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Phylogenetic placement, delimitation, and relationships among genera of the

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Phylogenetic placement, delimitation, and relationships among genera of the enigmatic
Nelsonioideae (Lamiales: Acanthaceae)
Author(s): Lucinda A. McDade, Thomas F. Daniel, Carrie A. Kiel and Agneta Julia Borg
Source: Taxon, Vol. 61, No. 3 (June 2012), pp. 637-651
Published by: International Association for Plant Taxonomy (IAPT)
Stable URL: http://www.jstor.org/stable/41679278
Accessed: 31-08-2017 16:01 UTC

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TAXON 61 (3) • June 2012: 637-651 McDade & al. • Phylogenetic placement and relationships among Nelsonioideae

Phylogenetic placement, delimitation, and relationships among genera

of the enigmatic Nelsonioideae (Lamíales: Acanthaceae)

Lucinda A. McDade,1'2 Thomas F. Daniel,3 Carrie A. Kiel1'2 & Agneta Julia Borg4

1 Rancho Santa Ana Botanic Garden, 1500 N. College Ave., Claremont, California 91711 U.S.A.
2 Department of Botany, Claremont Graduate University, Claremont, California 91711 U.S.A.
3 Department of Botany, California Academy of Sciences, 55 Music Concourse Dr., Golden Gate Park, San Francisco,
California 94118, U.S.A.
4 Department of Botany, Stockholm University, Lilla Frescativägen 5, 106 91 Stockholm, Sweden
Author for correspondence: Lucinda A. McDade, lucinda.mcdade@cgu.edu

Abstract We took a two-tiered approach to test monophyly of Nelsonioideae and place the group within Lamiales, and to deter-
mine relationships among taxa within the group. Phylogenetic analysis of a molecular dataset ( ndhF+trnL-F ) for a broad sample
of Lamiales supports monophyly of Nelsonioideae and places the clade with strong support as sister to a lineage composed of
all other plants treated as Acanthaceae ( Avicennia , Thunbergioideae, Acanthoideae). We propose to treat this entire group as
Acanthaceae s.l. and hypothesize that indurate, explosively dehiscent capsules are a synapomorphy for the family, albeit with
autapomorphic fruit types in Avicennia and Mendoncia. These results further support monophyly of family-level groups that
have emerged from recent studies of Lamiales but are largely unsuccessful in resolving relationships among these groups, as
also encountered by other workers. Our results contradict some aspects of relationships that have seemed resolved by earlier
studies, notably among Byblidaceae, Scrophulariaceae, Thomandersia , and other Lamiales. Among Nelsonioideae, analysis of
sequence data from rapidly evolving genie regions ( trnS-G , ndhF-rpl32+rpl32-trnL(]JAG' nrlTS) and a larger sample of nelso-
nioids (i.e., all genera and multiple taxa to represent the diversity of species-rich genera) indicates that Nelsonia and Elytraria
are monophyletic with strong support, but with only moderate support for Nelsonia as the first branching clade and Elytraria
sister to the remaining nelsonioids. An African clade comprising monospecific Saintpauliopsis sister to Anisosepalum (two
of three species sampled) is sister to a clade that includes all sampled members of pantropical Staurogyne plus New World
Gynocraterium and Asian Ophiorrhiziphyllon. Gynocraterium is sister to all sampled members of New World Staurogyne ; this
last clade is sister to a clade comprising the other sampled Staurogyne plus Ophiorrhiziphyllon , which is nested among Asian
Staurogyne. The taxonomie implications of these patterns of relationship are discussed. Our results suggest that Nelsonioideae
have a complex history of inter-continental dispersals compared to other lineages of Acanthaceae of similar to much larger
size in terms of number of species, making it an interesting group for biogeographic study.

Keywords Acanthaceae; Bayesian analysis; Lamiales; maximum likelihood analysis; Nelsonioideae; parsimony;
phylogenetic relationships

■ INTRODUCTION of taxonomie outliers together compose the recently recognized

Linderniaceae, Albach & al., 2005; Rahmanzadeh & al., 2005)
Lamiales, with as many as 24,000 species classified
whereasinothers remain as isolated lineages (e.g., Paulownia
Siebold & Zucc., Oxelman & al., 2005; Thomandersia Baili.,
more than 20 families, have been supported as monophyletic
in several modern phylogenetic studies (reviewed Wortley
by Judd & al., 2007). Here we examine one of the enigmatic
& Olmstead, 2004; Schäferhoff & al., 2010) although groups of Lamiales, termed nelsonioids.
many as-
pects of relationships within the order remain uncertain.ASome
morphologically similar group of genera including
Elytraria , Nelsonia , and Staurogyne has been recognized as
family-level taxa of Lamiales have a long record of taxonomie
taxonomically coherent since Nees von Esenbeck (1832) treated
recognition and have been affirmed by modern phylogenetic
work (e.g., Acanthaceae s.str., Scotland & al., 1995; Schwarz-
them as tribe Nelsonieae of Acanthaceae (note that species now
bach & McDade, 2002; Bignoniaceae, Olmstead & considered
al., 2009; Staurogyne were treated by Nees von Esenbeck in
Lentibulariaceae, Müller & al., 2006) whereas other traditional
Ebermaiera Nees and Erythracanthus Nees). Subsequently, this
assemblage
families have been rather dramatically recast in recent years, often has been treated as subfamily Nelsonioideae
notably Lamiaceae (Wagstaff & al., 1998) and Scrophularia-
of Acanthaceae (e.g., Lindau, 1895; Scotland & Vollesen, 2000).
ceae (reviewed by Tank & al., 2006). Lamiales also Theinclude
most detailed examination of the group is the Ph.D. dis-
a number of lineages that have been challenging to sertation
associ- of Hossain (1971), the results of which have been
ate with other plants. Some of these have been resolved
published
by only partially (Hossain, 1972, 1984, 2004). Hossain
phylogenetic research (e.g., Phrymaceae includes Mimulus L.,
(2004) preferred recognition at the tribal level within Acantha-
Beardsley & Olmstead, 2002; Oxelman & al., 2005; aceae. However, Bremekamp (1953, 1965) cited morphological
number

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McDade & al. • Phylogenetic placement and relationships among Nelsonioideae TAXON 61 (3) • June 2012: 637-651

differences between nelsonioids and members of Acanthaceae nelsonioids lack the modified funiculi or retinacula (Fig. IP)
s.str. (i.e., plants with retinaculate fruits, see below), and ar- that are present in fruits of most Acanthaceae (also lacking in
gued that the group is more appropriately treated as a tribe Thunbergioideae, Avicennia L.); lack cystoliths that are present
(Nelsonieae) of Scrophulariaceae than as Acanthaceae. Taxo- in the epidermal cells of most other Acanthaceae (absent also
nomically, nelsonioids have also been recognized as a distinct in Thunbergioideae, Avicennia , Acantheae); have more ovules
family (Sreemadhavan, 1977; Lu, 1990), or classified together per ovary (Fig. IP) than the four that are typical of Acantha-
with thunbergioids as Thunbergiaceae (Van Tieghem, 1908). ceae; and produce seeds with endosperm (vs. exalbuminous).
The delimitation of nelsonioids has also been problematic in Knowledge of phylogenetic placement of nelsonioids is central
that two genera, Ophiorrhiziphyllon and Saintpauliopsis , were to understanding patterns of morphological evolution in Acan-
originally described in Scrophulariaceae (Kurz, 1871) and thaceae and in Lamíales as a whole.
Gesneriaceae (Staner, 1934), respectively. Similarly, some spe- To date, phylogenetic work on nelsonioids has been limited.
cies of Staurogyne (as Neozenkerina Mildbr.) were described Representatives of nelsonioids have been included in studies
in Scrophulariaceae (Mildbraed, 1921). Thus, whereas the ex- of phylogenetic relationships among Acanthaceae s.l. (Hedrén
istence of a nelsonioid taxon has not been controversial, its & al., 1995; Scotland & al., 1995; McDade & Moody, 1999;
constituent genera, placement and rank have been so. Borg & al., 2008; McDade & al., 2008), but with insufficient
As treated by Hossain (2004), nelsonioids putatively com- sampling of other Lamíales to test rigorously the hypotheses
prise seven genera and about 175 species (Table 1). Staurogyne that nelsonioids are most closely related to Acanthaceae and
is by far the largest genus with about 140 species and a pantropi- that nelsonioid genera are together monophyletic. Similarly,
cal distribution. Elytraria has about 20 species naturally oc- nelsonioids have been represented in some broad-scale studies
curring in the New World, Africa, Madagascar, and the Indian of Lamiales (e.g., Olmstead & al., 1993; Bremer & al., 2001;
subcontinent (some species have been introduced elsewhere). Oxelman & al., 2005; Wortley & al., 2005; Schäferhoff & al.,
Nelsonia is very widely distributed (tropical Africa, Madagas- 2010), but with only a single representative and with insuffi-
car, southern Asia, Australia, southern North America, Central cient sampling of other Acanthaceae to constitute a rigorous test
America, tropical South America, the West Indies), although of the monophyly and placement of nelsonioids. Wenk & Daniel
it has been suggested that its occurrence in the New World (2009) sampled 15 nelsonioids (11 of which were Elytraria ),
is the result of introduction (Bentham, 1869; Hossain, 1984; but included only a single other Acanthaceae as outgroup. In
cf. Daniel, 2001). Interestingly, taxonomists differ in terms of this paper we test (1) monophyly of Nelsonioideae, (2) the tra-
species circumscriptions for Nelsonia with some treating these ditional association of Nelsonioideae with Acanthaceae s.l.
plants as one variable and somewhat weedy species whereas with a sample that includes a broad range of taxa of Lamiales
others recognize as many as seven (Nees von Esenbeck, 1847; (including Acanthaceae) and five of seven genera tradition-
Hossain, 1984; Vollesen, 1994). The remaining genera have one ally considered nelsonioids, and (3) the phylogenetic status of
to three species and limited distributions (Table 1). Two of these each of the seven genera that putatively constitute nelsonioids
( Anisosepalum , Saintpauliopsis) have been treated as part of (Table 1) and relationships among them using a denser sample
Staurogyne by some authors. Bremekamp (1955) also pointed to of Nelsonioideae.
similarities between Gynocraterium and Staurogyne. Nelsoni-
oids clearly present a number of taxonomie issues at the genus
and species levels. The group is also biogeographically com- ■ MATERIALS AND METHODS
plex, with three genera that show inter-continental disjunctions.
Nelsonioids share descending-cochlear aestivation of Taxon selection for placement of nelsonioids among
corolla lobes (Fig. 1A-D) (Scotland & al., 1994; Scotland Lamiales. - Within Lamiales, our sampling was guided by
& Vollesen, 2000), a pattern thus far unknown among other results that have consistently shown that several families are
Acanthaceae. However, unambiguous synapomorphies for basally branching (e.g., Oleaceae, Gesneriaceae; reviewed
nelsonioids and other Acanthaceae have not been identified: by Tank & al., 2006; see also Soltis & al., 2011) relative to

