Content uploaded by Alessandro Rapini
Author content
All content in this area was uploaded by Alessandro Rapini on Jun 17, 2015
Content may be subject to copyright.
1
American Journal of Botany 102 ( 6 ): 1 – 16 , 2015 ; http://www.amjbot.org/ © 2015 Botanical Society of America
AMERICAN JOURNAL OF BOTANY RESEARCH ARTICLE
South America harbors the largest biodiversity hotspots in
the world ( Myers et al., 2000 ). Its geographic isolation during
much of the Cenozoic, until the closure of the Isthmus of Pan-
ama at the end of the Pliocene ( Iturralde-Vinent and MacPhee,
1999 ; Coates et al., 2004 ; but see Montes et al., 2012 for an
earlier connection between South and North America), fa-
vored the appearance of a unique biota. The formation of a land
bridge with North America and the Andes orogeny were geo-
logic events that, together with climatic changes since the
Neogene—particularly pronounced in the Pleistocene ( Zachos
et al., 2001 )—appear to have signifi cantly affected the history
of South American vegetation and patterns of species distri-
bution, resulting in a complex and extremely diversifi ed bi-
ota ( Antonelli et al., 2009 ; Rull, 2011 ; Hughes et al., 2013 ).
Nevertheless, the sequence and relative importance of these
events during the diversifi cation of lineages is questionable
and deserves attention.
South American moist forests, including those in the Ama-
zon region to the north and Atlantic region to the east [including
open, mixed and closed evergreen, semideciduous and decidu-
ous forests ( IBGE, 2012 )], are separated by a corridor of drier
vegetation in the central part of South America, comprising
the Caatinga, Cerrado, and Chaco (e.g., Pennington et al., 2000 ;
Santos et al., 2007 ; see Werneck, 2011 for characterizations
of these vegetation types/biomes). These moist forest biomes
(Amazonian and Atlantic forests) would have been essentially con-
tinuous until the middle Miocene ( Bigarela et al., 1975 ; Morley,
2000 ). Due to the increased aridity that marked the end of the
Neogene, the moist forests would have become segregated into
1 Manuscript received 23 December 2014; revision accepted 28 April
2015.
The authors are grateful to P. Ribeiro, U. Silva, L. de Queiroz, P. Fiaschi,
and C. van den Berg, for comments on the manuscript and assistance in the
analyses, and to R. Olmstead and two anonymous reviewers for important
contributions to improve the paper. This study is a result of the following
projects: Pronex (FAPESB-PNX0014/2009), Refl ora (CAPES and CNPq),
and AuxPe-PNADB (CAPES). It is also part of the fi rst author’s thesis
developed in the Programa de Pós-Graduação em Botânica (UEFS) and
supported by grants from FAPESB, CAPES, and SWE CAPES at
California Academy of Sciences, San Francisco, California, USA. AR is a
CNPq researcher (Pq-1D).
4 Author for correspondence (email: analuiza.cortes@gmail.com)
doi:10.3732/ajb.1400558
T HE T ETRAMERIUM LINEAGE (ACANTHACEAE: JUSTICIEAE)
DOES NOT SUPPORT THE PLEISTOCENE ARC HYPOTHESIS FOR
SOUTH AMERICAN SEASONALLY DRY FORESTS 1
A NA L UIZA A . C ÔRTES 2,4 , A LESSANDRO R APINI 2 , AND T HOMAS F . D ANIEL 3
2 Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, Av. Transnordestina s/n, Novo Horizonte
44036-900, Feira de Santana, Bahia, Brazil; and
3 Department of Botany, California Academy of Sciences, 55 Music Concourse
Dr., Golden Gate Park, San Francisco, California 94118 USA
• Premise of the study: The Tetramerium lineage (Acanthaceae) presents a striking ecological structuring in South America, with
groups concentrated in moist forests or in seasonally dry forests. In this study, we investigate the circumscription and relation-
ships of the South American genera as a basis for better understanding historic interactions between dry and moist biomes in
the Neotropics.
• Methods: We dated the ancestral distribution of the Tetramerium lineage based on one nuclear and four plastid DNA regions.
Maximum parsimony, maximum likelihood, and Bayesian inference analyses were performed for this study using 104 termi-
nals. Phylogenetic divergences were dated using a relaxed molecular clock approach and ancestral distributions obtained from
dispersal-vicariance analyses.
• Key results: The genera Pachystachys , Schaueria , and Thyrsacanthus are nonmonophyletic. A dry forest lineage dispersed
from North America to South America and reached the southwestern part of the continent between the end of the Miocene and
beginning of the Pleistocene. This period coincides with the segregation between Amazonian and Atlantic moist forests that
established the geographic structure currently found in the group.
• Conclusions: The South American genera Pachystachys , Schaueria , and Thyrsacanthus need to be recircumscribed. The con-
gruence among biogeographical events found for the Tetramerium lineage suggests that the dry forest centers currently dis-
persed throughout South America are relatively old remnants, probably isolated since the Neogene, much earlier than the Last
Glacial Maximum postulated by the Pleistocene Arc hypothesis. In addition to exploring the Pleistocene Arc hypothesis, this
research also informs evolution in a lineage with numerous geographically restricted and threatened species.
Key words: Amazonian forest; Atlantic forest; biogeography; Neotropics; phylogenetics; refuges.
http://www.amjbot.org/cgi/doi/10.3732/ajb.1400558The latest version is at
AJB Advance Article published on June 15, 2015, as 10.3732/ajb.1400558.
Copyright 2015 by the Botanical Society of America
2 • VOL. 102 , NO. 6 JUNE 2015 • AMERICAN JOURNAL OF BOTANY
Amazonian and Atlantic forest ‘blocks’ connected only by gal-
lery forests and moist enclaves in Caatinga ( Brejos ) ( Oliveira-
Filho and Ratter, 1995 ; Oliveira et al., 1999 ; Costa, 2003 ;
Santos et al., 2007 ; Batalha-Filho et al., 2013 ).
