP ¼1.0; Fig. 1), excluding the spiny Bolivian endemic I.
cardenasianum, which apparently is not a member of
Iochroma. Within Iochrominae, ITS resolved only small
clades, such as Vassobia (BS ¼76%, PP ¼1.0; Fig. 2) and
the C clade (BS ¼93%, PP ¼1.0, see Fig. 2 legend for
explanation of clade names). One interesting feature of the ITS
phylogeny was the placement of the U group, which appeared
as a clade (BS ¼52%; not shown) sister to the rest of
Iochrominae (BS ¼33%; not shown) in parsimony analyses or
as a basal grade in likelihood and Bayesian analyses (Fig. 2).
waxy—Most taxa were directly sequenced for waxy and only
a few (Lycianthes,Larnax,Iochroma parvifolium,I. fuch-
sioides, and the three putative hybrids) required cloning. In
TABLE 2. Summary statistics and analysis parameters for individual and combined data sets for phylogenetic analysis of Iochrominae. The number of
most parsimonious trees includes only the unique trees after collapsing zero-length branches.
Region No. characters
uninformative characters) g1 Tree length
Temperature used in
ITS 803 239 138 0.42/0.54 0.35* 670 16 353 GTR þCþI 0.07
489 167 0.64/0.84 0.67* 687 264 HKY þC0.05
LFY 1806 764 322 0.63/0.78 1.49* 1230 458 HKY þC0.2
Combined ITS and waxy 2275 728 305 0.48/0.66 0.47* 1393 180 GTR þCþI
Combined all (excluding
4023 1204 511 0.57/0.73 1.22* 1991 12 HKY þCþI
Notes:CI¼consistency index; RI ¼retention index. *, significant phylogenetic signal (P,0.01) according to the g1 statistic (Hillis and Huelsenbeck,
For likelihood searches of combined data sets, we used the indicated model, but for Bayesian searches, we applied the best fitting model for each
individual data set to its partition.
Fig. 1. Maximum likelihood trees showing placement of Iochrominae within the Solanoideae. Regions analyzed are listed to the upper left of each tree.
All trees are shown with branch lengths proportional to the estimated average number of substitutions per site under the models indicated in Table 2.
Bootstrap support (BS) values .50% are shown above branches or before the slash, and posterior probabilities (PP) .0.75 (shown as percentages) are
below branches or after the slash. Asterisks indicate a PP of 1.0. The rightmost tree is labeled with Olmstead et al. (1999) tribal and subtribal groupings.
Solid vertical lines label monophyletic groups; dashed vertical lines indicate non-monophyly.
August 2006] SMITH AND BAUM—PHYLOGENETICS OF IOCHROMINAE 1143
Fig. 2. Maximum likelihood trees of Iochrominae for individual and combined analyses. All trees are shown with branch lengths proportional to the
estimated average number of substitutions per site under the models indicated in Table 2. Outgroups for ITS and waxy include all taxa shown in Fig. 1;
outgroups for LFY and combined include only Physaleae (Physalis,Leucophysalis,Witheringia,Tubocapsicum,Cuatresia and Larnax; Fig 1.). Bootstrap
support (BS) values .50% are shown above branches or before the slash, and posterior probabilities (PP) .0.75 (shown as percentages) are placed below
branches or after the slash. Asterisks indicate a PP of 1.0. To facilitate comparison of relationships among trees, groups of interest are labeled with vertical
1144 AMERICAN JOURNAL OF BOTANY [Vol. 93
those cases, we conducted initial parsimony analyses to see if
the separate clones formed a clade. When they did, we either
created a consensus sequence (when clones differed by fewer
than five bases per kilobase) or selected a single exemplar
sequence. For I. ayabacense and I. ‘‘sagasteguii,’’ two distinct
sequence variants were found that did not form a clade. Both
divergent alleles were retained in the final data set. We found
no evidence of intragenic recombination among ingroup waxy
sequences, either by visual inspection or by use of MaxChi and
GENECONV methods in RDP (P.0.05). Characteristics of
the data set are given in Table 2.
Bayesian analyses of the waxy data set mixed well as
indicated by the convergence diagnostics and the low variation
among independent runs. The waxy analyses strongly supported
the monophyly of Iochrominae (BS ¼100, PP ¼1.0; Fig. 1) and
its inclusion in Physaleae (BS ¼83, PP ¼1.0; Fig. 1), perhaps as
sister to Physalinae plus Tubocapsicum (BS ¼75, PP ¼1.0;
Fig. 1). Like ITS, waxy showed I. cardenasianum to be distantly
related from other iochromas. Further, all waxy analyses divided
Iochrominae into a principally northern Andean clade contain-
ing Acnistus and Iochroma (A, C, L, F, and U clades; Fig. 2) and
a mixed northern, central, and southern Andean clade
containing members of Dunalia,Eriolarynx,Saracha, and
Vassobia (D, E, S, and V; Fig. 2).
LFY—This region was more variable than waxy (Table 2),
and could not be directly sequenced for many taxa. Neverthe-
less, most clones constituted minor sequence variants that were
represented in the final matrix by consensus sequences.
However, three of 49 taxa (Eriolarynx lorentzii,I. ayabacense,
and I. ‘‘sagasteguii’’ ) contained two alleles that did not form
a clade with others from the same accession. These divergent
alleles were kept in the final matrix for phylogenetic analysis.
We found no evidence of intragenic recombination in
Iochrominae, either by visual inspection or with MaxChi and
GENECONV methods implemented in RDP (P.0.05).
Although LFY sequences were completed for all taxa, this
intron could not be aligned outside Physaleae due to the
enormous length variation (2.2 kb in Capsiceae vs. 1.4 kb in
Iochrominae). Characteristics of the final data set of 43 taxa are
given in Table 2. Similar to waxy, final Bayesian analyses
mixed well as judged by acceptance rates and agreement
among runs. Although LFY was too variable to be informative
outside of Physaleae, it provided a good resolution within
Iochrominae. Using other Physaleae as outgroup taxa, as
indicated by waxy and ITS (Fig. 1), LFY produced an ingroup
topology with many of the same well-supported clades that
appear in ITS and waxy, but with some differences in
relationships among the groups. Unlike waxy but similar to
ITS, LFY placed the U clade sister to the rest of Iochrominae
(BS 70%, PP 1.0; Fig. 2). As in waxy analyses, LFY supported
a northern Andean clade with Acnistus and most of Iochroma
(A, C, L, and F; Fig. 2) and a clade with Dunalia,Eriolarynx,
Saracha, and Vassobia (D, E, S, and V; Fig. 2). LFY supported
a monophyletic group of Acnistus and Acnistus-like iochromas
(the A clade, Fig. 2) sister to a clade comprising other
Iochroma subclades (C, L, and F; Fig. 2). This is in contrast
with waxy, which placed the F clade sister to a clade
comprising A, L, and C (but with A unresolved).