Table 1. Nelsonioideae: Genera, numbers of species (numbers sampled for study of relationships among nelsonioids), and geographic distr
Species
Genus (sampled) Distribution
Anisosepalum E. Hossain 3 (2) Tropical Africa
Elytraria Michx. -20 (9) N. America, West Indies, S. America, tropical & subtropical Africa, Madagascar, Indian subcontinent
Gynocraterium Bremek. 1(1) Northern S. America
Nelsonia R. Br. 1-5 (3^4) Africa, Madagascar, Asia, Australia, New World (disputed whether native)
Ophiorrhiziphyllon Kurz 2? (1) Southeast Asia
Saintpauliopsis Staner 1(1) Tropical Africa, Madagascar
Staurogyne Wall. -140 (25) Pantropical (including northern Australia)

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TAXON 61 (3) • June 2012: 637-651 McDade & al. • Phylogenetic placement and relationships among Nelsonioideae

Fig.1. Selected morphological traits of Nelsonioideae. A-D, Corolla aestivation: A, apex of floral bud of Elytraria caroliniensis showing descend-
ing cochlear aestivation; scale bar = 360 'im ; photo by Robert Scotland, used with permission; B, apex of floral bud of Nelsonia canescens show-
ing descending cochlear aestivation; scale bar = 225 |iim; photo by Robert Scotland, used with permission; C, close-up of bud of N. canescens ;
scale bar = 178 |im; photo by Robert Scotland, used with permission; D, schematic drawing of descending cochlear aestivation of corolla lobes in
bud ofis. caroliniensis (Daniel s.n., cultivated, CAS). E-l, Pollen; scale bars = 4 jim: E, 3-aperturate pollen ofis. marginata (Daniel 11158 , CAS),
apertural view; F, 3-aperturate pollen of Staurogyne concinula (Bartholomew & Boufford 6215 , CAS), interapertural view; G, 3-aperturate pollen
of N. canescens (Daniel 11186 , CAS), apertural view; H, pollen of Anisosepalum humbertii (de Witte 7121 , CAS), apertural view (3-colporate),
with arrow indicating somewhat indistinct endoaperture (os); I, pollen of S. glauca (Maxwell 94-76 , CAS), polar view (3-colporate), with arrow
indicating one of three somewhat indistinct endoapertures (ora). J-L, Seeds; scale bars = 90 jim: J, E. marginata (Daniel 11158 , CAS); K, N. ca-
nescens (Lewalle 3921 , CAS); L, S. miqueliana (Lott & al 3226 , CAS). M-O, Seed surface ornamentation; scale bars = 20 Jim: M, E. marginata ,
lacking trichomes; N, N. canescens , hooklike trichomes; 0, S. miqueliana , looped and branched trichomes. P, Interior of capsule of S. obtusa
(Maxwell 90-362 , CAS) showing numerous seeds, some seeds removed at base and apex of capsule to show (arrows) darkened hilar regions,
which lack conspicuous hook-like outgrowths (retinacula), scale bar = 1 mm.

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McDade & al. • Phylogenetic placement and relationships among Nelsonioideae TAXON 61 (3) • June 2012: 637-651

a core group. Resolving relationships within this core group Molecular methods. - Fresh leaf material, leaf material
dried in silica gel, or recently collected herbarium specimens
has proven extremely challenging. Recently, Schäferhoff & al.
(2010) found evidence from rapidly evolving chloroplast re- were used as sources of DNA. Total genomic DNA was ex-
gions for exclusion of Plantaginaceae and Scrophulariaceaetracted using the modified CTAB method of Doyle & Doyle
(1987) except that QIAGEN DNeasy kits (QIAGEN, Valencia,
from a group referred to by these authors as 'higher core Lamía-
les'. All five sampled members of Acanthaceae s.L, including California, U.S.A.) for plant tissue were usually used for her-
one nelsonioid ( Elytraria imbricata ), were part of this latter
barium samples. Procedures to amplify the trnL-F spacer and
clade. Our sampling therefore included representatives of trnL all intron were as described by McDade & Moody (1999); the
constituent clades of Schäferhoff & al.'s (2010) 'higher core trnS-G spacer and nrlTS regions were amplified as described
Lamiales' (Appendix 1). We also included representatives of by McDade & al. (2005) and McDade & al. (2000), respec-
Scrophulariaceae and Plantaginaceae because of the earliertively. Additional primers were sometimes needed to complete
hypothesis of a relationship between nelsonioids and Scrophu- the nrlTS sequences, as reported in Daniel & al. (2008). The
lariaceae (sensu earlier authors to indicate a much larger group
intergenic spacer regions ndhF-rpl32 and rpl32-trnL^AG) were
than currently recognized, and including both Scrophularia- amplified using the primers described by Shaw & al. (2007):
ceae and Plantaginaceae as presently treated; Stevens, 2001 5'ndhF+yrpl32R , and 5 ' rpL32F+ 3 r trnL{X]kQ' respectively, and
onwards). One Gesneriaceae and three Oleaceae were used as using the conditions employed for trnS-G by McDade & al.
outgroups for rooting purposes and also because one nelsoni- (2005). The chloroplast ndhF gene was amplified in two parts
using primer pairs 1F+1318R and 1318F+2110R as described
oid, Saintpauliopsis , was originally described in Gesneriaceae.
Representatives of all major lineages (sensu McDade & al.,
by Olmstead & Sweere (1994). When reactions failed to amplify
2008) of Acanthaceae s.l. were included (37 taxa) and five with of primer pair IF + 1318R, we used primer 32F as described by
seven putative genera of nelsonioids were sampled (1 1 taxa). Terry & al. (1997) instead of the IF primer. Amplification condi-
Because this sampling density is considerably higher than for tions were as for trnS-G (McDade & al., 2005) except that we
any of the other sampled lineages of Lamiales, we conducted used an annealing temperature of 50°C and a 2 min extension
experiments to examine the impact of sampling density of nel- time. For ndhF ' the purified PCR products were sequenced with
sonioids and other Acanthaceae on their phylogenetic place- the same forward and reverse primers as used in amplification.
ment, as described below. Sequencing with additional internal primers (536F and 803R
Many of the sequences analyzed for the placement of Nel-as described by Olmstead & Sweere, 1994) was occasionally
sonioideae study were downloaded from GenBank or EMBL necessary as to achieve complete bidirectional sequences.
indicated in the Appendices. When possible we selected records To optimize sequencing results, PCR products were
that reported an accessioned voucher specimen and that had cleaned using PEG precipitation (Morgan & Soltis, 1993) or
been included in a published study. In some cases, a composite Antarctic Phosphatase (New England BioLabs, Beverly, Mas-
'taxon' was assembled combining data from two different spe- sachusetts, U.S.A.). Sequences were generated on Beckman
(Beckman Coulter, Fullerton, California, U.S.A.) or ABI
cies of the same genus. In all such cases, we first verified that
(Applied Biosystems, Foster City, California, U.S.A.) auto-
the two taxa were each other's closest relatives (given the taxon
sample used here) by including them as taxa with missing data mated sequencers; both strands were sequenced for verification
for one locus in preliminary analyses. and to complete the sequences. Electropherograms of all se-
Relationships among Nelsonioideae. - Representatives quences were proofread manually in 4Peaks v. 1.7.2 (Griekspoor
of all genera that have been treated as Nelsonioideae were in- & Groothuis, 2006). Overlapping portions were reconciled
cluded, with numbers of species sampled proportionate to size by reverse-complementing one, aligning the two, and double-
of the genus (Table 1; Appendix 2). For Staurogyne , the larg- checking any inconsistencies against the electropherograms;
est genus, the sampling scheme reflected the morphological ambiguities that could not be resolved were coded as uncertain.
diversity of the group (e.g., rosette forming plants, erect herbs, Alignment and analysis. - Sequences for immediately
and shrubs were included as were taxa with a wide range adjacentof cp regions were treated together for purposes of align-
corolla sizes and shapes), as well the overall geographic rangement and analysis (i.e., trnL intron + trnL-F spacer = trnL-F ;
(i.e., species from the Americas, Africa, and Asia were in- ndhF-rpl32+rpl32-trnL spacers = ndhF-rpl32-trnL). Sequences
cluded). Likewise, for Elytraria, sampling included New World, for each DNA region were aligned separately by eye in Se-Al
African, and Malagasy plants. As outgroups, we included two 2.0all (Rambaut, 1996-2002) following methods discussed by
Acanthaceae (one Thunbergioideae and one Acantheae), oneMorrison (2006) and Graham & al. (2000) for manual align-
Martyniaceae, and one Pedaliaceae. ment of sequence data for phylogenetic analysis. As noted by a
Selection of genie regions. - To place nelsonioids withnumber of authors (e.g., McDade & Moody, 1999; Shaw & al.,
confidence among Lamiales, we chose three chloroplast (cp) 2007), the non-coding cp regions have a relatively high fre-
quency of parsimony-informative indels (i.e., length mutations).
loci that should offer a range of rates of evolution: trnL-F spacer
(relatively fast), trnL intron (medium), protein-coding ndhF Indels were scored for phylogenetic analysis following Graham
& al. (2000: S86); overlapping gaps were not scored. Indels
(relatively slow). For relationships among Nelsonioideae, we as-
were
sembled a dataset of three rapidly evolving cp spacers (> trnS-G , added to the dataseis as presence/absence characters. For
ndhF-rpl32 , rpl32-trnL^KG' plus nuclear ribosomal (nr) ITS
the placement of nelsonioids within the Lamiales dataset, 33
(ITS1, 5.8S, ITS2). and 6 indels were added to the trnL-F and ndhF data matrices,