Seasonally dry forests, on the other hand, occur disjunctly in
numerous regional centers: in the northeast of Brazil (Caat-
inga), along the watersheds of the Paraguay and Paran á Rivers
(Missiones), southwestern Bolivia and northwestern Argentina
(Piedmont), Chiquitano forests in Bolivia, in Andean valleys, in
the Central-West region of Brazil, and at the Caribbean coast of
Colombia and Venezuela, spreading into several centers in
Central America, along the Pacifi c coast, and Mexico ( Prado
and Gibbs, 1993 ; Pennington et al., 2000 , 2009 ; Queiroz, 2006 ;
Mogni et al., 2015 ). Lineages currently found in this biome are
usually distinguished by their high phylogenetic niche conser-
vatism and limited dispersal among centers, resulting in a sig-
nifi cant spatial arrangement and diversifi cation by geographical
isolation ( Pennington et al., 2004 , 2006 , 2009 ; Lavin, 2006 ;
Särkinen et al., 2012 ). Despite the presence of arid and semiarid
zones in South America before the Quaternary, there is no evi-
dence that these centers formed a corridor, and they would
probably have been even more isolated than the current centers
( Raven and Axelrod, 1974 ). However, based on woody species
that are widely distributed in this biome, Prado and Gibbs
(1993) suggested a continuous arc of dry forests in South
America during the Last Glacial Maximum (LGM), which is
known as the Pleistocene Arc hypothesis. Conditions in dry for-
est biomes do not favor fossilization ( Queiroz, 2006 ) and the
available paleo-ecological data are insuffi cient to infer their
distribution during the Last Glacial Maximum ( Mayle, 2006 ).
One of the most emblematic theories to explain Neotropical
diversifi cation, particularly in Amazonia, is the theory of Pleis-
tocene refuges ( Haffer, 1969 ; Pennington et al., 2000 ). Accord-
ing to this theory, moist forests would have been continuous
during interglacial periods, but would retract into isolated cen-
ters, establishing moist forest refuges, during the drier glacial
periods. In such refuges, vicariant populations tend to diverge,
leading to the establishment of new species. The succession of
pulses of diversifi cation governed by climatic fl uctuations dur-
ing the Pleistocene would therefore explain the high species
diversity in areas of Amazonia. In a similar and complementary
manner, it is postulated that South American seasonally dry for-
ests constitute refuges of a more extensive and continuous for-
mation in the past, and which would have formed a corridor
during the colder and drier periods of the Quaternary, coincid-
ing with the retraction of moist forests ( Raven and Axelrod,
1974 ; Prado and Gibbs, 1993 ; Pennington et al., 2000 ).
In recent decades, the refuge theory for Amazonia has been
disputed by studies using different approaches. Indeed, the het-
erogeneous pattern of diversity in Amazonia may simply refl ect
a sampling error, and the refuges would not necessarily be spe-
cies-rich areas, but merely areas of higher collecting density
( Nelson et al., 1990 ). Paleo-ecological data ( Colinvaux and
Oliveira, 2001 ; Mayle and Power, 2008 ) suggest that moist for-
ests are resilient to drier climatic conditions and probably cov-
ered the largest part of Amazonia throughout the Pleistocene.
Moreover, studies using dated phylogenies have not documented
a considerable increase in speciation rates during the Pleistocene
( Hoorn et al., 2010 ; but see Rull, 2008 , 2011 ). Regarding dry
forests, dated phylogenies of South American lineages suggest
diversifi cation by geographic isolation, with divergences gener-
ally preceding the Pleistocene ( Pennington et al., 2004 ; Särkinen
et al., 2012 ) and biome paleodistribution simulation suggests that
dry forest areas in the LGM would be similar to, or even more
restricted than, their current distribution ( Mayle, 2004 ; Werneck
et al., 2011 ; but see Collevatti et al., 2013 ).
In this study, we reconstructed the history of the Tetramer-
ium lineage (Acanthaceae) in South America with the objective
of testing whether genera are monophyletic and investigating
the historic relationships between moist and dry biomes in this
continent. The phylogeny of the group was reconstructed from
plastid and nuclear data, clades were dated and ancestral distri-
butions estimated. The congruence of results from our phyloge-
netic and biogeographic analyses, and their temporal and spatial
agreement with studies carried out using numerous other groups
of organisms and with different approaches allowed for rele-
vant conclusions to be made regarding relationships between
dry and moist biomes in South America. Most of all, they do
not support the classic Pleistocene Arc hypothesis which is
widely used to explain the distribution and diversifi cation of
numerous groups of unrelated plants from seasonally dry for-
ests in South America.
The Tetramerium lineage— The Tetramerium lineage belongs
to the tribe Justicieae of Acanthaceae, and consists of approxi-
mately 170 species in 23 genera ( Daniel et al., 2008 ). The lineage
includes considerable morphological and ecological diversity,
is especially rich in dry biomes, and its constituent taxa attract
diverse fl oral visitors and pollinators. The lineage originated in
the Old World and dispersed to the New World, where it radiated
into at least 125 species ( Daniel et al., 2008 ). In the New World,
the Tetramerium lineage consists of three large clades, i. e.,
two essentially restricted to southern North America and Central
America, mainly in dry biomes, encompassing approximately
59% of the Neotropical species, and the third, which occurs in both
moist and dry environments almost exclusively in South America,
but which also includes a few species reaching Central America
and Mexico ( Daniel et al., 2008 ).
The South American clade consists of fi ve main genera: Pa-
chystachys Nees, Schaueria Nees, Fittonia Coem., Streblacan-
thus Kuntze, and Thyrsacanthus Moric. (formerly species of
Anisacanthus Nees from South America) comprising approxi-
mately 25% of species in the lineage ( Fig. 1 ). Each genus, as
traditionally circumscribed, is essentially restricted to a single
biome and has a relatively low number (< 20) of species ( Nees,
1847 ; Wasshausen, 1986 ; Smick, 2004 ; Côrtes et al., 2010 ).
Pachystachys , Streblacanthus , and Fittonia are almost exclu-
sively distributed in lowland regions of the Amazon basin [ex-
cluding Streblacanthus monospermus Kuntze, which diverges
close to the root of the Tetramerium lineage and does not per-
tain to this group ( Daniel et al., 2008 ), and Streblacanthus cor-
datus Lindau, which occurs in the moist lowlands to the west of
the Andes and in southern Central America]. They are mainly
found in the western part, with species concentrated in Peru, but
a few occur in Guyana. Schaueria is endemic to the Atlantic
forest (Bahia state, in Brazil, to southern South America), while
Thyrsacanthus usually occurs in dry forests and restingas [coastal
lowlands formed by Quaternary sandy sediments ( Silva, 1999 )],
in northern (Venezuela and Guyana), northeastern (Bahia, Piauí,
and Rio Grande do Norte states, in Brazil) and southwestern
South America (Bolivia). The lineage does not contain any
characteristic savanna species, but Thyrsacanthus ramosus
(Nees) A. Côrtes & Rapini occurs in dry forests at the interface
of the Cerrado and Atlantic forest domains (Goi ás and São
THE TETR AMERIUM LINEAGE—CÔRTES ET AL. • VOL. 102 , NO. 6 JUNE 2015 • 3
Paulo states, in Brazil) ( Nees, 1847 ; Wasshausen, 1986 ; Smick,
2004 ; Daniel et al., 2008 ; Côrtes et al., 2010 ; Côrtes, personal
observation).