Divergent alleles in LFY and waxy—Three species,
Eriolarnyx lorentzii,Iochroma ‘‘sagasteguii,’’ and I. ayaba-
cense, had divergent LFY alleles, and I. ayabacense also had
divergent waxy alleles. In the case of E. lorentzii, one LFY allele
formed a clade with E. fasciculata and the other with I. australe
(Fig. 2). When the two alleles are constrained to be sister, the
resulting trees are significantly longer than the optimal trees
(WSR, P¼0.0001–0.0017), suggesting that E. lorentzii alleles
are not exclusive and that there may be true genealogical
discordance (e.g., due to lineage sorting or hybridization).
One LFY allele of Iochroma ‘‘sagasteguii’’ was sister to
a sample of Acnistus arborescens, whereas the other fell in the
distantly related U clade (Fig. 2). The LFY alleles of I.
ayabacense were split between the C and L clades. When either
I. ‘‘sagasteguii’’ or I. ayabacense alleles were forced to form
a clade, the resulting trees were significantly longer than
unconstrained trees (WSR, P¼0.0001–0.004 for I. ayabacense
and P,0.0001 for I. ‘‘sagasteguii’’ ). Iochroma ayabacense
also showed divergent waxy alleles, with one allele in the C
clade and the other in the L clade, consistent with the LFY
analysis (Fig. 2). Constraining the two waxy alleles from I.
ayabacense to form a clade resulted in some significantly
longer trees (WSR, P¼0.025–0.096). The distant placement of
I. ‘‘sagasteguii’’ LFY alleles and I. ayabacense LFY and waxy
alleles points to a hybrid origin for these taxa, a possibility that
will be explored in more detail in the discussion.
Discordance among genes—The ILD (Farris et al., 1994)
was used as an initial test of ‘‘ global’’ congruence among and
within data partitions. An ILD test indicated that the assign-
ments of characters to the three waxy partitions (first and
second codon positions, third codon positions, and introns) was
not significantly different from random (P¼0.70), suggesting
that waxy can be treated as a single data partition. In contrast,
pairwise comparisons of the ITS, LFY, and waxy (excluding
non-Physaleae outgroups and putative hybrids) all yielded
significant ILD tests (P,0.01), indicating that the three data
sets are not drawn from the same population of characters (but
see Darlu and Lecointre, 2002; Hipp et al., 2004). We
attempted to localize the discordance by repeating the ILD
test with successively pruned data sets (Table 3). We divided
the data set into three parts, the ACLF group, the DESV group
and the U group, and we found that only the DESV returned
significant P-values (Table 3). However, simply deleting the
DESV taxa from the larger clade did not result in insignificant
ILD results (not shown), suggesting that it was not the sole
source of incongruence.
Templeton tests—Although ILD tests suggested significant
differences in signal among data sets, inspection of the
brackets on each tree. The labels D, E, S, and V indicate members of the traditional genera Dunalia,Eriolarynx,Saracha and Vassobia, respectively.
Iochroma has been divided into smaller clades: A, including Acnistus and Acnistus-like iochromas; C, containing the type I. cyaneum; L, after I. lehmannii;
F, after I. fuchsioides; and U after I. umbellatum. Solid bracket lines indicate monophyly; dashed bracket lines indicate non-monophyly. The D’s in the
combined tree designate points of difference between the combined likelihood tree and combined Bayesian consensus; PP for clade ACL is 0.97 and PP for
clade AL is 1.0 (see text, Results, Combined analysis).
August 2006] SMITH AND BAUM—PHYLOGENETICS OF IOCHROMINAE 1145
individual trees revealed only eight points of hard in-
congruence (conflicting clades with BS .70; Mason-Gamer
and Kellogg, 1996) among the three gene trees (Fig. 2). Three
of these cases were due to differences in the placement of
divergent LFY or waxy alleles. We used Templeton tests to
compare the remaining five sources of hard incongruence
(Table 4). In all cases, one or the other partition failed to reject
the conflicting resolution at the P,0.05 level. This suggests
that the incongruence detected by ILD tests is ‘‘ diffuse’’ rather
than due to particular points of discordance.
Combined analysis—Before conducting combined analyses,
we removed all of the putative hybrids, Iochroma stenanthum,
I. ayabacense, and I. ‘‘sagasteguii,’’ as they appeared to be
a source of conflict among data sets. We chose not to remove
E. lorentzii despite its divergent alleles because that would
severely reduce our sampling of Eriolarynx. Instead, we
removed the E. lorentzii LFY allele B, whose position conflicts
with that supported by ITS and waxy. In addition, we reduced
the outgroup sampling to include only other Physaleae
tresia, and Larnax; Fig. 1).
Parsimony analysis of the combined data set of 40 taxa and
4023 characters yielded 12 MPTs (Table 2) and increased
support for many of the clades observed in individual data sets
(Fig. 2). Similar results were obtained for ML and Bayesian
analyses. For example, among individual analyses, the A clade
only appeared in the LFY tree (BS ¼74%; PP ¼1.0; Fig. 2), but
it appeared in the combined analysis with a BS of 90% and PP
of 1.0. Likewise, the placement of the U clade sister to the rest
occurred with moderate support in LFY (BS ¼70%, PP ¼1.0;
Fig. 2) and weak support in analyses of ITS (BS ¼33%, PP ¼
0.12; not shown), but appeared strongly supported in the
combined analysis (BS ¼90%, PP ¼0.99; Fig. 2). Nonetheless
several areas on the combined tree remain unresolved, most
notably within the DESV clade and within the A clade. Also,
there were differences among modes of analysis. Clades C, L,
and F together formed a clade in parsimony and ML searches
of the combined data (Fig. 2), but Bayesian analyses showed
clade F as sister to an A, C, and L clade and clade A sister to
clade L with high posterior probability (PP ¼0.96–1.0) at all
relevant nodes (tree not shown, but see Fig. 2 caption).