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TAXON 61 (3) • June 2012: 637-651 McDade & al. • Phylogenetic placement and relationships among Nelsonioideae

respectively (all ndhF indels were multiples of three in length);

tested as alternative hypotheses of relationships, asking whether
for the nelsonioids dataset, 10, 22 and 5 indels were addedone dataset could reject the results supported by the other. Al-
to the trnS-G , ndhF-rpl32-trnL , and nrlTS data matrices, re-ternative topologies, each with only one branch constrained,
spectively. Table 2 compares the five DNA regions in terms of were loaded as constraint trees into PAUP* and the program
length, variability, number of taxa sequenced, and missing data.was asked to find the shortest tree consistent with that constraint
For analysis, data matrices were prepared as Nexus files (TBR branch swapping, 50 random addition sequence repli-
in MacClade v.4.08 (Maddison & Maddison, 2005). We tested cates). One randomly chosen most parsimonious tree consistent
for congruence among genie regions using the incongruencewith the constraint was compared to one randomly selected MP
length difference (ILD) test (Farris & al., 1994) implementedtree using Templeton's (1983) test (reported as z statistics). The
in PAUP* v.4.0 (Swofford, 2002) as the partition homogeneity two dataseis disagreed significantly and positively (i.e., each
test (200 replicates, 50 random addition sequences, maxtreesdataset rejected the topology supported by the other) only with
= 5000). The two cp regions sequenced for the placement of regard to details of relationships among Asian species of Stauro-
nelsonioids among Lamíales, trnL-F and ndhF ' were congru-gyne , as described further below. As a result, we combined
ent and the data were combined for further analysis. Because the data for analysis, but discuss the contrasting hypotheses
we were unable to generate ndhF sequences for a number ofsupported by cp versus nrlTS data for Asian Staurogyne below.
our target taxa of Lamiales (see Results), we also explored The combined datasets for each level of analysis (i.e.,
the sensitivity of the results to these missing data. We con-placement of Nelsonioideae in Lamiales and relationships
ducted two parsimony bootstrap analyses (100 replicates, 10 among nelsonioids; archived in TreeBASE as study ID TB2:
random addition sequences, tree bisection-reconnection [TBR])S12503) were analyzed using maximum parsimony (MP) as the
in PAUP*: (1) including all taxa; and (2) excluding taxa of optimality criterion in PAUP*. All parsimony analyses used
Lentibulariaceae and Byblidaceae for which ndhF sequencesrigorous heuristic search strategies designed to find all islands
were missing. Aside from the absence of the omitted taxa, the(sensu Maddison, 1991) of most parsimonious trees (i.e., >50
random addition sequences, TBR branch swapping); results
results were identical (not shown) and we consider further only
the results from analyses that included all taxa. were summarized as strict consensus trees. Branch support
was evaluated using non-parametric bootstrap values (BS;
Results from the partition homogeneity test indicated that
Felsenstein, 1985) from at least 100 replicates with 10 random
the two cp regions sequenced for analysis of relationships among
nelsonioids were congruent, but these together were incongru-addition sequences and TBR branch swapping.
ent with the nrlTS data (P < 0.01). As a result, we analyzed The datasets were also analyzed using Bayesian Mar-
the cp and nuclear data separately using parsimony and non- kov chain Monte Carlo inference (Yang & Rannala, 1997) in
parametric bootstrapping, and compared the results to identifyMrBayes v.3.0 beta 4 (Ronquist & Huelsenbeck, 2003). Bayes-
taxa or clades that were placed differently by data from the ian analyses used a mixed model approach, with a one-rate
two genomes. All resolved differences in topology between themodel applied to the indel characters and the model of se-
quence evolution selected by Akaike information criterion in
majority-rule bootstrap trees for the cp versus nrlTS data were

Table 2. Characteristics of five DNA regions used here: trnL-F and ndhF for placement of Nelsonioideae among Lamiales; trnS-G , ndhF-rpl32-
/rař/UAG), and nrlTS for relationships among Nelsonioideae.

Placement of Nelsonioideae Relationships among Nelsonioideae

(65 in-group taxa) (43 in-group taxa)
ndhF-rpl32-
Characteristic trnL-F region ndhF trnS-G spacer /ra£(UAG) nrlTS region
a. Aligned length 1515 2237 962 2020 861
b. Variable sites (proportion) 697 (0.460) 1112 (0.497) 255 (0.265) 614 (0.304) 495 (0.575)
c. Parsimony informative sites (proportion) 421 (0.279) 690(0.308) 145 (0.151) 400(0.198) 324(0.376)
d. Parsimony informative indels 33 6 10 22 5
e. Pairwise distances (range) 0.3-19.3% 0.8-17.0% 0.0-14.1% 0.2-14.6% 0.8-33.6%
f. Consistency index 0.563 0.466 0.784 0.717 0.567
g. Retention index 0.469 0.419 0.925 0.887 0.787
h. Missing data (# in-group taxa) 1 9 0 0 0
i. Missing data (not including taxa for
which entire sequence is missing) 1.4% 11.7% 0.5% 3.5% 2.8%
Note that taxon sampling differs between levels of analysis (see text and Appendices). Ali
locus; the remaining statistics are for the in-group only (i.e., including all but Oleaceae an
Nelsonioideae for relationships among that group). Consistency (f) and retention indices (
Missing data are reported as (h) number of total of 65 and 43 in-group taxa, respectively,
taxa for which data are entirely lacking for that locus. Statistics for the trnL-F region includ
intron; those for the nrlTS region include 28 bp of the 26S ribosomal gene that flanks ITS

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McDade & al. • Phylogenetic placement and relationships among Nelsonioideae TAXON 61 (3) • June 2012: 637-651