Phylogenetic studies ( Daniel et al., 2008 ) contributed to
generic recircumscription within the Tetramerium lineage
( Côrtes et al., 2010 ) and showed that colonization in Mexico
( Mirandea clade) probably proceeded diversifi cation of the
lineage in South America. However, none of the analyses to
date adequately sampled the diversity of the South American
clade. Therefore, we provide here new evidence to improve
our understanding of the systematics and biogeography of the
Tetramerium lineage in South America. The phylogenetic
framework presented in this study reveals the need for several
taxonomic realignments and will serve as a basis for taxonomic
revisions of South American genera, which are currently in
preparation.
MATERIALS AND METHODS
Taxon sampling — Collecting efforts were concentrated in South America
(Brazil, Bolivia, and Peru), during the years of 2009 to 2011. Approximately 30
species from the Tetramerium lineage were collected. We sequenced 31 silica-
gel dried specimens and eight herbarium specimens, representing almost the
totality of known South American species in the lineage: Thyrsacanthus (5 spe-
cies sampled/5 species in the genus), Streblacanthus (4/4), Schaueria (11/20),
Pachystachys (7/12), and Fittonia albivenis (Lindl. ex Veitch) Brummitt, in
addition to fi ve undescribed species (all ined.): Pachystachys “linearibracte-
ata”, P. “ gracilis”, Schaueria “hirta”, S. “pyramidalis”, and S . “thyrsifl ora”
(Côrtes et al., in prep.). Additional sequences of the Tetramerium lineage ( Daniel
et al., 2008 ) and justicioids, Diclipterinae, Isoglossinae, and Pseuderanthemum
lineages ( McDade et al., 2000 ; Kiel et al., 2006 ) were obtained from GenBank.
The names of clades of the Tetramerium lineage ( Henrya clade, Carlowrightia
parvifl ora (Buckley) Wassh. clade, Carlowrightia clade, Mirandea clade, Tet-
ramerium clade, Anisacanthus clade) used here follows those delimited by
Daniel et al. (2008) . The analyses consist of 104 terminals, which include three
species of Ruellieae (outgroups), 41 South American species and 28 terminals
representing ca. 23 South American species that were newly sequenced for this
study (Appendix 1).
Molecular data — Total genomic DNA was extracted using the CTAB 2%
method ( Doyle and Doyle, 1987 ) adapted for microtubes. For herbarium
specimens ( Justicia gonzalezii (Greenm.) Henrickson & P. Hiriart, J. zopilotensis
Henrickson & P. Hiriart, Carlowrightia sulcata (Nees) C. Ezcurra, Schaueria
azaleifl ora Rusby, S. hirsuta Nees, S. malifolia Nees, S. parvifl ora (Leonard) T.F.
Daniel, and Pachystachys “gracilis”, we used the Qiagen Dneasy kit (Qiagen Sci-
ences, Germantown, Maryland, USA. For amplifi cations, we selected the DNA
markers used by Daniel et al. (2008) , i. e., the plastid intergenic spacers trnT-trnL ,
trnL-trnF ( Taberlet et al., 1991 ) and trnS-trnG ( Hamilton, 1999 ), the plastid in-
trons trnL ( Taberlet et al., 1991 ) and rps16 ( Oxelman et al., 1997 ), and the nuclear
region ITS ( Sun et al., 1994 ). To amplify the majority of regions we used 1 µL of
total DNA, 1 × buffer, 50 mM MgCl
2 , 10 mM dNTP, 0.2 mM primer, 10 ng BSA,
5U/ µL Taq DNA polymerase (Phoneutria Biotecnologia Serviços Ltda, Belo
Horizonte, Brazil), adding ultrapure water to a total volume of 25 µl for plastid
reactions and 30 µl for reactions with ITS; for the ITS primers, 1.0 M betaine and
2% DMSO was also added. For plastid reactions, we used TopTaq (Qiagen Sci-
ences) with 0.2 mM primer for each region and 1 µL of total DNA.
PCR products were cleaned with PEG 20% (polyethylene glycol) and se-
quenced using the same primers as for amplifi cation, except for trnL , trnL-trnF ,
which were amplifi ed using primers “c” and “f” and sequenced in two parts,
one with “c” and “d” and other with “e” and “f” ( Taberlet et al., 1991 ). For ITS,
we tested primers ITS17, ITS26 ( Sun et al., 1994 ), ITS4, ITS5 ( White et al.,
1990 ), ITS92, ITS75 ( Desfeux et al., 1996 ), ITS9, and C26A ( Wen and Zim-
mer, 1996 ; Daniel et al., 2008 ). In all cases, long G + C chains limited complete
reading of sequences for a few taxa, as cited by Daniel et al. (2008) ; in such
cases, we cut off this part of the sequences. Sequences were generated in an
automatic sequencer (ABI 3130XL Genetic Analyzer, Applied Biosystems,
Life Technologies, Grand Island, New York, USA) using the Big Dye Termina-
tor Kit (Applied Biosystems), edited in the Staden program ( Staden et al., 2003 )
and aligned in Mesquite version 2.74 ( Maddison and Maddison, 2010 ). For a
few specimens unsatisfactory sequences of certain markers were obtained (Ap-
pendix 1) and sections of ITS are missing for Pachystachys badiospica Wassh.
and Thyrsacanthus microphyllus A. Côrtes. Regions were aligned by eye; only
the ITS matrix exhibited regions of ambiguous alignment, which were excluded
from the analyses.
Alignment and phylogenetic analyses — Sequences were grouped into
three datasets: 1) ITS matrix, with 98 terminals and 866 characters; 2) plastid
matrix ( trnL , trnL-F , trnT-L , rps16 , and trnS-G ), with 97 terminals and 4088
characters; and 3) nuclear and plastid combined data matrix, with 104 terminals
and 4921 characters. The incongruence length difference test (ILD; Farris et al.,
1994 ) was performed in PAUP* version 4.0b10 ( Swofford, 2000 ) to assess con-
gruence among plastid regions and between the combined plastid and nuclear
regions, using a heuristic search with 500 replicates, random taxon addition,
and TBR algorithm, saving 15 trees per replicate. Incongruence was considered
signifi cant when p < 0.01 ( Cunningham, 1997 ).