Exploration of pruned data sets (not shown) established that the
resolution among the A, C, L, and F clades in a Bayesian
framework is very sensitive to the inclusion or exclusion of L
and to model choice (e.g., whether data partitions were allowed
to evolve under different models or whether they were linked
as in traditional likelihood searches).
Congruence in a Bayesian framework—As an additional
assessment of congruence, we compared the results of the
Bayesian analysis of the combined data set (described
previously) with the results from Bayesian analyses of
individual data sets that had been pruned to the same 40 taxa
(Buckley et al., 2002). The results of these runs are provided in
Appendix S1 (see Supplemental Data accompanying the online
version of this article). We found that there were no trees that
were shared between the posterior distributions (post burn-in)
of the individual and combined data sets. This is perhaps not
surprising given that there are 1.3 310
trees for 40 taxa and thus a fairly small chance that different
data sets would sample exactly equivalent topologies.
We next examined localized points of disagreement among
data sets in the Bayesian framework. Within the ACLF group,
we observed that LFY had a PP of 0.0 for F sister to ACL,
whereas waxy had a PP of 0.0 for the F sister to C topology.
This suggests that there may be true genealogical discordance
between LFY and waxy within the ACLF clade (Table 4,
conflict 5). A contrasting result was found with respect to the
placement of the U clade (sister to the rest in LFY and sister to
A, C, L, and F in waxy). We found that trees with U sister to the
rest of Iochrominae (the ‘‘U-sister’’ topology), as suggested by
LFY, appeared in waxy posterior distributions with a PP of
0.0078. Similarly U was sister to A, C, L, and F (the ‘‘ U-nested’’
topology) in the LFY posterior with a PP of 0.001. While these
values are lower than the traditional 0.05 threshold, the fact that
both topologies were present in the posterior distributions for
both data sets suggests that there may not be hard incongruence
(consistent with the WSR tests, Table 4). On the other hand, the
fact that the combined analysis supports the U-sister topology
more strongly than does LFY alone, suggests that U-sister is
a more plausible hypothesis at this time than U-nested.
Position of Iochrominae in Solanaceae—Our goal in
outgroup sampling was to confirm the monophyly of
Iochrominae and verify that it belongs in Physaleae as indicated
by plastid data. Indeed, once Iochroma cardenasianum is
excluded, Iochrominae appears to be monophyletic. Plastid data
(Olmstead et al., University of Washington, personal commu-
nication) confirm the distant relationship of I. cardenasianum to
Iochrominae and place it within the Datureae.
Our data support the inference that Iochrominae is part of
Physaleae, but its relationship to other taxa remains unclear. Of
the three markers, the LFY intron could not be readily aligned
with the more distant outgroups, and ITS provided little
resolution among Physaleae (Fig. 1). However, analysis of
waxy alone and combined analysis of waxy and ITS strongly
supported Iochrominae as sister to Physalinae sensu Olmstead et
al. (1999) plus Tubocapsicum. This result disagrees with the
most recent plastid phylogeny, which places Deprea plus
Larnax sister to Iochrominae, albeit with weak support
(Olmstead et al., University of Washington, personal commu-
nication). Resolving the lineages that comprise Physaleae and
the relationships among them will require increased sampling
and perhaps additional markers.
Taxonomic implications for genera of Iochrominae—
Acnistus—In Hunziker’s (1982) revision of Acnistus,he
acknowledged that Acnistus has greatest affinity to the genus
TABLE 3. Pvalues from incongruence length difference (ILD) tests of
combined data sets. In each case, the putative hybrids, Iochroma
stenanthum,I. ayabacense, and I. ‘‘sagasteguii,’’ were already
Data set ITS vs. waxy ITS vs. LFY LFY vs. waxy
Iochrominae þPhysaleae outgroups 0.001** 0.001** 0.001**
Iochrominae 0.001** 0.002** 0.007**
Clades A, C, L, F only 0.446 0.057* 0.479
Clades D, E, S, V only 0.206 0.01** 0.001**
Clade U only 1.0 1.0 1.0
Notes: Clade names (A, C, etc.) are explained in caption for Fig. 2. **,
significant at P,0.01; *, marginally significant values.
1146 AMERICAN JOURNAL OF BOTANY [Vol. 93
Iochroma. The important differences he noted between them
were the small flowers and anthers of Acnistus, the calyx
(accresent in Iochroma but not in Acnistus), and the bud
aestivation (induplicate in Iochroma and valvate in Acnistus).
Confusing this demarcation are a few species currently placed in
Iochroma that have the latter two characteristics of Acnistus.For
example, I. ellipticum and I. confertiflorum, two large-flowered
species that were transferred from Acnistus by Hunziker (1977,
1982), have valvate bud aestivation and lack a strongly
accresent calyx. This combination of traits is also found in
two recently named species, I. edule and I. salpoanum (Leiva,
1995; Leiva et al., 2003) and in I. peruvianum. Furthermore,
field observations of these five iochromas (S. D. Smith, personal
observation) indicate that they share with Acnistus a conspicuous
green mark on the inner surface of the corolla lobe, which fades
to yellow as the flower ages (Fig. 3). Thus, it is not surprising
that Acnistus and these five other species form a well-supported
clade in our analyses (clade A), but whether this group should
be officially segregated from Iochroma deserves careful
consideration and will be discussed further (see Iochroma).
The small-flowered form traditionally named Acnistus
arborescens occurs from Argentina to Mexico and the
Caribbean and is morphologically variable, with 28 synonyms
in the taxonomic literature (Hunziker, 1982). The three
accessions representative of this traditional species do not
form a monophyletic group on any of the gene trees (though
a clade is not contradicted by waxy). One possible explanation
is that there has been incomplete lineage sorting within the A
clade. This seems unlikely because our screen of clones
revealed no allele sharing among other species in the A clade:
all alleles from a given group A species formed a clade at all
loci. Another interpretation is that A. arborescens refers to
a lowland progenitor form that has given rise to multiple novel
higher-elevation forms, similar to the case of Lisianthus
skinneri (Sytsma and Schaal, 1985). Alternatively, because A.
arborescens may occasionally hybridize in nature with related
higher-elevation taxa such as Iochroma confertiflorum (S. D.