jModeltest v.0.1.1 (Posada, 2008) applied to each partition of ■ RESULTS

sequence data. For analyses of both levels of relationships, two
replicates of three heated and one cold chain were run for five Datasets and molecular evolution. - Whether expressed
million generations, with trees saved every 1000 generations.in terms of taxa for which sequences for entire genie regions
Output files were inspected to determine that log-likelihoodare missing or as percent missing data, our datasets are nearly
stationarity was achieved and when (i.e., after how many complete except for ndhF (Table 2). It proved impossible to
generations). Bayesian posterior probability (BPP) values for obtain sequences for ndhF for a number of taxa, notably four
branches were determined by opening the tree files produced of five Lentibulariaceae and both Byblidaceae (the others were
by MrBayes in PAUP*, filtering to remove the pre-burn-in distributed among families for which we had ndhF sequences
trees, and then constructing majority-rule consensus trees. for multiple other taxa). In addition, we were able to generate
Because parsimony and Bayesian inference (BI) may bothonly about half of the ndhF sequence for a few additional taxa.
be affected by long-branch attraction (Kolaczkowski & Thorn- More generally, ndhF was more challenging to sequence than
ton, 2009), the dataseis were also analyzed using maximum the other loci that we have worked with regardless of source of
likelihood (ML) in GARLI v. 1.0 (Zwickl, 2006). Models ofmaterial (i.e., even when DNA was extracted from fresh leaf
evolution were specified as described above for the Bayesianmaterial). As noted above, preliminary analyses indicated that
analysis. Support for individual branches was evaluated using these missing data did not alter our results although they may
100 bootstrap replicates. The ML topologies for both levels of have contributed to some problems with resolution as described
analysis were identical to those that resulted from BI suggest- below. The two genie regions used to place Nelsonioideae in
ing that the parsimony and Bayesian results were not biasedLamiales were roughly comparable in terms of proportionate
by long-branch attraction. variability although trnL-F contributed many more informative
Results of our analyses of relationships among Lamiales indels than ndhF ' which is unsurprising given that the former is
differed from those of Schäferhoff & al. (2010) in the placementnon-coding whereas the latter is protein coding. nrlTS contrib-
of Byblidaceae, Scrophulariaceae, and Thomandersia. We thusuted proportionately more parsimony-informative characters
tested whether our data could reject the Schäferhoff & al. (2010)(PICs) for the study of relationships among nelsonioids than
results. We also asked whether our data could reject placement either cp region. However, owing to its length and relatively
of nelsonioids with Orobanchaceae. This last family is one ofhigh variability, ndhF-rpl32-trnL^AG) contributed the most
several clades that comprised the former Scrophulariaceae and PICs; this last region also had the most indels.
includes genera assigned to Rhinantheae, the tribe identified by Placement of nelsonioids among Lamiales. - Results
Bremekamp as most likely related to nelsonioids; of clades of were entirely congruent across all sampling densities of Acan-
former Scrophulariaceae, Orobanchaceae are also the most phy-thaceae s.l. (i.e., 2-11 nelsonioids, 8-37 other Acanthaceae;
logenetically proximate to Acanthaceae in our analysis (see be-
results not shown). We thus present results from analyses using
low). Analysis of relationships among nelsonioids indicated thatrepresentatives of five putatively nelsonioid genera plus eight
two small genera (i.e., Gynocraterium , Ophiorrhiziphyllon ) were other Acanthaceae. This sampling density enables us to test
nested among species of Staurogyne (see below). We thus testedmonophyly and placement of nelsonioids and is roughly similar
the monophyly of Staurogyne exclusive of each of these genera.to that used for other lineages of Lamiales. Results from the
These alternative hypotheses of relationships were evaluated bythree methods of analysis (MP, BI, ML) were identical except
in weakly supported or unresolved aspects of relationships.
constructing a set of constraint trees with only a single branch
resolved. These trees were loaded into PAUP* and the program
The topology presented in Fig. 2 depicts the majority-rule tree
was set to find the shortest trees consistent with the constraint. from the post-burn-in trees sampled by the Bayesian analysis.
As indicated by the ML and parsimony bootstrap values, these
One randomly chosen MP tree consistent with the constraint was
analytical methods failed to resolve some aspects of relation-
compared to one randomly selected MP tree using Templeton's
(1983) test (reported as z statistics). The same strategy was usedships that were resolved by Bayesian inference.
to compare likelihood scores of trees reflecting the alternative Results of all analyses indicate that nelsonioids are mono-
phylogenetic hypothesis with all likelihood parameters (except phyletic with strong support and are sister to other Acantha-
base frequencies for which empirical values were used) esti- ceae, again with strong support (Fig. 2). Henceforth, we use
mated using one randomly selected MP tree. These parameters Nelsonioideae to refer to nelsonioids, Acanthaceae s.l. for the
were then used as the model to compare likelihood scores of thelineage that includes Nelsonioideae and all other Acanthaceae,
and Acanthoideae to refer to the clade marked by retinaculate
most likely tree to that of the tree consistent with the alternative
phylogenetic hypothesis using the Kishino-Hasegawa RELL fruit. There is support for monophyly of sampled taxa of Bigno-
test (Kishino & Hasegawa, 1989; K-H RELL) as implemented in niaceae, Byblidaceae, Lamiaceae, Lentibulariaceae, Lindernia-
PAUP*. Tests were one-tailed because an optimal tree was one ceae, Martyniaceae, Orobanchaceae, Pedaliaceae, Phrymaceae,
of the trees being compared (Felsenstein, 2004: 369). Plantaginaceae, Schlegeliaceae, Scrophulariaceae, Stilbaceae,
For nelsonioids, biogeographic ranges were assigned as and Verbenaceae, although few aspects of relationships among
Africa, Madagascar, Asia, Americas, or polymorphic combina- these are resolved. Notably, our results do not identify the sister
tions as appropriate, and optimized onto the Bayesian majority group for Acanthaceae s.l. The optimal ML tree places Bigno-
rule consensus tree using parsimony in MacClade (Maddisonniaceae in this position (results not shown), but there is no ML
& Maddison, 2005). bootstrap support for that relationship. One of the islands of MP

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TAXON 61 (3) • June 2012: 637-651 McDade & al. • Phylogenetic placement and relationships among Nelsonioideae

trees likewise places Bignoniaceae sister to Acanthaceae, but

Lentibulariaceae, Martyniaceae, Mazus , Orobanchaceae +Pau-
others place Martyniaceae, Lentibulariaceae, or combinationslowniaceae, Pedaliaceae, Phrymaceae, Schlegeliaceae, Ver-
benaceae) are part of a large polytomy, with BPP of 0.99 but
of these in that position, but again without support. Bayesian
only weak (60% ML BS) or no (<50% parsimony BS) support
inference resolves no group as sister to Acanthaceae s.l. with
from analyses using other optimality criteria. Linderniaceae,
>0.5 posterior probability. Among other Lamiales, our results
support a lineage that includes Mazus , Phrymaceae, Paulow- Scrophulariaceae, and Stilbaceae form a polytomy with the
niaceae, and Orobanchaceae s.l., and weakly support the as-just described clade, with strong support from all analytical ap-
proaches (BPP = 1.00, parsimony BS = 100%, ML BS = 100%).
sociation of Lamiaceae with that lineage. Most of the sampled
lineages of Lamiales (Acanthaceae, Bignoniaceae, Lamiaceae, Thomandersia is sister to this last large clade, and Byblidaceae

Fig. 2. Relationships among Lamiales. Majority-rule consensus tree of the post-burn-in sample of trees from the Bayesian analysis of the com-
bined dataset; values above branches are Bayesian posterior probability/parsimony bootstrap/likelihood bootstrap; * = 1.0 or 100%, - = <50%.
Species names given only when more than one congener included here or in Fig. 3; see Appendix 1 for full names of all taxa.

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McDade & al. • Phylogenetic placement and relationships among Nelsonioideae TAXON 61 (3) • June 2012: 637-651

and Plantaginaceae are basal to all sampled members of higher P < 0.0001; K-H RELL P < 0.01). Our data also reject placement
Lamíales (i.e., all but those used as outgroups). of Nelsonioideae with Orobanchaceae (z = -2.121, P = 0.034;
The results of Schäferhoff & al. (2010) placed Thomander-K-H RELL P < 0.01), thus refuting the notion of a relation-
sia more distally in Lamíales, in a polytomy with Acanthaceae, ship between Nelsonioideae and Rhinantheae of the former
Bignoniaceae, Lentibulariaceae, Martyniaceae, Pedaliaceae,Scrophulariaceae.
and Schlegeliaceae. The dataset assembled for our study rejects Relationships among Nelsonioideae. - Results of the
this placement (Templeton: z = -3.173, P = 0.0015; K-H RELL:three approaches to analysis of the combined cp and nrlTS
P < 0.01). Similarly, Schäferhoff & al. (2010) placed Bybli- dataset (i.e., MP, ML, BI) were entirely congruent except with
daceae as part of those authors' 'higher core Lamíales', with regards to weakly supported or unresolved aspects of relation-
Scrophulariaceae sister to that group. In contrast, our resultsship. Figure 3 shows the majority-rule consensus tree from the
place Byblidaceae together with Plantaginaceae basal to all ofBayesian analysis with support values from all three analytical
the other sampled Lamíales (except outgroups) and the Schäfer-approaches (note that Fig. 3B presents results from the analysis
hoff & al. (2010) result is rejected by our data (z = -4.799, of the nrlTS data for Asian Staurogyne as discussed below).

A */*/*¡- Staurogyne zeylanica - i* B

Fig. 3. Relationships among
Nelsonioideae. A, majority-rule
consensus tree of the post- */*/* I I - S. spathulata - I I 74
burn-in sample of trees from */-/66

the Bayesian analysis of the

I* - S. helferi
combined dataset; values
0.91 /-/-I i - S. elongata
adjacent to branches are Bayes-
*/-/63 0.58/597621 - s. expansa
ian posterior probability/parsi-
*/97/99| S.jaherii ~190.
mony bootstrap/likelihood */Qi/Qyil II - S. sandakanica - "I92
bootstrap; * = 1.0 or 100%,
Q-79J± */Qi/Qyil I - |o.53«t_ II - S. s sandakanica axillaris -
- = <50 %. **Nelsonia Lewalle
Asian clade */53/831
4734 has been identified by ^ * 1*1 * */53/831 '
D. Champluvier (BR) as a new - 0.84/-/53 i- S. dispar

species (manuscript in review). I - S. lasiobotrya

*/91/94 0.99/-/56 1 - S. citrina
Abbreviations: Afr, Africa; Aus,
Australia; Mad, Madagascar; African clade II L- Ophiorrhiziphyllon
*/*/* *1*1* ' i
NW, New World. B, Boot-
strap majority-rule tree from l*/*/¡j - S. letestuana (Afr)
analysis of the nrlTS data for
Staurogyne s.l. African clade I -
'
Asian Staurogyne only; values /*/* */*/* i - S. mandioccana (NW)
on branches are parsimony
bootstrap.
r TÃ^h~sfiava{uw) n
n s miquenana (NW)
td i 1 - s- lepidagathoides (NW)
n i

0.80/84/791 - s

*1*1* i Saintpauliopsis (Afr)

It/1 - Anisosepalum alboviolaceum (Afr)
0.94/75/66 A- lewallei (Afr)
r~ */*/* i - Elytraria imbricata (NW)
*/96/93 1 ' - E. mexicana (NW)
- I */82/82 1 - E. macrophylla (NW)
*/88/84 » I ' - E. caroliniensis (NW)
p */89/92|
*/*/* */*/* i - E ivorensis (Afr)
*/99/* ' - E marginata (Afr)
- */98/* i - E acaulis (Afr)
■ - E. nodosa (Mad)

*,*/* XII */59/811

- |~| i - N. smithii [Daniel 11125] (Afr)
*/687831 * ; - N- smithii [Daniel 11176] (Afr)