Maximum parsimony (MP), maximum likelihood (ML), and Bayesian in-
ference (BI) analyses were conducted for the three datasets. Editing and view-
ing of trees was done in FigTree version 1.3.1 ( Rambaut, 2009 ).
The MP analysis was performed in PAUP* version 4.0b10 ( Swofford, 2000 ),
using a heuristic search with 1,000 replicates, random taxon addition, and TBR
algorithm, saving up to 15 trees per replicate. Characters were equally weighted
and unordered. Support for clades was estimated using the nonparametric boot-
strap method (MP_BS) in PAUP*, with a heuristic search of 1,000 replicates,
with simple taxon-addition and TBR algorithm, saving 15 trees per replicate.
Fig. 1. Representatives of genera in the Tetramerium lineage. (A) Fittonia
albivenis . (B) Pachystachys lutea . (C) Pachystachys spicata . (D) Schaueria ca-
lycotricha . (E) Streblacanthus dubiosus . (F) Thyrsacanthus ramosissimus . Pho-
tos A, D, and E: T. F. Daniel; B, C, and F: A. L. A. Côrtes.
4 • VOL. 102 , NO. 6 JUNE 2015 • AMERICAN JOURNAL OF BOTANY
For the BI analyses, the best-fi tting models for each partition— trnL-F ( trnL
intron and trnL-F intergenic spacer), trnT-L and rps16 (GTR+G), trnS-G
(HKY+G), and ITS (GTR+I+G)—were selected using MrModeltest version 2.3
( Nylander, 2008 ). The analysis was performed in MrBayes version 3.1.2 ( Ron-
quist and Huelsenbeck, 2003 ), with two simultaneous replicates, using one cold
and three hot chains each, for 5 million generations, saving one tree per 1,000
generations. Trees prior to stabilization of likelihood values were discarded
(burn-in). The remaining trees were then used to obtain posterior probabilities
(PP) of clades using majority consensus in PAUP*. ML analyses were carried out
in PhyML version 3.0 ( Guindon et al., 2010 ; http://atgc.lirmm.fr/phyml/ ), using
the models previously selected in MrModeltest version 2.3 ( Nylander, 2008 ).
Alternative topologies obtained with ITS and combined plastid dataset were
tested using the Shimoidaira-Hasegawa test (SH; Shimodaira and Hasegawa,
1999 ) and evaluated individually based on the majority consensus tree of the
Bayesian Inference analysis of the combined dataset. The test was conducted in
PAUP*, using RELL optimization and 100 replicates.
Dating — Age estimates for nodes were based on the combined dataset (ITS,
trnL , trnL-F , trnT-L , trnS-G , and rps16 ). Only terminals with sequences for all
markers were used, comprising a matrix with 47 terminals and 45 taxa, includ-
ing the Tetramerium and justicioid lineages. As there are no known fossils of
the Tetramerium lineage, we restricted the minimum age divergence of the
American justicioids ( Justicia caudata A. Gray and Poikilacathus macranthus
Lindau) from the African justicioids ( Justicia adhatoda L.) based on Aeropolis
insularis Mautino, a pollen fossil of New World Justicia from the middle Mio-
cene (11.62 Ma; Mautino, 2011 ). Due to its narrow stratigraphic range, the ar-
eoles surrounding the apertures, and the Argentinean origin, this fossil provides
a safe calibration and was assessed as the highest utility by Tripp and McDade
(2014) . The relationships between geological epochs and absolute ages fol-
lowed the time scale provided by Cohen et al. (2013, updated). Divergence time
estimates were obtained in BEAST version 1.8 ( Drummond and Rambaut,
2007 ), using a relaxed molecular clock approach. Across the fi ve partitions,
models of nucleotide substitution and clock were set to unlinked, models of
sequence evolution were applied as in phylogenetic analysis, and base frequen-
cies were estimated for all loci. The analysis was conducted with the Yule spe-
ciation model, a random starting tree, and uncorrelated lognormal distribution
(UCLD; Mean = 1.6; Stdev = 1.5; Offset = 11.5, 5% quantile 11.5; 95% quan-
tile 17.6) following priors used by Tripp and McDade (2014) . The uniform
prior for UCLD means were used for each partition (default deviations). The
Markov Chain Monte Carlo (MCMC) chain was run for 50 million generations,
saving one tree per 1,000 generations. The main clades of the Tetramerium
lineage were predefi ned based on the BI tree with complete sampling. The log
archive was analyzed in Tracer version 1.5 to evaluate the effective size of the
sample for all parameters (ESS ≥ 200) and the maximum credibility tree was
reconstructed in TreeAnnotator version 1.8 after exclusion of the fi rst 10% of
saved trees (burn-in) ( Drummond and Rambaut, 2007 ).
Ancestral distribution — Species distributions were obtained from herbar-
ium collections and literature ( Wasshausen, 1986 ; Hilsenbeck, 1989 ; Ezcurra,
1994 ; Smick, 2004 ; Côrtes et al., 2010 ). For a defi nition of phytogeographic
areas, georeferenced data were plotted on a map of ecoregions ( Olson et al.,
2001 ; available at: http://www.worldwildlife.org/science/data/terreco.cfm ).
Two biomes were specifi ed: (1) moist or wet forests with two main blocks,
Amazonian (including forests in northern and northwestern South America)
and Atlantic (east coast of South America); and (2) seasonally dry forests with
fi ve main centers delimited as: (1) dry forests of North America (extending to
Costa Rica); (2) dry forests of northern South America; (3) Caatinga (northeast-
ern Brazil); (4) Piedmont dry forests; and (5) Chiquitano and Missiones (south-
western South America). Three additional areas representing the distribution of
the Tetramerium lineage were specifi ed, based on geographic features: Old
World, Central America, and the Antilles.
To reconstruct the ancestral distribution of the Tetramerium lineage in
South America, we used statistical dispersal-vicariance (S-Diva) analysis in
RASP version 2.1 ( Yu et al., 2010 ), which calculates the probabilities of an-
cestral distributions ( Ali et al., 2012 ) and estimates vicariance, dispersal, and
extinction events for each node. This reconstruction was calculated based on
the BI tree of the combined dataset, and for two alternative trees, one from
ITS and another from plastid analyses, that, despite presenting distinct to-
pologies, were not considered statistically different according to the SH test.
Each reconstruction was carried out using the 7,402 trees obtained in the BI
of the combined dataset with complete sampling, limiting species distribu-
tions to four ancestral areas. And lastly, ancestral distribution reconstructions
were plotted onto the chronogram.