Smith, personal observation), it is possible that different A.
arborescens populations have acquired different introgressed
alleles from other members of the A clade.
Dunalia—Hunziker’s (1960) delimitation of Dunalia cen-
tered on a single character, the presence of enlarged and showy
‘‘stapets,’’ which appear as winged or toothed lateral
appendages emerging from the filament bases at the point of
their insertion on the corolla tube. Our analyses suggest that
Dunalia sensu Hunziker (1960) is not monophyletic. Notably,
the type species, D. solanacea appears more closely related to
Saracha than to other Dunalia species. Whereas other Dunalia
species are xerophytes of the central and southern Andes, D.
solanacea is a northern Andean cloud forest shrub with a dense
indumentum of stellate hairs, anisogeminate leaves, and small,
yellow-green, trumpet-shaped flowers. Although its placement
within Saracha could be a phylogenetic artifact (note that D.
solanacea has a long terminal branch for all genes), there is no
evidence of an association between this species and the other
The remaining four Dunalia species are similar to each other
in morphology, distribution, and habit; however, they do not
form a clade in any of the trees. Furthermore, one species
traditionally placed in Iochroma,I. parvifolium, appears more
closely related to some dunalias. However, the association of I.
parvifolium with D. brachyacantha and D. spinosa is reason-
able given its spiny xerophytic habit and tubular purple
flowers. Iochroma parvifolium was placed in Iochroma as
opposed to Dunalia because it lacks the showy stapets
(Hunziker, 1977). Nevertheless, close examination of fresh
flowers of I. parvifolium in the field revealed small, tooth-like
expansions of the stapets, which are hard to detect in dried
specimens (S. D. Smith, personal observation). Also, during
the course of collection trips, one population of I. parvifolium
was found to be gynodioecious, a condition found in some
Dunalia species (S. D. Smith, personal observation). Iochroma
species (members of A, C, L, F, and U) are invariably
hermaphroditic and never spiny, making I. parvifolium an
unlikely Iochroma. The epithet ‘‘parvifolia’’ does not exist in
Dunalia, but transferring I. parvifolium to Dunalia is
confounded by the fact that D. solanacea, the type species, is
not associated with the other ‘‘Dunalia’’ species, making the
taxonomic future of Dunalia uncertain.
TABLE 4. Wilcoxon signed-ranks tests of conflicting phylogenetic hypotheses. The instances of conflict described can be observed in the gene trees in
Fig. 2. See Fig. 2 legend for explanation of clade names.
Conflicting topologies Constraint Result
1. I. stenanthum in clade C (ITS: BS 93%) vs. outside clade
Force I. loxense,I. cyaneum and I. cornifolium
to form a clade in ITS
Force I. loxense,I. cyaneum and I. cornifolium
and I. stenanthum form a clade in LFY and waxy
2. A. arborescens (Peru) sister to I. confertiflorum (ITS: BS 82%)
vs. sister to A. arborescens (Ecuador) (LFY:BS94%)
Force A. arborescens (Peru) sister to
A. arborescens (Ecuador) in ITS
Force I. confertiflorum sister to A. arborescens
(Peru) in LFY
3. D. spathulata sister to Vassobia (LFY:BS71%) vs. sister
to D. obovata (waxy:BS70%)
Force D. spathulata sister to D. obovata in LFY
Force D. spathulata sister to Vassobia in waxy
4. Clade U sister to rest of Iochrominae (LFY:BS75%) vs. sister
to clade with A, C, L, and F (waxy:BS70%)
Force U to form a clade with A, C, L, and F
(including putative hybrids) in LFY
Force all members of A, C, L, and F
(including I. stenanthum and I. ayabacense)
and D, E, S, and V form a clade in waxy
5. C forms a clade with F (LFY:BS92%)
vs. with A and L (waxy:BS71%)
Force A, L, and C (including I. ayabacense)
to form a clade in LFY
Force C (including I. ayabacense A) and
clade F to form a clade in waxy
August 2006] SMITH AND BAUM—PHYLOGENETICS OF IOCHROMINAE 1147
Fig. 3. Floral diversity, biogeography, and hybridization in Iochrominae. Cladogram showing relationships from combined analysis with the well-
supported (BS .70%, PP .0.95) branches bolded. See Fig. 2 caption for explanation of clade names (L, C, F, etc.); members of Dunalia and Saracha are not
labeled as the genera are non-monophyletic. Colored boxes indicate entire geographic distribution with the exception of A. arborescens (widespread, with
1148 AMERICAN JOURNAL OF BOTANY [Vol. 93
Eriolarynx—The three species of Eriolarynx, recently
segregated from Vassobia, can be distinguished from other
Iochrominae by the dense ring of trichomes inside the corolla
(Hunziker, 2000). Our analysis upholds the monophyly of
Eriolarynx, with the addition of I. australe. This species was
originally described in Iochroma (Grisebach, 1874), but later
transferred to Acnistus (Grisebach, 1879) and then to Dunalia
(Sleumer, 1950). Iochroma australe was not a good fit in
Iochroma because its variable flowers can sometimes be short
and funnel-shaped and because the corolla interior is densely
pubescent near the base, whereas other Iochromas are typically
glabrous. Further, it lacks the valvate aestivation of Acnistus
and the characteristic filament appendages of Dunalia. The
hairy flowers suggest a better fit with Eriolarynx despite the
fact that the three described species typically have rotate or
campanulate flowers, while I. australe has a funnel-shaped or
tubular corolla. Geography also argues for this placement
because both I. australe and Eriolarnyx are restricted to Bolivia
and Argentina. There is no good argument against creating the
new combination E. australe, except that this may prove to be
only a temporary solution if it becomes necessary to combine
the entire DESV clade into a single genus (with or without
other elements of Iochrominae).
Iochroma—Species currently identified as Iochroma were
not found to form a clade, even after the misplaced I. australe
and I. parvifolium are ignored. One group of iochromas, the U
clade appears as sister to remainder of Iochrominae. We
consider this ‘‘U-sister’’ position to be strongly supported by
our study for three reasons. First, two of the three loci sampled,
ITS and LFY, support or are compatible with the ‘‘ U-sister’’
topology. Second, heuristic searches using the waxy data
constrained to be consistent with ‘‘U-sister’’ topology do not
result in trees that are significantly longer than unconstrained
trees (Table 4). Last, despite the differences in topology among
loci, support for a ‘‘U-sister’’ relationship is highest in the
combined analysis. Specifically the combined analysis of all
three genes yielded a 90% bootstrap, as contrasted with a 78%
bootstrap support for this relationship in a two-gene combined
analysis of LFY and ITS (not shown). This pattern suggests that
even though waxy does not return U as sister to the rest of
Iochrominae, the waxy data do contain some support for this
topology (Olmstead and Sweere, 1994).