0.57/-/52 I - ' - Stenandrium

: |*/*/j- ' ' Martynia
- Sesamum

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TAXON 61 (3) • June 2012: 637-651 McDade & al. • Phylogenetic placement and relationships among Nelsonioideae

familial placement of Nelsonioideae: Saintpauliopsis is not a

Nelsonia and Elytraria are monophyletic with strong support,
but there is only moderate support (BPP = 0.94, parsimony Gesneriaceae
BS and Nelsonioideae are distant from all lineages o
= 75%, ML BS = 66%) for relationships between them and other
plants formerly classified as Scrophulariaceae. No study to date
of relationships among Lamiales that has sampled Nelsonioi-
Nelsonioideae. Nelsonia is the first branching clade, with Ely-
traria sister to the remaining Nelsonioideae. Within Nelsonia ,
deae has supported a different placement for the group although
there is little indication that accessions identified as conspe-
sampling has been sparse as noted above and poor resolution
cific are monophyletic. African accessions are monophyletic, has been a frequent result. Still, given continuing uncertainties
with New World, Australian, and Asian collected plants basal about many aspects of relationships among lineages of Lamia-
to the African clade. Among Elytraria , Old World taxa are les, this result merits further testing. We urge colleagues to
paraphyletic, forming two clades that are serially basal to employ
a sampling strategies that will permit further testing of
clade that includes all sampled species from the New World. placement of Nelsonioideae and offer support for such projects
An African clade comprising monospecific Saintpauliopsisin the form of DNA samples.
sister to Anisosepalum (two of three species sampled) is sister Our results are not congruent with those of Schäferhoff
to a group that includes all sampled members of pantropical & al. (2010) with regard to relationships of Thomandersia and
Staurogyne plus New World Gynocraterium and Asian Ophior- Byblidaceae, with both placed here among the basal-most lin-
rhiziphyllon. Gynocraterium is sister to all sampled members eages
of of Lamiales that were sampled (exclusive of those used
New World Staurogyne ; this last clade is sister to a clade com-
as outgroups). Although support values were not provided, th
prising the other sampled Staurogyne and Ophiorrhiziphyllon. results of Wortley & al. (2007) also place Thomandersia more
African Staurogyne are paraphyletic, forming two clades that distally although branch lengths are very short (Byblidaceae
are serially basal to Asian members of the genus plus Ophi- were not sampled by these last authors). These incongruences
orrhiziphyllon. Not surprisingly, given the relationships just are interesting in that all three studies used cp data and had
described, our data reject exclusion of Ophiorrhiziphyllon fromthe trnL-F region (Schäferhoff & al., 2010) or ndhF (Wortley
Staurogyne (z = -6.746, P < 0.0001; K-H RELL: P < 0.01), but & al., 2007) in common. In fact, we were unable to gener-
cannot reject exclusion of Gynocraterium (z = 0.289, P = 0.377;ate ndhF data for either of the sampled taxa of Byblidaceae.
K-H RELL: P = 0.42). The placement of this group of odd plants certainly merit
Biogeography. - Uncertainty about the sister group to further examination with more data. On the other hand, in
Acanthaceae s.l. plus the extreme wide range (i.e., essentiallyaccord with both earlier studies, our results indicate relatively
pantropical and subtropical) and taxonomie confusion in Nel- early branching positions for Plantaginaceae and Scrophu-
sonia makes inference of the ancestral range for Nelsonioideae lariaceae. Also consistent between our analysis and that of
and Nelsonia impossible. The common ancestor of all other Schäferhoff & al. (2010) is the clade that includes Lamiaceae,
Nelsonioideae is optimized as African. Elytraria is ancestrallyMazus , Phrymaceae, Paulowniaceae, and Orobanchaceae s.l.
an Old World (African) lineage, and our results support one (i.e., including Cyclocheilon Oliv., Lindenbergia Lehm., and
dispersal event to the New World. At least three colonizations Rehmannia Libosch. ex Fisch. & C.A. Mey.). This lineage has
of Madagascar occurred by Nelsonioideae: one by Nelsoniabeen resolved in other studies of Lamiales as well (reviewed
by Tank & al., 2006). Excluding Thomandersia , our results
canescens , at least one by Elytraria (with two species there, one
of which, E. madagascariensis (Benoist) E. Hossain, is known certainly do not refute Schäferhoff & al.'s (2010) clade com-
from very few collections and apparently has not been found prised of Acanthaceae s.l., Bignoniaceae, Lentibulariaceae
in about 50 years), and one by Saintpauliopsis , with Malagasy Martyniaceae, Pedaliaceae, and Schlegeliaceae (unresolved
plants putatively conspecific with African ones. Early in the in our results; Fig. 2). Lamiales clearly constitute a very chal-
evolutionary history of Staurogyne s.l. (i.e., including Gynocra-
lenging problem from the perspective of deducing phylogenetic
terium and Ophiorrhiziphyllon ), there was apparently a disper-relationships and very large dataseis (in terms of numbers of
sal event from Africa to the New World. Lineage diversificationinformative characters) will likely be necessary. Notable in
in Africa was followed by dispersal to Asia. One widespread this regard is Wortley & al.'s (2005) estimate that about 10 kb
African species of Elytraria , E. acaulis , has also spread as of sequence data would be necessary to provide strong suppor
far east as the Indian subcontinent and Sri Lanka. Nelsonia is for relationships among Lamiales (for comparison, Rokas & al.,
likewise known from Asia although a great deal remains to be 2003 and Hackett & al., 2008 have found that about 24 kb and
32 kb were necessary to deduce relationships with confidence
learned about the native range of plants of that genus.
among yeast and birds, respectively).
Monophyly of Acanthaceae s.l. and Nelsonioideae. - Nel-
■ DISCUSSION sonioideae are placed with confidence as the basally branching
lineage of Acanthaceae s.l. by both of our datasets. These results
Placement of Nelsonioideae among Lamíales. are -consistent
Our with the hypothesis that indurate, explosively de
data provide strong support for a clade that includeshiscent capsules are synapomorphic for Acanthaceae s.l., albei
all genera
tentatively assigned to Nelsonioideae and also forwith the sister
autapomorphic fruit types in Avicennia and Mendoncia
Veil,asex
relationship between this clade and other plants treated Vand. However, our inability to identify the closest
part
relatives of Acanthaceae s.l. among Lamiales makes it impos-
of Acanthaceae (Acanthoideae plus Avicennia and Thunbergioi-
deae). There is no evidence supporting earlier ideassible
regarding
to reconstruct evolutionary transitions in fruit morphology

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McDade & al. • Phylogenetic placement and relationships among Nelsonioideae TAXON 61 (3) • June 2012: 637-651

in the ancestors of Acanthaceae. Several other characters thatsupport monophyly of the two species for which more than
are synapomorphic for the vast majority of Acanthaceae (e.g.,one accession was included in the present study. It is, however,
presence of retinacula, presence of cystoliths, ovules reducedimportant to note that more than one of our accessions has been
to four, exalbuminous seeds) are lacking in Nelsonioideae. determined differently by students of Acanthaceae, including
The conditions of these traits in Nelsonioideae (absence of specialists on the group. Hossain (1984) found great varia-
retinacula and cystoliths, numerous ovules, seeds with persis-tion in features of vegetative traits among and within Nelsonia
tent endosperm) are almost certainly symplesiomorphic and do species from different ecological habitats, but no correlation
not provide evidence supporting any alternative placement ofamong these. He also noted very little variation in floral and
Nelsonioideae. In fact, among basally branching Acanthaceae fruit traits across species. He argued that variation in vegeta-
(Nelsonioideae, Thunbergioideae, Avicennia , Acantheae), our tive traits likely reflected plasticity rather than distinct species
current hypothesis of phylogenetic relationships indicates that and doubted the validity of recognizing more than one highly
these synapomorphies have evolved both asynchronously andvariable species. Our results are consistent with Hossain's hy-
with some further autapomorphic changes yielding an overall pothesis.
pattern that has obscured relationships. All sampled members of Elytraria comprise a clade, con-
In addition to molecular characters, monophyly of Nelso-firming the results of Wenk & Daniel (2009). These plants have
nioideae is supported by shared descending-cochlear aestiva- scaly scapes (Fig. 4A-C) and touch-sensitive stigmas (Fig. 4D),
tion of corolla lobes (Fig. 1 A-D). Corolla aestivation patternsboth putative synapomorphies for the genus, and androecia of
are phylogenetically informative among Acanthaceae, and the2 or 4 stamens, with staminodes present or absent. Compared
descending-cochlear pattern is thus far known only from Nel- to the earlier study of Wenk & Daniel (2009), the increased
sonioideae (Scotland & al., 1994; Scotland & Vollesen, 2000). sequence data assembled here provide strong support for re-
Other synapomorphies for the lineage have not been identifiedlationships among sampled taxa. There is strong support for a
and the group is markedly heterogeneous (see below, Fig. 4).sister relationship between E. acaulis (widespread in the Old
Notably, pollen ultrasculpturing and especially aperture typesWorld) + Malagasy E. nodosa and these are together sister to
are phylogenetically informative at many levels across Acan-a clade composed of the remaining taxa. African E. ivorensis
thaceae but results to date suggest that pollen varies among and E. marginata are sister taxa and these together are sister
Nelsonioideae in a pattern that is not phylogenetically informa-to a clade comprising all sampled New World taxa.
tive (see below). However, additional palynological study will Our results confirm Hossain's (2004) proposal and Wenk
be necessary for a full assessment of pollen traits. It should also& Daniel's (2009) results that there is a closer relationship
be noted that aperture type can be difficult to assess for pollenamong Anisosepalum , Gynocraterium , Ophiorrhiziphyllon ,
of these plants unless it is fully expanded (i.e., fresh, collected Saintpauliopsis , and Staurogyne , than between these and Ely-
into liquid fixative, or rehydrated). traria and Nelsonia. However, Hossain (2004) pointed to no
Relationships among Nelsonioideae. - Our results pro- feature uniquely shared among the five former genera. For
vide strong support for all aspects of intergeneric relationshipsexample, although many of these species have pollen with com-
among Nelsonioideae except that the placement of Elytraria
pound apertures (see Fig. IH, I), others have pollen with simple
sister to the clade that includes Anisosepalum , Saintpauliopsis ,apertures like those of Elytraria (fide Hossain 2004: 28). It
Gynocraterium , Staurogyne , and Ophiorrhiziphyllon is mod-should be noted that apertural type can be difficult to ascertain
erately supported (BPP = 0.94, parsimony BS = 75%, ML BS unless pollen grains are fully expanded, and further study of
= 66%; Fig. 3). The additional data included here (both taxa pollen traits across Nelsonioideae is warranted. We can identify
and characters) provide stronger support for resolution among no clear morphological synapomorphies for the group. Nota-
genera than in the Wenk & Daniel (2009) study, which showedbly, Hossain (2004) may have based his view on the deviating
little resolution of relationships among genera. features found in Nelsonia and Elytraria , as described above
Nelsonia is monophyletic and is consistently supported (e.g., lack of bracteoles in Nelsonia , scaly scapes in Elytraria ),
as the first-branching lineage of Nelsonioideae. These resultsrather than similarities among the other Nelsonioideae.
agree with the morphology-based work on Nelsonioideae pre- Our analyses confirm that Anisosepalum is monophyletic
sented by Hossain (2004), who described Nelsonia as morpho-and, together with Saintpauliopsis , it forms a clade that is sister
logically rather distinct. Nelsonia consists of low, spreading to the large group consisting of all Staurogyne plus Gynocrate-
herbs with spirally arranged bracts (Fig. 4E) that differ from rium and Ophiorrhiziphyllon. Anisosepalum was originally de-
all other Nelsonioideae by lacking bracteoles and by anthers scribed in Staurogyne (Mildbraed, 1937). Likewise, Saintpauli-
that often have a sub-basal flap-like appendage that seems toopsis , which can be distinguished from Staurogyne only by few
be associated with dehiscence. Wenk & Daniel (2009) pointedmorphological characters (Champluvier, 1991), was transferred
out that Nelsonia shares some similarities with Elytraria (e.g.,to Staurogyne by Burtt (1958). In contrast, Hossain (2004)
prominent fusion of the anterior calyx lobes), whereas other proposed maintaining Saintpauliopsis as a distinct genus and
characters connect it to other nelsonioid genera (e.g., seedsseparated Anisosepalum from Staurogyne , referring in both
with hygroscopic trichomes that are often branched or uncinate;cases to the absence of acicular fibers, presence of prophylls
compare Fig. IK, L, N, O to Fig. 1 J, M). As many as seven adnate to the calyx, and a calyx with the lateral lobes much
species have been treated in Nelsonia (e.g., Sprengel, 1825; shorter and narrower than the adaxial and abaxial lobes. Thus
Nees von Esenbeck, 1847; Vollesen, 1994). Our results do notthe close relationship between the two genera that is supported