RESULTS
Phylogenetic analyses — ITS has a higher percentage of
parsimony-informative characters compared to individual
plastid regions, with 32.7% informative characters. However,
it presented the highest rate of homoplasy, indicated by the
low consistency and retention indices ( Table 1 ). The BI, MP,
and ML analyses with ITS support the monophyly of the Tet-
ramerium lineage (PP = 1/ BS_MP = 60% / BS_ML = 94%),
but did not recover the Neotropical clade. A few clades were re-
covered in at least two methods, i. e., Schaueria clade (1/94/93),
Henrya clade (0.78/-/88), Anisacanthus clade (0.89/86/86),
Carlowrightia (1/84/93), Tetramerium (1/98/99), and the Car-
lowrightia parvifl ora clade (1/91/94). The ML tree with ITS
supported a few deeper relationships and clades such as the
Neotropical clade (−/−/95), the Thyrsacanthus clade (0.56/-/92),
and Mirandea clade + Schaueria parvifl ora (−/−/69). More-
over, this method supported a single dry forest lineage (( Miran-
dea clade + core Tetramerium lineage) Thyrsacanthus clade)
(−/−/65) (−/−/91), whereas the Pachystachys clade appeared
segregated, partly as sister to Schaueria clade (−/−/76) form-
ing two clades and, together with species of Streblacanthus ,
partly as a grade of the dry forest lineage clade ( Table 1 ; Ap-
pendix S1 (see Supplemental Data with the online version of
this article)).
T ABLE 1. Summary of phylogenetic analyses for the Tetramerium lineage showing incongruences of the different datasets analyzed; results of the individual
analyses are restricted to maximum parsimony. Support: posterior probability / bootstrap of the maximum parsimony / bootstrap of the maximum
likelihood. N = number of taxa; Alinh. = length of the aligned molecular matrix; PI = number of parsimony informative characters; L = length of
MPT; CI = consistency index; RI = retention index. The columns 1 to 11 are support value [1 – clade Pachystachys ; 2 – clade Schaueria ; 3 – clade
Thyrsacanthus ; 4 – clade ( Mirandea + (remainder)); 5 – clade ( Mirandea ( Schaueria + remainder)); 6 – clade ( Pachystachys ( Thyrsacanthus + core
Tetramerium lineage)); 7 – clade ( Thyrsacanthus + core Tetramerium lineage); 8 – clade (core Tetramerium lineage ( Thyrsacanthus + Pachystachys ));
9 – ( Thyrsacanthus + Pachystachys ); 10 – clade ( Mirandea ( Thyrsacanthus + core Tetramerium lineage)); 11 – clade ( Schaueria + Pachystachys )].
DNA marker N Alinh PI L CI RI 1 2 3 4 5 6 7 8 9 10 11
trnL+trnL-trnF 68 1086 155 395 0.8 0.9 79 86 95 71 — — — — —
trnT-trnL 84 934 112 306 0.86 0.89 — 54 — — — — — — —
rps16 80 1064 135 352 0.8 0.86 50 — 69 — — — — — —
trnS-trnG 86 1017 147 442 0.81 0.85 77 — — — — — — — —
ITS 98 866 284 1508 0.51 0.66 −/−/- 1/94/94 56/-/92 −/−/- −/−/- −/−/- 51/-/91 −/−/- −/−/- 56/56/- −/−/-
Plastid Combined 99 5917 652 1796 0.81 0.87 1/96/99 96/-/1 1/97/1 96/-/74 93/67/89 — −/−/- 57/-/91 91/-/73 — —
(ITS + plastid
combined)
104 6783 936 3341 0.67 0.77 1/1/1 1/1/99 1/99/99 1/91/94 100/-/93 95/−/− 98/−/− — — — −/−/22
THE TETR AMERIUM LINEAGE—CÔRTES ET AL. • VOL. 102 , NO. 6 JUNE 2015 • 5
The majority of the plastid regions were equally informative,
trnL-F being the most informative, with relatively low levels of
homoplasy. The MP results for individual plastid regions are
only slightly resolved, with support for only a few clades ( Table
1 ). On the other hand, combined analyses of plastid regions re-
solved relationships among the main clades of the Tetramerium
lineage, most of them with high to average support in the three
methods (PP ≥ 0.95 and BS ≥ 80%). These analyses show the
Pachystachys and Thyrsacanthus clades as sister (0.97/-/92),
forming a clade (1/59/100) with the core Tetramerium lineage
(1/86/100). In the Thyrsacanthus clade, four species of the ge-
nus appeared resolved in the ML (BS = 99%), but collapsed in
the BI ( Table 1 ; Appendix S2 (see Supplemental Data with the
online version of this article)).
Partition homogeneity tests did not indicate any signifi cant
incongruence. However, topological confl icts between nuclear
and plastid datasets were observed with regard to the internal
nodes of Thyrsacanthus , Pachystachys , and Schaueria clades.
The ITS tree (Appendix S1 (see Supplemental Data with the
online version of this article)) generally agrees with morpho-
logical affi nities, whereas plastid data appear to be strongly in-
fl uenced by the geographic proximity of species (Appendix S2
(see Supplemental Data with the online version of this article)):
(1) T. ramosissimus Moric. forms a clade with Justicia angus-
tissima A. Côrtes & Rapini and Schaueria humulifl ora Nees
(0.97/61/99), the fi rst two species are from Caatinga vegetation
in Bahia and the latter one is from deciduous forests in Bahia;
(2) T. secundus , from dry forests and restingas of northern
South America, appears more closely related to T. microphyl-
lus , from the Caatinga in Piauí (northeastern South America)
(1/99/99); (3) the two accessions of Pachystachys spicata (Ruiz
& Pav.) Wassh. are not closely related; and (4) Schaueria mar-
ginata Nees is in a unresolved clade with other species from
Bahia (1/71/100). In the topology of the combined data ( Fig. 2 ),
the internal relationships obtained from plastid data prevail.
Topological confl icts are also present in the relationships
among the main clades of the Tetramerium lineage. The Pa-
chystachys clade is sister to a dry forest lineage ( Thyrsacanthus
clade + core Tetramerium lineage) in the combined analyses
( Fig. 2 ), but it emerges as the sister group to Thyrsacanthus
clade with plastid data (Appendix S2 (see Supplemental Data
with the online version of this article)). This clade is not re-
solved with ITS alone, which shows part of the clade weakly
related to the Schaueria clade in the ML tree (Appendix S1 (see
Supplemental Data with the online version of this article)). Ac-
cording to the SH test, the best tree recovered was that with the
combined plastid and nuclear data ( Fig. 3A ). However, alterna-
tive trees, either with ITS or plastid dataset, were not rejected
and alternative scenarios for ancestral distribution reconstruc-
tions were considered: one of the scenarios establishes an
initial dichotomy between moist forest lineages ( Pachystachys
clade and Schaueria clade) and dry forest lineages ( Thyrs-
acanthus clade and core Tetramerium lineage + Mirandea
clade), as obtained from the ITS data in the BI and ML analyses
( Fig. 3B ); the other considers Pachystachys and Thyrsacanthus
clades forming a clade, as obtained with the plastid data ( Table 2 ;
Fig. 3C ).