The U group is distinguished from species in the ACLF
clade by the form of the corolla and the androecium. Flowers of
ACLF (excluding Acnistus) are funnel-shaped or tubular,
whereas those of the U group are salverform. Also, the
filaments are attached near the base in ACLF, while in the U
group they are attached near the middle of the corolla tube
(often with a visible bump at the point of attachment, e.g., I.
grandiflorum, Fig 3.). The most extreme example of filament
adnation in the U group is I. ‘‘tingoense’’ in which the anthers
are more or less sessile on the corolla. Thus, even if one
doubted the sister group relation between the U clade and other
Iochrominae, there is reason to believe that the U clade is
divergent from other traditional iochromas.
If the U clade (and I. australe and I. parvifolium) were
excluded from Iochroma and if Acnistus were expanded to
include the entire A clade (discussed previously), then one
could imagine assigning only members of clades C, L, and F to
Iochroma. However, this decision would be premature
considering that it is not certain from these data that C, L,
and F form a clade. Furthermore, there are no clear
morphological differences between Acnistus and Iochroma,
largely because I. squamosum and I. lehmannii (clade L)
possess a mixture of traits from clade A on the one hand and
clades C and F on the other; the bud aestivation is induplicate,
resulting in wide corolla lobes and plaits in the corolla tube,
like C and F, but the yellow flowers lack anthocyanins
(Hunziker, 1982) and have the green markings on the corolla
lobes, as in clade A. The other alternative, if we are to only
recognize monophyletic groups, is to sink Acnistus into
Iochroma. We also note that in a rank-independent system of
nomenclature, Acnistus could be defined as a monophyletic
group within a monophyletic Iochroma.
Saracha—This small genus of high-elevation treelets is
morphologically well defined, including two species of pa´ramo
treelets with small coriaceous or subcoriaceous leaves and
funnel-shaped or campanulate flowers that can be purple or
yellow with purple spots (Alvarez, 1996). Dunalia solanacea,
which often appears nested within Saracha, does not share any
obvious features with Saracha except for its high-elevation
distribution and occurrence in the northern Andes. As noted, D.
solanacea has a long terminal branch for all tree genes, raising
the possibility that its placement within Saracha is an artifact.
Moreover, although Saracha only appears monophyletic in ITS
trees and not in LFY or waxy trees, we note the sister
relationship of S. quitensis and S. punctata does appears in
Bayesian analyses of LFY with PP 0.08 and in those of waxy
with PP 0.33 (Appendix S1, see Supplemental Data accompa-
nying the online version of this article). Thus, we consider it
premature to conclude that Saracha is nonmonophyletic.
Vassobia—Among the genera of Iochrominae, Vassobia is
the only one that appeared monophyletic in all analyses.
Vassobia includes two southern Andean species with small,
purple, campanulate, glabrous flowers: V. dichotoma a cloud
forest tree restricted to Bolivia, and V. breviflora, a widespread
spiny shrub (Hunziker, 1984, 2001). The stapets of Vassobia are
expanded to form small ‘‘auricles’’ similar to the appendages
found in Eriolarynx (Hunziker, 2001). Considering that species
of Eriolarynx formerly belonged to Vassobia, one might have
expected a sister relationship between the genera. These data
neither support nor strongly contradict this inference.
Hybridization in Iochrominae—Identifying hybrid taxa is
a challenge for phylogenetics because reticulation erodes the
strictly tree-like process of evolution assumed by most
phylogenetic methods (McDade, 1990). Nonetheless, even
when species trees are reticulate, gene trees will be strictly
divergent structures so long as the rate of intragenic
samples from Ecuador, Peru, and Costa Rica included in this analysis) and V. breviflora, (widespread through southern South America). The samples of A.
arborescens from Peru and Ecuador are condensed to a single line because they appear to be sister taxa in the combined analysis; the A. arborescens from Costa
Rica is abbreviated ‘‘A. arb.’’ Gray, curved lines connect the putative hybrids, I. ayabacense,I. stenanthum, and I. ‘‘ sagasteguii’’ to their putative parents.
August 2006] SMITH AND BAUM—PHYLOGENETICS OF IOCHROMINAE 1149
recombination is low relative to the rate at which lineage
sorting occurs. Given that homoploid hybrid taxa potentially
carry genetic contributions from one or both parents, we may
observe divergent alleles on a single gene tree (with alleles
associated with each parent) or disagreement among gene trees,
with hybrid alleles appearing related to one parental lineage on
one tree and to the other parent on a different gene tree. Thus,
we can test hypotheses of hybrid ancestry by identifying
divergent alleles or points of conflict among gene trees (Doyle,
1992; Maddison, 1997). In this study, our sampling included
three taxa, Iochroma ayabacense,I. ‘‘sagasteguii,’’ and I.
stenanthum, which we had hypothesized to be of hybrid origin
due to their distribution and morphology.
Iochroma ayabacense was hypothesized to be an in-
terspecific hybrid between I. cyaneum and I. squamosum.
Iochroma ayabacense occurs in at high elevations (2600–2700
m a.s.l.) around the city of Ayabaca in northern Peru, often in
proximity to populations of its putative parents, I. squamosum
and I. cyaneum. The infrequent I. squamosum favors mildly
disturbed habitats like forest gaps or riparian areas, whereas the
widespread I. cyaneum tolerates drier conditions and open
habitats like roadsides and pastures. The two putative parents
are, however, found occasionally in close proximity, for
example, when a road passes through a patch of forest. Several
morphological features pointed to the possibility that I.
ayabacense was a hybrid between these two. It has peculiar
yellowish-purple flowers intermediate between the yellow I.
squamosum and the purple I. cyaneum, and it has yellow-green
markings inside the corolla, which are signatures of clades A
and L. Our phylogenetic analyses revealed divergent alleles of
I. ayabacense in both waxy and LFY trees, and in each case,
one I. ayabacense allele fell in clade C and one in clade L (Fig.