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TAXON 61 (3) • June 2012: 637-651 McDade & al. • Phylogenetic placement and relationships among Nelsonioideae

by our data is also reflected in morphological traits. The two describing Gynocraterium , Bremekamp (1955) had noted its
genera can be distinguished by several characteristics, per-close resemblance to Staurogyne , but considered the fewer
haps most notably the bifurcately appendaged anther thecae ofovules and distinctive crateriform stigma (versus bifid and
Saintpauliopsis vs. oblique thecae on an expanded, triangular lobed in Staurogyne) sufficient basis for placing it in a separate
connective and with flap-like but not bifurcating appendagesgenus. Stigma morphology certainly merits further compara-
in Anisosepalum. Hossain (2004) also pointed to corolla, ovuletive study across Staurogyne as the trait is neither described
number and seed differences: Saintpauliopsis has pendulousnor illustrated in detail in most descriptions of species (e.g., the
stigma of New World S. stolonifera (Nees) Kuntze appears to
corollas, locules with 14-18 ovules and seed surfaces with elon-
gate, often branched, trichomes, whereas Anisosepalum has be bifid, Fig. 4F). In our analyses, Gynocraterium is sister to
non-pendent corollas, 6-8 ovules per locule, and seed surfaces New World Staurogyne. If this placement holds with denser
without trichomes. Plants of this small clade are African, withsampling of New World species (only 6 of about 30 species
Saintpauliopsis also occurring on Madagascar. sampled here), three alternatives would achieve monophyletic
The remaining Nelsonioideae {Gynocraterium, Ophior- genera: (1) this species could be transferred to Staurogyne ,
rhiziphyllon , and all sampled members of Staurogyne) are(2) New World Staurogyne could be transferred to Gynocra-
monophyletic with strong support, with New World Gynocra- terium , or (3) a new genus could be described for New World
terium sister to a clade comprising all sampled New WorldStaurogyne. At present, because we are unaware of any marked
Staurogyne , and Ophiorrhiziphyllon nested among Asian morphological distinctions between species of Staurogyne in
Staurogyne. Hossain (2004) stated that Gynocraterium and the Old World and those in the New World, we favor the first
Ophiorrhiziphyllon have very close affinity to Staurogyne alternative
, but are undertaking a more densely sampled study
and can only be distinguished by a few minor characters. Inof New World taxa before taking that step formally.

Fig. 4. Living plants and flowers of Nelsonioideae. A ,Elytraria caroliniensis , acaulescent perennial herb; B, E. imbricata , monocaulous perennial
herb with aerial cluster of leaves; C, E. nodosa , branched shrub with leaves distributed throughout plant; D, E. imbricata , corolla with touch-
sensitive stigma in "cocked" position; E, Nelsonia canescens , spirally arranged bracts and flowers; F, Staurogyne stolonifera (photo by Kris Wein-
hold, used with permission); G, Ophiorrhizphyllon macrobotryum , long-exserted style and stamens (photo by E. A. Tripp, used with permission).

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McDade & al. • Phylogenetic placement and relationships among Nelsonioideae TAXON 61 (3) • June 2012: 637-651
Ophiorrhiziphyllon macrobotryum differs from Stauro-
gyne principally by stamens that are conspicuously exserted
(Fig. 4G) and have elongate anthers apparently pointing up-
wards from the filament and appearing V-shaped at anthesis.
The genus was earlier separated from Staurogyne by three
characters: possession of only two stamens with two stami-
nodes (compared to four in Staurogyne ), exserted stamens,
and thecae dehiscent by short slits. Another supposed differ-
ence between the genera is the stigma, which is described as
bifid in Ophiorrhiziphyllon rather than bifid and lobed as in
Staurogyne. However, at least two of these characters are not
unique to Ophiorrhiziphyllon , and Hossain (2004) questioned
whether it should be recognized as a distinct genus. Recently,
Xin & al. (2010) transferred a species of Staurogyne described
by Hossain (1972) to Ophiorrhiziphyllon , on the basis of having
two stamens although the anthers of this species are not like
those of O. macrobotryum (cf. Hossain, 2004). Stamen number
has undergone numerous evolutionary transitions among Acan-
thaceae and it is not clear to us that reduction to two stamens is
sufficient basis for moving S. diandra E. Hossain to Ophior-
rhiziphyllon. There is clearly considerable diversity of stigma
and anther forms in Staurogyne and close relatives that merits
further study ideally from fresh material. It is extremely diffi-
cult to study many of these traits (notably anther dehiscence and
stigma morphology) in dried material. In any case, our results
support transfer of Ophiorrhiziphyllon to Staurogyne within
which it is part of a clade that comprises all Asian species; other
taxonomie solutions are possible but will require recognition
of multiple genera for clades within what is now Staurogyne.
As noted above, the cp and nrlTS datasets differ with regard
to relationships among Asian species of Staurogyne (although
both place Ophiorrhiziphyllon among these). We first confirmed
these results via re-extraction or re-sequencing of some of the
taxa in contention (e.g., Ophiorrhiziphyllon of which we initially
had DNA only from a somewhat old herbarium specimen but
later acquired two accessions that yielded higher-quality DNA,
one of which was from tissue collected fresh into silica [Tripp
1557]). We could find no evidence that contamination or poor
quality sequences were responsible for the conflict. The cp
topology is entirely congruent with that recovered by analysis
of the combined data (i.e., Fig. 3A), albeit with somewhat less
resolution. In contrast, the nrlTS topology only weakly sup-
ports monophyly of Asian Staurogyne (parsimony BS = 59%;
Fig. 3B) and the clade that includes S. setigera , S. spatulata, and
S. zeylanica is sister to a clade composed of the remaining sam-
pled taxa (versus nested distally within the S. argentea clade;
compare Fig. 3A, B). Similarly, whereas Ophiorrhiziphyllon
+S. citrina are sister to all other Asian Staurogyne in the cp
result, these two taxa are nested in a more distal polytomy made
up of most Asian Staurogyne in the nrlTS analysis. It is likely
that nrlTS alone has too few characters to resolve these relation-
ships fully and with confidence. On the other hand, it is also
possible that the conflicting patterns we observe are due to
biological processes that affect nuclear vs. cp data differently
(e.g., hybridization). In the future, we will explore relationships
among Asian Staurogyne with more taxa sampled and ideally
with at least one additional nuclear locus sampled.
648
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Biogeography. - The small genus Nelsonia is remarkable
for its nearly pantropical and subtropical distribution; with the
possible exception of the extremely weedy Ruellia blechum L.,
we know of no other Acanthaceae so widely distributed. How-
ever, as noted above, it is not clear that the group's present
range is entirely non-anthropogenic. The recently confirmed
report of Nelsonia as naturalizing in southern Florida (Franck
& Daniel, 2012) makes it clear that these plants are very agile
dispersers. The plants are not especially attractive and not in
the horticultural trade such that natural dispersal - or dispersal
aided at most passively by humans - is likely responsible for the
wide range of the group. Other Nelsonioideae are optimized as
African in origin with at least two dispersal events to the New
World (one within Ely traria, one in Staurogyne). This 'out of
Africa' pattern has been shown in a number of other lineages
of Acanthaceae (e.g., Acantheae, McDade & al., 2005; Tetrame-
rium lineage, Daniel & al., 2008; Ruellia , Tripp 2007). Taken as
a whole, our results indicate that Nelsonioideae are remarkable
among Acanthaceae in terms of numbers of intercontinental
disjunctions. For example, Daniel & al. (2008) showed that a
single dispersal event from the Old to the New World explains
the current distribution of the Tetramerium lineage, a group
with about as many species as Nelsonioideae; a single disper-
sal event likewise is sufficient to explain the distribution of
Acantheae in the New World (McDade & al., 2005).
Conclusions. - Our results provide strong support for
monophyly of Nelsonioideae and placement of the group sis-
ter to all other Acanthaceae, but otherwise contribute little to
the larger problem of relationships among core Lamiales. In
fact, some aspects that appeared to be resolved in the results
of Schäferhoff & al. (2010) (e.g., placement of Thomandersia ,
Byblidaceae) are back in contention. Notably, our continuing in-
ability to identify the closest relatives of Acanthaceae s.l. makes
it challenging to discuss morphological synapomorphies for
the family. We hypothesize that indurate explosively dehiscent
fruits are such a synapomorphy (with autapomorphic transitions
in Avicennia and Mendoncia), but can make no inferences about
the fruits from which those capsules evolved. Within Nelsoni-
oideae, our results provide strong support for monophyly of
Nelsonia , Elytraria , and Anisosepalum. The largest genus of
Nelsonioideae, Staurogyne is also monophyletic provided that
Gynocraterium and Ophiorrhiziphyllon are treated within it.
Continued recognition of these last two genera would require
description of additional genera to accommodate clades within
Staurogyne. The likely first branching member of Nelsonioi-
deae, Nelsonia , apparently has dispersal capacities that make it
difficult to be certain about its native range, which has been in
contention for some time. Other Nelsonioideae appear to have .
originated in Africa with at least two dispersal events to the
New World, three to Madagascar, and two to Asia. It is tempt-
ing to hypothesize that the woody retinacula of Acanthoideae
have adaptive value in enhancing seed dispersal distance by
propelling seeds away from the parental plant at dehiscence.
This idea, however, would seem to be refuted by the fact that the
small genus (i.e., 1-7 species) Nelsonia has a wider range than
many large lineages of Acanthoideae. Clearly much remains to
be learned about dispersal capabilities in Acanthaceae.
TAXON 61 (3) • June 2012: 637-651 McDade & al. • Phylogenetic placement and relationships among Nelsonioideae