The three methods produced similar topologies with the
combined nuclear (ITS) and plastid ( TrnL - F , TrnT-L , TrnS-G ,
and rps16 ) data, when taking into account only the highly sup-
ported relationships. The BI is the only analysis that recovers
relationships among the main clades of the Tetramerium lineage
with signifi cant support (PP = 0.95–1). Our results ( Fig. 2 ) con-
fi rm that the Tetramerium lineage is made up of a basal grade
composed of Old World species from which the Neotropical
clade, with high support (1/90/96), emerged; and the Mirandea
clade is sister to the rest of the Neotropical clade. South Ameri-
can genera are sustained in distinct clades. Schaueria parvifl ora
and Fittonia albivenis collapsed into a basal polytomy of the
Tetramerium lineage with ITS. However, S. parvifl ora appears
as sister to the Mirandea clade in the combined analysis
(0.90/−/−) and F. albivenis is sister to the Schaueria clade with
plastid (0.5/−/−) and combined data sets (0.99/70/87). Schaue-
ria forms a clade (1/100/99) if S. azaleifl ora , S. hirsuta , S. hu-
mulifl ora , S. malifolia , and S. parvifl ora are excluded and
Justicia paranaensis (Rizzini) Wassh. & L.B. Sm. and three
new species are included. The genus is endemic to the Atlantic
forest and forms a clade with the Amazonian Fittonia albive-
nis . Together, the two genera appear as sister (1/-/92) to the
group made up of the Pachystachys clade and one dry forest
lineage ( Thyrsacanthus clade + core Tetramerium lineage)
(0.94/–/64).
The Pachystachys clade is supported (0.98/68/96), including
Schaueria azaleifl ora and species of Streblacanthus (except S.
monospermus ), as a basal grade in relation to Pachystachys s.s.
The Thyrsacanthus clade is also well supported (1/98/99); in-
cludes a lineage from Mexico ( Yeatesia mabryi Hilsenb., Mi-
randea hyssopus (Nees) T.F. Daniel, Justicia gonzalenzii , and
J. zopilotensis ); and otherwise consists of species of Thyrs-
acanthus , Carlowrightia sulcata , Justicia angustissima , and
Schaueria humulifl ora ( Table 1 ; Fig. 2 ). Unlike other species of
South American Anisacanthus that are now placed in Thyrs-
acanthus , the taxonomic affi nities of A. trilobus Lindau appear
to be close with Harpochilus , among the New World justicioids
( Fig. 2 ). The relationship between the Pachystachys clade and the
dry forest lineage is highly to moderately supported (0.94/–/64),
depending on the method.
Dating — The chronogram based on the sum of the maxi-
mum credibility clades is presented in Appendix S3 (see Sup-
plemental Data with the online version of this article); the
average ages and highest probability density (HPD) 95% are
summarized in Table 3 . According to this chronogram, the Tet-
ramerium lineage dispersed to the New World (nodes 3–4),
probably at the middle and end of the Miocene, between
(18.72–)11.4 and 9.11(–4.88) Ma, and the main South American
clades ( Schaueria , Pachystachys , and Thyrsacanthus clades:
nodes 9’–11) began to diversify most likely close to the Mio-
cene/Pliocene boundary, between (8.8–)5.1 and 3.3(–1.4) Ma
( Fig. 3A ; Table 3 ).
Fig. 2. Majority-rule consensus tree derived from the Bayesian ITS and plastid ( trnL-F , trnT-L , trnS-G , and rps16 ) combined analysis showing relationships in
the Tetramerium lineage, with emphasis on the South American genera Schaueria , Pachystachys , and Thyrsacanthus . Names in bold indicate terminals with incon-
gruent positions between plastid and nuclear results. Bayesian posterior probability (PP) support values are reported above branches; parsimony (BS_MP) and likeli-
hood bootstrap (BS_ML) support values are below branches, respectively; branches in bold are supported PP ≥ 95% and BS ≥ 80%; * = 100%. The arrows show
clades collapsed in the strict consensus of the MP. The phylogram derived from the maximum likelihood analysis of the combined dataset is shown on the left.
→
6 • VOL. 102 , NO. 6 JUNE 2015 • AMERICAN JOURNAL OF BOTANY
THE TETR AMERIUM LINEAGE—CÔRTES ET AL. • VOL. 102 , NO. 6 JUNE 2015 • 7
Ancestral reconstruction — The Tetramerium lineage origi-
nated in the Old World (A, Fig. 3A , node 1) with one dispersal
event to the New World. The arrival of the ancestor in the Neo-
tropical region and the order of colonization events in North
America and South America are uncertain ( Fig. 3A , nodes 3–6),
and initially show an expanded ancestral area in the Neotropical
region. One ancestral area in North America (B, Fig. 3A ) is
suggested for the dry forest clade (node 7), which dispersed to
South America, occupying the semiarid regions: Caatinga (H)
and the dry forests of Missiones (J). This dry forest lineage thus
segregated (node 11 and 14) and thereafter lineages began to
disperse into neighboring areas (node 13), with migrations to
restingas and deciduous forests (node 16), both in the Atlantic
forest, but the former close to the coast and the latter more to
the interior. The South American clades Schaueria and Pachys-
tachys diversifi ed in their respective current areas of distribu-
tion: Schaueria clade in Atlantic forest (E, Fig. 3A , node 9),
after a divergence with an Amazon forest lineage, represented
by Fittonia (node 9’, Appendix S4 (see Supplemental Data with
the online version of this article)), and Pachystachys clade in
Amazon forest (F, Fig. 3A , node 10), following a divergence
with a dry forest lineage from western South America, repre-
sented by Schaueria azaleifl ora (Appendix S4, node 10’), a
species that appears to be confi ned to more humid mountainous
areas. Figure 3A summarizes the reconstruction of ancestral
distributions through time; the complete reconstruction, includ-
ing Fittonia albivenis , whose ancestral distribution is also
shown in this fi gure (node 9’) is present in Appendix S4 (see
Supplemental Data with the online version of this article).