2). In ITS trees, I. ayabacense appeared to be sister to I.
squamosum in the L clade (Fig. 2). Considering these gene
trees together with its distribution and morphology, we
conclude that I. ayabacense is a hybrid between I. cyaneum
and I. squamosum. Further field research and genetic data
would be needed to determine if I. ayabacense is best
interpreted as a hybrid species or a transient hybrid form that
lacks sufficient permanence to warrant species status.
Iochroma ‘‘sagasteguii’’ has small white flowers with greenish
markings inside the corolla that resemble Acnistus. However, the
pubescence on the calyx and corolla, the slightly induplicate bud
aestivation, and the extended area of filament adnation are
reminiscent of species in the U group. Although the distribution
of I. ‘‘sagasteguii’’ is not well known, in some localities in
northern Peru, it grows within a few kilometers of populations of
I. stenanthum,I. cornifolium,I. grandiflorum,andI. cf.
peruvianum and within 15 km of populations of Acnistus
arborescens. Similar to I. ayabacense, genetic evidence
supported the hypothesis of hybrid ancestry in I.‘‘ sagasteguii.’’
We found divergent alleles in the waxy tree, with one allele in the
U group and another in clade A. The genetic data, the
morphology, and the geography point to I. grandiflorum and
A. arborescens as the most likely parental species.
Iochroma stenanthum was the third suspected hybrid. It
occurs in northern Peru and has long, tubular, pubescent flowers,
most similar to I. cornifolium (Leiva et al., 1998), but with more
triangular corolla lobes and yellow-green markings inside the
corolla lobes as in clade A. The corolla color, which fades from
cream at the base to purple at the apex, suggests that it is the
result of crossing a white-flowered species (e.g., Acnistus
arborescens) and a purple-flowered species. Iochroma sten-
anthum occurs in close proximity of populations of the putative
parents, I. cornifolium and A. arborescens. However, our data
were insufficient to resolve the relationship of I. stenanthum to
other Iochrominae. Its position varied among gene trees and was
generally poorly supported. This pattern might be ascribed to
lineage sorting, but its morphology is so strongly indicative of
a hybrid ancestry that we favor the hypothesis that I. stenanthum
is the product of a more ancient hybridization event whose
genetic signatures have been blurred by subsequent evolution.
Biogeographical context of the Iochrominae radiation—
Simpson (1975) recognized that phytogeographical distributions
in the Andes tend to coincide with the geologically defined
structural units of the Cordilleras. Many subsequent authors have
observed such a relationship (e.g., Berry, 1982; Luteyn, 2002),
although the exact delimitation of the structural units and
associated phytogeographic zones varies slightly among studies.
For instance, Berry (1982) modified Simpson’s (1975) structural
units by recognizing the Amotape–Huancabamba zone (A–H
zone), an area of low elevation between the northern and central
Andes (4–88S), as a separate unit. Weigend (2002; Weigend et
al., 2004) supported Berry’s distinction, noting the large number
of A–H zone endemics, and additionally suggested distinguish-
ing the Andes below 188S as the southern Andes (as in Fig. 3). As
a basis for discussing the biogeography of Iochrominae, it is
useful to divide the tropical Andes into northern, central, and
southern regions, to recognize the A–H zone as a distinct unit,
and to divide the central Andes into a region north of the Pisco
deflection (148S; Berry’s Cordillera Central and Occidental) and
a region south of the deflection (Berry’s Cordillera Oriental).
Similar to other plant groups that have radiated in the Andes
(e.g., Fuchsia and Nasa), distribution patterns of Iochrominae
species and clades strongly reflect the structural units of the
Andes (Fig. 3). The diverse ACLF clade, excluding the weedy
Acnistus arborescens and the Galapagos endemic Iochroma
ellipticum, is restricted to the Andes from 58Nto88S, the
southern boundary of the A–H zone. The DEV group contains
taxa that only occur below 88S, while its probable sister group,
clade S, is widely distributed from 98Nto168S. Clade U
straddles the ACLF and the DEV groups, with a distribution
from 48S (the northern limit of A–H zone) to 108S.
Despite the clear patterns along the latitudinal gradient, we
do not observe strong east–west separation of clades as has
been the case in many Andean groups (Berry, 1982; Slade and
Moritz, 1998; Brower, 1994). Although there is some tendency
for greater species richness on the western cordilleras, several
taxa, e.g., I. calycinum and Dunalia solanacea, are known to
occur on both sides of the Andes. However, the distribution of
many species remains poorly characterized. With increased
collecting effort, it may eventually be possible to determine if
Iochrominae distributions follow east–west structural units as
closely as they do north–south units.
Iochrominae show a center of diversity in the A–H zone,
where 16 of 33 (48%) species (excluding I. cardenasianum)
occur, 11 of which are restricted to this zone. This enhanced
diversity can be attributed to the overlap of the ACLF and U
clades. The A–H zone is characterized by fragments of the
Cordilleras, usually less than 3500 m a.s.l, separated by valleys
that dip down to ca. 1000 m a.s.l. Iochrominae prefer cloud
forest or Andean scrub forest between 2300 and 2800 m a.s.l and
are abundant in the high elevation valleys of the A–H zone. In
some areas, as many as five species may occur over the distance
of a few kilometers. The proximity coupled with the ease of
1150 AMERICAN JOURNAL OF BOTANY [Vol. 93
crossing has resulted in several hybrid taxa, as revealed by this
study, all of which are confined to this A–H zone (Fig. 3).
Here we have examined the three putative hybrid taxa with
three loci, but this represents only a first attempt at exploring
hybridization in Iochrominae. Further investigation into the
potential hybrid ancestry of all Iochrominae should include
samples of multiple individuals and populations per taxon,
additional chromosome counts, statistical morphometric stud-
ies, characterization of species distributions, and analysis of
mitochondrial or plastid markers. Greater sampling of
individuals, taxa, and genes will permit a more fine-tuned
estimate of the frequency of hybridization and introgression in
As documented in this study, episodes of hybridization have
clearly impacted the evolutionary history of Iochrominae.