■ ACKNOWLEDGEMENTS Microstructural changes in noncoding chloroplast DNA: Inter-

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Appendix 1. Placement of Nelsonioideae among Lamiales: Taxon, origin, voucher, GenBank or EMBL accession numbers (trnL-F, ndhF; - = sequence not
obtained; * = sequence newly generated for this study). Taxa are in alphabetical order by family (sensu Stevens 2001 onwards and as depicted in Fig. 2). Nels
oids are listed under Acanthaceae. Brackets enclose pairs of congeneric species that were combined to form composite sequences (see text for full explanatio

Acanthaceae. Anisosepalum lewallei Bamps (Nelsonioideae): Tanzania, Mpanda, Bidgood & al. 4570 (CAS), JQ728995*, JQ712668*; Aphelandra leonar
McDade, Costa Rica, San José, McDade 310 (DUKE), AF063112, JQ712669*; Avicennia marina (Forssk.) Vierh.: Australia, New South Wales, Schwarzba
97-A1 (KE), JQ728990*, JQ712670*; Barleria lupulina Lindl.: native to Madagascar and Mascarene Islands, cultivated San Francisco Conservatory of Flower
California, U.S.A., Daniel s.n. (CAS), AF289758, JQ712671*; Gynocraterium guianense Bremek. (Nelsonioideae): Guyana, Rupununi, Jansen-Jacobs & al. 234
(US), JQ728991*, JQ712672*; Elytraria imbricata (Vahl) Pers. (Nelsonioideae): U.S.A., Arizona, McDade & Jenkins 1155 (ARIZ), AF061819, JQ712673*; Justic
caudata A. Gray: Mexico, Sonora, Faivre 64 (ARIZ), AF063134 , JQ712674*; Mendoncia phytocrenoides Benoist: Cameroon, Southwest region, Schönenberge
50 (K), AF167330, JQ712675*; Nelsonia canescens Spreng. (Nelsonioideae): Panama, Canal Area, Daniel & al. 5452 (CAS), AF363668, JQ712676*; Neur
canthus africanus T. Anderson ex S. Moore: South Africa, Limpopo, McDade & al. 1258 (J), EU528936, JQ712677*; Phlogacanthus thyrsiflorus Nees: In
Uttar Pradesh, Lindburg 200 (DAV), EU528938, JQ712678*; [Ruellia californica (Rose) I.M. Johnst.: AF063115, -; R. ciliosa Pursh: -, U12664]; Staurog

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 TAXON 61 (3) • June 2012: 637-651 McDade & al. • Phylogenetic placement and relationships among Nelsonioideae

Appendix 1. Continued.

trinitensis Leonard (Nelsonioideae): Surinam, Tumuc-Humac, Sastre 1472 (U), JQ728992*, JQ7 12679*. Bignoniaceae. Tabebuia striata A.H. Gentry: EF10511 1,
EF105049; Tecoma stans (L.) Juss. ex Kunth: AY008826, AF102639; Tecomaria capensis (Thunb.) Spach: AY008827, FJ887874. Byblidaceae. Byblis gigantea
Lindl.: Australia, Western Australia, L. DeBuhr 3892 (RSA-POM), JQ728993*, B. Uniflora Salisb.: AF482605, -. Gesneriaceae. Paliavana prasinata (Ker
Gawl.) Benth.: AY047140, AF130160. Lamiaceae. Callicarpa dichotoma (Lour.) K. Koch: AF363665, L36395; Lamium purpureum L.: AF363664, LPU78694.
Lentibulariaceae. Genlisea hispidula Stapf.: AF488528, Pinguicula ugnata Casper: AF482617, P. grandiflora Lam.: AF482623, Utricularia striatula
Srn.: AF482675, U. vulgaris L.: U.S.A., California, Meyer & Townesmith 148 (RSA-POM), JQ728994*, JQ712680*. Linderniaceae. [Lindernia brevidens Skan:
AY492182, L. dubia (L.) Pennell: -, EF527446]; [Stemodiopsis buchananii Skan: -, (AJ619570 + AJ619571 + AJ619572); S. ruandensis Eb. Fisch: FN794073,
-]; Torenia baillonii Godefroy ex André: AY492193, AJ617583. Martyniaceae. Martynia annua L.: Mexico, Sonora, P. Jenkins 97-149 (ARIZ), AF067065,
JQ712681*; [Proboscidea fragrans (Lindi.) Decne: AJ430921, -; P. louisiana (Mill.) Thell.: -, AF123690]. Mazus insertae sedis. M. spicatus Vaniot: FJ172689,
-; Mazus stachydifolius (Turcz.) Maxim.: -, AJ619559. Oleaceae. Fraxinus chinensis Roxb.: AF231827, DQ673275; Olea europea L.: HQl 17895, DQ673278;
[Syringa dilatata Nakai: EU281163, S. vulgaris L.: -, DQ673277]. Orobanchanceae s.l. Agalinis heterophylla (Nutt.) Small: EU828161, AY563928; [Bartsia
alpina L.: -, AF123678; B. inaequalis Benth.: EU040319, -]; Cyclocheilon somátense Olive: AJ430923, AJ429117; Euphrasia strida Kunth: FJ600681, -; Lin-
denbergia sp. ( Thulin 8079): AJ430924, AF027286; [Melampyrum cristatum L.: -, AM503842; M. lineare Desr.: AF482608, -]; [Pedicularis cheilanthifolia
Schrenk: AY881133, -; P. foliosa L.: -, AF123689]; Rehmannia chingii H.L. Li: DQ856494, EF522187. Paulowniaceae. Paulownia tomentosa (Thunb.) Steud.:
AJ430926, L36406. Pedaliaceae. Ceratotheca triloba (Bernh.) Hook, f.: AF482611, AY919281; Sesamum indicum L.: AF067067, L36413; Uncarina grandidieri
(Baili.) Stapf.: AF363667, -. Phrymaceae. Mimulus aurantiacus Curtis: AF478982, AF188186; Phryma leptostachya L.: AF478988, AJ4291 18. Plantaginaceae.
Antirrhinum majus L.: AJ492270, L36392; [Callitriche verna L.: -, L47330; C. sp. {Albach 491): AY492172, -]; Globularia cordi/olia L.: AJ430930, AF027282;
[Plantago argentea Chaix: AJ430931, -; P. lanceolatea L.: -, AF130151]; Veronica persica Poir.: AF513336, L36419. Schlegeliaceae. Exarata chocoensis A.H.
Gentry: FJ870031, FJ887860; Schlegeliaparviflora (Oerst.) Monach.: AY008825, L36410; Synapsis ilici/olia Griesb.: -, AY919282. Scrophulariaceae. [Buddleja
davidii Franch.: -, AF130143; B. marrubi/olia Benth.: AF363666, -]; [Leucophyllum frutescens (Berland.) I.M. Johnst.: -, AF123685; L. laevigatum Standi.:
AF363669, -]; [Myoporum mauritianum A. DC.: -, L36403; M. parvifolium R. Br.: AF363670, -]; [Scrophularia arguta Sol. ex Aitón: AJ430936, -; S. sp.
( DePamphilis SS-20): -, L36411]. Stilbaceae. Halleria lucida L.: AJ621109, AF188185; [Stilbe albiflora E. Mey.: -, AF027287; S. ericoides L.: AJ430937, -].
Thomandersiaceae. Thomandersia hensii De Wild. & T. Durand: FN794075, AY919284. Verbenaceae. [Lontana camara L.: AY008824, -; L. hórrida Kunth:
-, AF130152]; Petrea volubilis L.: FJ870052, FJ887872; Stachytarpheta dichotoma (Ruiz & Pav.) Vahl: AY008823, L36414; [Verbena bracteata Lag. & Rodr.:
-, L36418; V urticifolia L.: AY008822, -].