Considering the alternative reconstructions, in the fi rst scenario
( Fig. 3B ), the Pachystachys clade appears as sister to the Schaueria
clade. The initial dispersal sequence of the Tetramerium lineage in
the New World is uncertain, but a striking dichotomy exists (node
6) between dry and moist forests. The dry forest lineage (node 7)
would have initially radiated in North America with dispersal to
South America in the Thyrsacanthus clade, whereas the moist for-
est lineage would have arisen in the Amazon region, where the
Pachystachys clade radiated, and from there would have dispersed
to the Atlantic forest, giving rise to the Schaueria clade. In the
second alternative scenario ( Fig. 3C ), the Pachystachys clade ap-
pears as sister group to the Thyrsacanthus clade. In this alternative,
the distribution of the Tetramerium lineage is equally broad and
uncertain at the beginning of its expansion in the New World, indi-
cating one extinction and three dispersal events at node 6, and 12
possible ancestral areas in node 7.
DISCUSSION
The main clades recovered in our study generally agree with
those in Daniel et al. (2008) . The primary difference is that
among the South American species three (vs. one) clades are
recovered here and new generic circumscriptions are needed to
establish monophylesis for Schaueria ( sensu Nees, 1847 ), Pa-
chystachys ( sensu Wasshausen, 1986 ), and Thyrsacanthus ( sensu
Côrtes et al., 2010 ). These lineages are morphologically distinct
and geographically structured between dry and moist forest bi-
omes. With the combined data ( Fig. 3A ), the moist forest clades
( Fittonia - Schaueria and Pachystachys ) appear to form a grade in
relation to a dry forest lineage ( Thyrsacanthus clade + core Tet-
ramerium lineage). An alternative tree ( Fig. 3B ), with moist for-
est lineages making up the sister clade of the dry forest lineage
(also including the Mirandea clade), however, is not statistically
different and should also be considered.
Incongruence — The phylogenetic incongruence between mo-
lecular markers may be explained by branching over a short time
period, which could or could not be associated with introgressive
hybridization ( Dorado et al., 1992 ; Costa, 2003 ; Rokas et al.,
2003 ; Small et al., 2004 ; Martins et al., 2009 ; Palma-Silva et al.,
2011 ; Song et al., 2012 ). The accuracy of analyses may not be
suffi cient to recover relatively ancient relationships resulted from
rapid radiation. This is probably the main reason for uncertain-
ties regarding relationships among the main clades of the Tetra-
merium lineage, hindering the establishment of the sequence of
diversifi cation for the group during the end of the Miocene, when
the lineage seems to have undergone an ecological restructur-
ing, with the formation of clades predominantly restricted ei-
ther to dry or moist forests. Recent rapid diversifi cations, on the
other hand, may often result in incomplete lineage sorting, and
this is possibly an explanation for incongruences in the Tetra-
merium lineage at the species level.
Introgressive hybridization is relatively common among
plastid genes ( Rieseberg and Wendel, 1993 ; Small et al., 2004 ;
Okuyama et al., 2005 ) and has been highlighted as one of the
causes of the geographic structure between species that have
presumably exchanged portions of the plastid genome ( Kikuchi
et al., 2010 ; Palma-Silva et al., 2011 ). In the Schaueria and
Thyrsacanthus clades, plastid data present a geographically
structured pattern for a few sympatric species ( Figs. 2 and Ap-
pendix S4 (see Supplemental Data with the online version of
this article)). Species of Thyrsacanthus clade show variation in
fl oral morphology that appears to conform to different pollina-
tion syndromes (e.g., butterfl ies in S. humulifl ora and hum-
mingbirds in T. ramosissimus ). In spite of that, many species of
the Tetramerium lineage can be both visited and pollinated
by multiple types of animals (incl. bees, fl ies, butterfl ies, and
hummingbirds), and some of these have been shown to be at
least partially interfertile in Carlowrightia ( Daniel, 1983a ), An-
isacanthus ( Daniel, 1985 ), and Tetramerium ( Daniel, 1986 ).
Although hybrids were not identifi ed in natural populations
of Schaueria and Thyrsacanthus species, older interspecifi c
Fig. 3. (A) Chronogram (in million years) based on the combined data (for confi dence intervals of ages, see Appendix S3 (see Supplemental Data with the online
version of this article), including only terminals with sequence for all markers. * indicate PP ≥ 90% and ** PP = 100% in the ITS and plastid combined analyses
( Fig. 2 ). Alternative reconstructions based on results obtained from nuclear (B) and plastid (C) data are represented below. Numbered nodes correspond to major
clades discussed in the text; the pie graphs summarize ancestral distribution reconstructions, with letters indicating areas on the map: Old World (A), North America
(B), Central America (C), Antilles (D), Atlantic Forest (E), Amazon Forest (F), Dry Forests of northern South America (G), Caatinga (H), Piedmont Dry Forests and
Chiquitano (I) and Dry Forest of Missiones nucleus (J); circles around the pie graphs represent biogeographic events: blue = dispersion; red = vicariance; yellow =
extinction; the ancestral reconstruction with a star below (reconstructions 9’: divergence of Fittonia albivenis ) was recovered from the analysis with all terminals
(Appendix S4 (see Supplemental Data with the online version of this article)) and placed according to its relative phylogenetic position, but it was not dated; arrows
on the map and on the chronogram indicate dispersal routes; the red cross over arrows represents the interruption of the route: a vicariant event between the two areas;
gray band in the Miocene marks the period inferred for the expansion of dry forests separating Amazon and Atlantic rainforests.
→
8 • VOL. 102 , NO. 6 JUNE 2015 • AMERICAN JOURNAL OF BOTANY
THE TETR AMERIUM LINEAGE—CÔRTES ET AL. • VOL. 102 , NO. 6 JUNE 2015 • 9
hybridization events could be involved in the diversifi cation of
the two lineages, as recognized in other plant groups ( Fehrer et
al., 2007 ; Palma-Silva et al., 2011 ; Jabaily and Sytsma, 2013 ),
as well as in Acanthaceae ( Tripp et al., 2013 ).