However, considering the amount of agreement among the
three nuclear markers, it appears that these events have not
entirely obscured the underlying divergent phylogenetic
history, having only clouded the branching pattern in some
parts of the tree. Furthermore, the presence of leaky species
boundaries has not apparently precluded the diversification of
Iochrominae. In addition to being the most florally diverse
subtribe in Physaleae and perhaps Solanoideae, Iochrominae
also boasts the greatest diversity of pollination systems
(Cocucci, 1999). Perhaps the combination of pollinator-
mediated selection, microallopatry in dissected Andean
habitats and episodic hybridization have together permitted
the explosion of floral diversity seen in Iochrominae.
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APPENDIX 1. Taxon sampling within Solanoideae, GenBank accession numbers (ITS, LFY,waxy), and voucher information. Tribes (ending -eae) and
subtribes (ending -inae) are given when available (Olmstead et al., 1999; R. G. Olmstead et al., University of Washington, personal communication).
Voucher specimens have been deposited in the following herbaria: BIRM ¼University of Birmingham; CDS ¼Charles Darwin Research Station, NY
¼New York Botanical Garden, UT ¼University of Utah, WIS ¼University of Wisconsin-Madison.
Taxon—GenBank accession nos.: ITS, LFY,waxy; voucher information.
Nicandra physaloides (L.) Gaertn.—DQ314155, DQ309515, DQ309465;
Peru. Dept. Amazonas. Prov. Chachapoyas, 6.242918S 77.874438W,
2250 m, 11-II-04, Smith 369, WIS.
1152 AMERICAN JOURNAL OF BOTANY [Vol. 93
Iochroma cardenasianum Hunz.—DQ314156, DQ309516, DQ309466;
Bolivia. Dept. Potosı´. Carretera Potosi-Orkhola-Tumusla. 20.396388S
65.562878W, 3099 m, 18-II-04, Smith 385, WIS.
Solanum lycopersicum L.—DQ314157, DQ309517, DQ309467; UW–
Madison, Botany Living Collections s.n.
Capsicum lycianthoides Bitter—DQ314158, DQ309518, DQ309468;
Ecuador. Prov. Pichincha. 0.01578S 78.6808W, 2250 m, 23-XII-02,
Smith 203, WIS.
Lycianthes inaequilatera Bitter—DQ314159, DQ309519, DQ309469;
Ecuador. Prov. Pichincha. 0.3268S 79.0008W 800 m, 25-XII-02, Smith
Salpichroa tristis Walp.—DQ314160, DQ309520, DQ309470; Bolivia.
Dept. Potosı´. Ca. 19.58S 65.458W, 4020 m, 18-II-04, Smith 382, WIS.
Physalis peruviana L.—DQ314161, DQ301514, DQ309471; Ecuador.
Prov. Pichincha. Gardens of Herbario Nacional (QCNE), 2800 m, 1-I-
03, Smith 217, WIS.
Leucophysalis grandiflora (Hook.) Rydb.—DQ314162, DQ301515,
DQ309472; Olmstead S-30, WTU.
Witheringia solanacea L’Herit.—DQ314164, DQ301517, DQ309474;
D’Arcy 16399, MO.
Tubocapsicum anomalum (Franchet & Savat.) Makino—DQ314163,
DQ301516, DQ309473; Chen 231, MO.
[Subtribe not known]
Cuatresia harlingiana Hunz.—DQ314165, DQ301518, DQ309475;
Ecuador. Prov. Pichincha. 0.01578S 78.6808W, 2250 m, 24-XII-02,
Smith 204, WIS.
Larnax sachapapa Hunz.—DQ314166, DQ301519, DQ309476;
Ecuador. Prov. Pichincha. 0.01578S 78.6808W, 2250 m, 24-XII-02,
Smith 205, WIS.
Acnistus arborescens (L.) Schlecht.—DQ314173, DQ301528,
DQ309483; Costa Rica. Prov. Puntarenas. Las Cruces B. S., 1992,
Bohs 2428,UT.A. arborescens (L.) Schlecht.—DQ314181,
DQ301536, DQ309491; Ecuador. Prov. Pichincha. 0.32608S 79.
0008W, 750 m, 25-XII-02, Smith 209, WIS. A. arborescens (L.)
Schlecht.—DQ314183, DQ301538, DQ309493; Peru. Dept.
Cajamarca. 7.424098W 78.901118S, 1976 m, 10-I-04, Smith 312, WIS.
Dunalia brachyacantha Miers—DQ314172, DQ301527, DQ309482;
Argentina. Prov. Jujuy, 2100 m, 20-IX-02, Nee and Bohs 50811,
NY. D. obovata Dammer—DQ314192, DQ301547, DQ309499; Peru.
Dept. Junin. 11.349198S 75.574088W, 2679 m, 8-III-04, Smith 458,
WIS. D. spathulata (Ruı´z & Pav.) Braun & Aschers—DQ314198,
DQ301554, DQ309506; Peru. Dept. Huanuco. 9.838318S76.
115038W, 1842 m, 6-III-04, Smith 452, WIS. D. solanacea H. B. &
K.—DQ314174, DQ301529, DQ309484; Ecuador. Dept. Pichincha,
ca. 0.238S 78.758W, ca. 2200 m, 31-XII-02, Smith 211, WIS. D.
spinosa Dammer—DQ314188, DQ301543, DQ309495; Bolivia. Dept.
Potosi, ca. 19.68S 65.68W, 4020 m, 18-II-04, Smith 379, WIS.
Eriolarynx lorentzii (Dammer) Hunz.—DQ314171, DQ301525/
DQ301526 (allele A/allele B); DQ309481; Argentina. Prov.
Tucuman, 26.6338S 65.4678W, 1700 m, 12-II-1966, Hawkes et al.
3452, BIRM. E. fasciculata (Miers) Hunz.—DQ314196, DQ301552,
DQ309504; Bolivia. Dept. Cochabamba, 17.464778S 65.752178W,
3180 m, 26-II-04, Smith 432, WIS.
Iochroma australe Griseb.—DQ314189, DQ301544, DQ309496; Bolivia.
Dept. Chuquisaca. 20.784778S 65.040888W, 3038 m, 18-II-04, Smith
390,WIS.I. ayabacense S. Leiva—DQ314194,
DQ301549/DQ301550 (allele A/allele B), DQ309501/DQ309502
(allele A/allele B); Peru. Dept. Piura. 4.614628S 79.711788W, 2701
m, 15-I-04, Smith 337, WIS. I. calycinum Benth.—DQ314201,
DQ301557, DQ309512; Ecuador. Prov. Pichincha. 0.24577 S 78.