Appendix 2. Relationships among Nelsonioideae: Taxon, origin, voucher, GenBank or EMBL accession numbers (trnS-G, ndhF-rpl32-trnL^AQ' nrlTS),
phylogenetic placement (clade names as in Fig. 3); taxa are in alphabetical order. * = sequences newly generated for this study.

Anisosepalum alboviolaceum E. Hossain: native to Central Africa, National Botanic Garden of Belgium accession no. 95002535 (BR), JQ691766*, JQ764595*,
JQ691812*, Anisosepalum clade; A . lew aliei Bamps: Tanzania, Mpanda, Bidgood & al. 4570 (CAS), JQ691767*, JQ764596*, JQ691813*, Anisosepalum clade;
Elytraria acaulis (L.f.) Lindau: Malawi, Chikwawa, Goyder & Patton 3520 (K), JQ691774*, JQ764604*, JQ691820*, Ely traria; E. caroliniensis (J.F. Gmel.)
Pers., native to SE U.S.A., living collection, 1971292900 (BR), JQ691770*, JQ764600*, JQ691816*, Elytraria ; E. imbricata (Vahl) Pers.: U.S.A., Arizona, McDade
& Jenkins 1155 (ARIZ), EU528957, JQ764597*, AF 169852; Elytraria ; E. ivorensis Dokosi: Ivory Coast, Tabou, Jongkind 4529 (WAG), JQ691772*, JQ764602*,
JQ691818*, Elytraria ; E. macrophylla Leonard: Mexico, Querétaro, Rzedowski 43302 (CAS), JQ691769*, JQ764599*, JQ69181 5*, Elytraria ; E. marginata
Vahl: native to W Africa, living collection, 19860332 (BR), JQ691773*, JQ764603*, JQ691819*, Elytraria ; E. mexicana Fryxell & S.D. Koch: Mexico, Colima,
Daniel 5284 (CAS), JQ691768*, JQ764598*, JQ691814*, Elytraria ; E . nodosa E. Hossain: Madagascar, Antsiranana, Daniel & McDade 10452 (CAS), JQ691775*,
JQ764605*, JQ691821*, Elytraria ; E.planifolia Leonard: Cuba, Las Villas, Howard & al. 341 (K), JQ691771*, JQ764601* JQ691817*, Elytraria ; Gynocraterium
guianense Bremek.: Guyana, Rupununi, Jansen-Jacobs & al. 2346 (US), JQ691764*, JQ764593*, JQ691810*, New World Staurogyne ; Martynia annua L.: Mexico,
Sonora, Jenkins 97-149 (ARIZ), EU528979, JQ764616*, AF169854, out-group; Nelsonia campestris R. Br.: Australia, Northern Territory, Craven 3953 (CANB),
JQ691778*, JQ764609*, JQ691825*, Nelsonia', N. campestris R. Br.: Zambia, Luapula province or Northern province, Fries 761 (UPS), JQ691777*, JQ764608*,
JQ691824*, Nelsonia ; N. canescens Spreng.: Panama, Canal Area, Daniel & al. 5452 (CAS), EU528985, JQ764606*, JQ691822*, Nelsonia', N. canescens Spreng.:
Thailand, likely Mae Hong Son, Hansen & Smitinand 12739 ( C ), JQ691776*, JQ764607*, JQ691823*, Nelsonia', N. sp. nov. (fide D. Champluvier, BR, pers. comm.):
Burundi, Bubanza, Lewalle 4734 (BR), JQ691779*, JQ764610*, JQ691826*, Nelsonia; N. smithii Oerst.: Tanzania, Bukoba, Bidgood & al. 4863 (CAS), JQ691780*,
JQ76461 1*, JQ691827* Nelsonia', N. smithii Oerst.: São Tomé and Príncipe, São Tomé, Daniel 11125 (CAS), JQ691781*, JQ764612*, JQ691828*, Nelsonia', N. smithii
Oerst.: cultivated at San Francisco Conservatory of Flowers from seeds collected in São Tomé and Príncipe, Príncipe, Daniel 11176 (CAS), JQ691782*, JQ764613*,
JQ691829*, Nelsonia ; Ophiorrhiziphyllon macrobotryum Kurz: China, Yunnan, Wang Hong 3090 (MO), JQ691745*, JQ764573*, JQ691790*, Asian Staurogyne',
O. macrobotryum Kurz: China, Yunnan, Tripp 1557 (RSA-POM), JQ691746*, JQ764574*, JQ691791*, Asian Staurogyne; Saintpauliopsis lebrunii Staner: Tan-
zania, Morogo, Frimodt-Moller 161 (C), JQ691765*, JQ764594*, JQ69181 1*, Anisosepalum clade; Sesamum indicum L.: Mexico, Sonora, Jenkins 97-141 (ARIZ),
EU528998, JQ764617*, AF169853, out-group; Staurogyne argentea Wall.: Thailand, Ranong, Larsen 43188 (AAU), JQ691738*, JQ764566*, JQ691783*, Asian
Staurogyne; S. axillaris S. Moore: Malaysia, Borneo, Sabah, Tadong 542 (K), JQ691749*, JQ764577*, JQ691794*, Asian Staurogyne; S . bicolor (Mildbr.) Champí.:
Cameroon, Kupe-Muanenguba, Cheek 10363 (BR), JQ691755*, JQ764583*, JQ691800*, African Staurogyne clade II; S. capitata E.A. Bruce: Liberia, Grand Gedeh,
Jongkind 7241 (WAG), JQ691757*, JQ764586*, JQ691803* African Staurogyne clade I; S. citrina Ridi.: Sumatra, Asahan, Boeea 10655 (L), JQ691754*, JQ764582*,
JQ691799*, Asian Staurogyne; S. concinnula (Hance) Kuntze: Taiwan, Taipei Hsien, Bartholomew & Bufford 6215 (CAS), JQ691750*, JQ764578*, JQ691795*, Asian
Staurogyne; S. diantheroides Lindau: Bolivia, Santa Cruz, Wood 10052 (CAS), JQ691763*, JQ764592*, JQ691809*, New World Staurogyne; S. dispar J.B. Imlay:
Thailand, Tak, Floto 7718 (U), JQ691752*, JQ764580*, JQ691797*, Asian Staurogyne; S. elongata Kuntze: Malaysia, Pahang, Teo 773 (L), JQ691739*, JQ764567*,
JQ691784*, Asian Staurogyne; S. eustachya Lindau: Brazil, Curitiba, Ribas & Cordeiro 559 (US), JQ691758*, JQ764587*, JQ691804*, New World Staurogyne;
S. expansa Bremek.: Thailand, Nakhon, Hansen 11982 (U), JQ691744*, JQ764572*, JQ691789*, Asian Staurogyne; S. flava Braz & R. Monteiro: Brazil, Minas
Gerais, Stehmann 2377 (US), JQ691759*, JQ764588*, JQ691805*, New World Staurogyne; S. helferi Kuntze: Thailand, Surratthani, Larsen 40901 (AAU), JQ691740*,
JQ764568*, JQ691785*, Asian Staurogyne; S.jaherii Bremek.: Brunei, Temburong, Poulsen 28 (AAU), JQ691747*, JQ764575*, JQ691792*, Asian Staurogyne;
S. lasiobotrys Kuntze: Thailand, Phetchaburi, Larsen 45105 (AAU), JQ691753*, JQ764581*, JQ691798*, Asian Staurogyne; S. lepidagathoides Leonard: Venezuela,
Cojedes, Llamozas 146 (US), JQ691761*, JQ764590*, JQ691807*, New World Staurogyne; S. letestuana Benoist: native to tropical Africa, cultivated National
Botanic Garden of Belgium accession no. 200000119-77 (BR), EU528999, JQ764584*, JQ691801*, African Staurogyne clade II; S. lineari/olia Bremek.: Surinam,
Sipaliwini, Irwin & al. 55848 (U), JQ691762*, JQ764591*, JQ691808*, New World Staurogyne; S. miqueliana Kuntze: Mexico, Jalisco, Lott & al. 3226 (CAS),
JQ691760*, JQ764589*, JQ691806*, New World Staurogyne; S. pseudocapitata Champí.: Gabon, Woleu-Ntem, Tabak 90 (WAG), JQ691756* JQ764585*, JQ691802*
African Staurogyne clade II; S. rosulata Bremek.: Thailand, Prachinburi, Larsen 22 (U), JQ691751*, JQ764579*, JQ691796*, Asian Staurogyne; S. sandakanica
Bremek.: Malaysia, Borneo, Sabah, Beaman & al. 9662 (C), JQ691748*, JQ764576*, JQ691793*, Asian Staurogyne; S. setigera Kuntze: Thailand, Songkhla, Larsen
45735 (AAU), JQ691741*, JQ764569* JQ691786*, Asian Staurogyne 5. spathulata Koord.: Thailand, Phetchaburi, Larsen 45045 (AAU), JQ691742*, JQ764570*,
JQ691787*, Asian Staurogyne; S. zeylanica Kuntze: Sri Lanka, Kegalla district, Fagerlind 3918 (S), JQ691743*, JQ764571*, JQ691788*, Asian Staurogyne; Stenan-
drium pilosulum (S.F. Blake) T.F. Daniel: Mexico, Sonora, Van Devender & Reina G. 97-434 (ARIZ), DQ059270, JQ764615*, AF169758, out-group; Thunbergia
erecta (Benth.) T. Anders.: native to tropical Africa, cultivated Missouri Botanical Garden accession no. 802421, EU529001, JQ764614*, AF169851, out-group.

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