80903 W, 1834 m, 27-XII-04, Smith 471, WIS. I. confertiflorum
(Miers) Hunz.—DQ314176, DQ301531, DQ309486; Ecuador. Prov.
Loja, 4.13168S 79.92188W, 1582m, 15-I-03, Smith 237, WIS. I.
cornifolium Miers—DQ314177, DQ301532, DQ309487; Ecuador.
Prov. Loja, 4.08698S 79.93568W, 2570 m, 15-I-03, Smith 242, WIS. I.
cyaneum (Lindl.) M. L. Green—DQ314180, DQ301535, DQ309490;
Ecuador. Prov. Loja, 3.8498S 79.4278W, 2325 m, 4-I-03, Smith 223,
WIS. I. edule Leiva—DQ314193, DQ301548, DQ309500; Peru. Dept.
La Libertad, 7.9281838S 78.583688W, 2550 m, 8-I-04, Smith 300, WIS.
I. ellipticum (Hook. f.) Hunz.—DQ314199, DQ301555, DQ309507;
Ecuador. Galapagos, 0.22038S 90.76278W, 700 m, 6-X-02, Jager 622,
CDS. I. fuchsioides Miers—DQ314203, DQ301559, DQ309514;
Ecuador. Prov. Azuay. 2.751178S 78.886298W, 2828 m, 9-I-05,
Smith 488, WIS. I. gesnerioides Miers—DQ314179, DQ301534,
DQ309489; Ecuador. Prov. Pichincha, 00.03948N 78.5008W, 2500 m,
20-XII-02, Smith 200, WIS. I. grandiflorum Benth.—DQ314170,
DQ301523, DQ309480; Peru. Prov. Cajamarca, 7.379088W78.
893328S, 2781 m, 11-I-04, Smith 320, WIS. I. lehmannii Bitter—
DQ314200, DQ301556, DQ309511; Ecuador. Prov. Can˜ar, 2.31973 S
78.92679 W, 2475 m, 8-I-05, Smith 484, WIS. I. loxense Miers—
DQ314175, DQ301530, DQ309485; Ecuador. Prov. Loja, 3.9998S 79.
3068W, 2050 m, 3-I-03, Smith 220, WIS. I. nitidum S. Leiva &
Quipuscoa—DQ314168, DQ301521, DQ309478; Peru. Dept.
Amazons, 6.389608S 77.987958W, 2605 m, 11-I-04, Smith 371, WIS.
I. parvifolium (Roem. & Schult.) D’Arcy—DQ314195, DQ301551,
DQ309503; Peru. Dept. La Libertad, 7.94771678W 78.561958S, 2759
m, 8-I-04, Smith 303, WIS. I. cf. peruvianum (Dunal) J. F. Macbr.—
DQ314197, DQ301553, DQ309505; Peru. Dept. Cajamarca, 7.385978S
78.897748W, 2602 m, 16-I-04, Smith 353, WIS. I. salpoanum S. Leiva
& Lezama—DQ314187, DQ301542, DQ309509; Peru. Dept. La
Libertad. 8.008788S 78.639118W, 2696 m, 10-II-04, Smith 364, WIS.
I. squamosum S. Leiva & Quipuscoa—DQ314186, DQ301541,
DQ309494; Peru. Dept. Piura. 4.6596528S 79.740388W, 2730 m, 14-
I-04, Smith 330, WIS. I. stenanthum S. Leiva, Quipuscoa & N. W.
Sawyer—DQ314184, DQ301539, DQ309508; Peru. Dept. Cajamarca,
7.401168S 78.896588W, 1976 m, 10-I-04, Smith 313, WIS. I. sp.
nov.—DQ314202, DQ301558, DQ309513; Ecuador. Prov. Napo, 0.
367948S 78.104718W, 2811 m, 4-I-05, Smith 476, WIS. I. sp. nov.
‘‘sagasteguii’’—DQ314185, DQ301524/DQ301540 (allele A/allele B),
DQ309510; Peru. Dept. Cajamarca. 7.384898S 78.897208W, 2603 m,
10-I-04, Smith 317,WIS.I. sp. nov. ‘‘tingoense’’—DQ314167,
DQ301520, DQ309477; Peru. Dept. Amazonas, 6.379728S77.
909628W, 1800 m, 11-I-04, Smith 370, WIS. I. umbellatum (Ruı´z &
Pav.) D’Arcy — DQ314169, DQ301522, DQ309479; Peru. Dept. La
Libertad, 7.93441678S 78.5923678W, 2468 m, 8-I-04, Smith 301, WIS.
Saracha punctata Ruı´z & Pav.— DQ314182, DQ301537, DQ309492;
Bolivia. Dept. La Paz, 16.326948S 67.891678W, 2850 m, 12-V-01,
Nee 51804, NY. S. quitensis (Hook.) Miers—DQ314178, DQ301533,
DQ309488; Ecuador. Prov. Napo, 0.38248S 78.16008W, 3400 m, 21-I-
03, Smith 257, WIS.
Vassobia breviflora (Sendt.) Hunz.—DQ314190, DQ301545, DQ309497;
Bolivia. Dept. Chuquisaca, 18.893688S 65.115558W, 1922 m, 22-II-
04, Smith 412, WIS. V. dichotoma (Rusby) Bitter—DQ314191,
DQ301546, DQ309498; Bolivia. Dept. La Paz, 16.320338S67.
827008W, 2157 m, 28-II-04, Smith 440, WIS.
APPENDIX 2. PCR primers designed for this study. All primers listed 50to
Locus Primer Sequence
waxy F41 GGT GAT GTT CTT GCT GGA CTA C
waxy F420 CGT GGG GTT GAT CGT GTT TTT G
waxy R991 CCT TCG CAT TCA CAT AGA TTC C
waxy R1235 GCT TCT AAA CTT GGT GGT CTG A
LFY LFYSOL-F7 AAY GGY YTR GAT TAY YTG TTC CAT
LFY LFYSOL-F68 AGA MTA TTG CYA AGG AAC GRG GTG
LFY LFYSOL-R700 GAC AMT RTW GAR AAG TRC GTA GCA
LFY LFYSOL-R1000 CAA GGT TAC AGG TGG AGR TAC TYG
August 2006] SMITH AND BAUM—PHYLOGENETICS OF IOCHROMINAE 1153