Phylogenetic relationships of the South American genera —
Although the South American clades of the Tetramerium lineage
are well supported, relationships among them are as yet uncer-
tain. The Schaueria-Fittonia clade seems to be one of the fi rst to
diverge in South America. Both Schaueria and Fittonia have
fl owers with small, yellow or white corollas, potentially polli-
nated by bees/fl ies, and are distributed disjunctly in Atlantic
and Amazonian forests, respectively. However, Fittonia does not
share linear bracts, common in Schaueria ( Nees, 1847 ; Brummitt,
1978 ; Daniel et al., 2008 ; Côrtes, personal observation). The
Pachystachys clade seems to have diverged subsequent to the
Schaueria-Fittonia clade, with a noticeable radiation in western
Amazonia. In the combined analysis, it emerges as sister to a
lineage of dry forest taxa, consisting of the Thyrsacanthus clade
and the core Tetramerium lineage, which represents the largest
radiation of the group, with diversifi cation mainly concentrated
in seasonally dry forests of North America. The Thyrsacanthus
clade has nototribic fl owers (androecium dehiscing toward the
inferior lip of the corolla), a symplesiomorphy of the lineage,
whereas sternotribic fl owers (androecium dehiscing toward the
superior lip of the corolla) emerged as a synapomorphy of the
core Tetramerium lineage, which is distributed mainly in North
America ( Daniel et al., 2008 ).
Biogeographic relationships — According to our results, the
Tetramerium lineage dispersed to the New World in the middle
of the Neogene. Its radiation in the Americas must have been
relatively rapid and our analyses were not suffi cient to establish
the initial sequence of colonization in the New World. During
the Late Miocene, the lineage would already be possibly occu-
pying the Amazon basin in South America, and occurring in
North and Central America, probably in both moist and dry en-
vironments. During the late Miocene, its history was probably
marked by a period of dry forest expansion and segregation into
Amazon and Atlantic moist forests ( Fig. 3 ). These events seem
to be directly associated with a global decrease in temperature
and humidity ( Zachos et al., 2001 ). The Pachystachys and Fit-
tonia clades were confi ned to the western part of the Amazon
basin and the Schaueria clade to the Atlantic forest on the east
coast of South America. The modern Cerrado, associated with
a fi re regime in the central region of South America ( Simon et al.,
2009 ; Simon and Pennington, 2012 ), and where taxa of the Tet-
ramerium lineage do not occur, may have represented an insur-
mountable subsequent barrier that fragmented the dry forests
into two main blocks, one in southwestern South America and
the other in northeastern Brazil.
Dry forests— The disjunct distribution seen in the dry forest
lineages, usually with groups endemic to isolated centers in
South America and also among North American Acanthaceae
(e.g., Holographis , Daniel, 1983b ; Tetramerium , Daniel, 1986 ),
concurs with biogeographic patterns also found in other plant
groups ( Prado and Gibbs, 1993 ; Pennington et al., 2000 , 2004 ,
2009 ; López et al., 2006 ; Caetano et al., 2008 ; Werneck, 2011 ;
Werneck et al., 2011 ; Cardoso et al., 2013 ). Colonization of the
Caatinga from North America may have occurred via arid re-
gions on the northern periphery of the Amazon region. The
Thyrsacanthus clade could have dispersed by a system of dunes
as well as via restingas that would have expanded with the de-
crease in sea level during colder periods, reaching 130 m below
the current sea level in the Last Glacial Maximum (LGM)
( Lambeck et al., 2002 ), for example. Currently, Thyrsacanthus
secundus presents disjunct distribution in drylands and restin-
gas in northern South America. A similar distribution in the
past would possibly enable dry forest lineages from North
America to reach northwestern Brazil.
The Thyrsacanthus clade would have reached the dry forest
block in Missiones, southwestern South America, most likely
during the late Miocene. This dispersal must have been facili-
tated by the expansion of dry forests, starting from the Caatinga
T ABLE 3. Estimated ages for the Tetramerium lineage nodes (My = million
years) and the 95% credibility interval (HPD); nodes correspond to
clades in Fig. 3 and Appendix S3 (see Supplemental Data with the
online version of this article).
Estimated age
Node Average (My) 95% HPD (My)
1 14.04 7.3-22.92
3 11.4 6.08-18.72
4 9.11 4.88-14.87
5 8.25 4.35-13.39
6 7.91 4.15-12.78
7 7.41 3.96-12.01
8 5.15 1.9-9.89
9 3.29 1.38-6.43
10 3.41 1.38-6.53
11 5.16 2.54-8.82
12 6.53 3.45-10.64
13 4.11 1.84-7.31
14 4.28 1.94-7.98
16 3.28 1.41-5.99
17 1.99 0.55-4.13
T ABLE 2. Results of the Shimoidaira-Hasegawa test, comparing alternative topologies with the majority consensus tree of the Bayesian Inference; values
p ≤ 0.05 indicate that the alternative topology is statistically different and must be rejected. Alternative topologies constrain clades recovered with ITS
or plastid dataset on the combined analysis. Parenthetical notation is used to represent the fi rst clade.
Alternative topologies -lnL -lnL alternative topology Difference p
( Mirandea ( Thyrsacanthus + core Tetramerium lineage)) Not reject 30530.60362 30541.80358 11.19996 0.21
Pachystachys + Thyrsacanthus Not reject 30530.60362 30538.43570 7.83208 0.22
Schaueria + Pachystachys Not reject 30530.60362 30535.49404 4.89042 0.29
Fittonia + Pachystachys reject 30530.60362 30551.75331 21.14968 0.02
Schaueria (sensu Nees, 1847 ) reject 30530.60362 31600.32591 1069.7229 0.00
Streblacanthus reject 30530.60362 30577.12348 4651985 0.02
Thyrsacanthus (sensu Côrtes et al., 2010 ) reject 30530.60362 30612.08106 81.47744 0.01
10 • VOL. 102 , NO. 6 JUNE 2015 • AMERICAN JOURNAL OF BOTANY
in northeastern Brazil and forming a diagonal corridor of dry
forests between the two blocks. Such a dry corridor, diagonally
crossing South America, may have worked as an ecological
barrier for moist forests, segregating lineages in Amazon forest
from those in Atlantic forests (see below). A long-distance dis-
persal between discontinuous blocks of dry forest would have
similar reconstruction (node 11), but then we would expect cur-
rent species with disjunct distribution between isolated blocks
of dry forests, as shown by Prado & Gibbs (1993) for particular
woody species for instance, and this is not the case for the Tet-
ramerium lineage.
Vicariance between dry forests in the Caatinga and Missio-
nes blocks may have been caused by a subsequent expansion
of fi re-adapted savannas (3–8 Ma; Cerling et al., 1997 ; Simon
et al., 2009 ). Dry forest lineages, sensitive to fi res ( Pennington
et al., 2009 ), would have then been eliminated or extensively
fragmented in the central plateau of South America, which
would explain the absence of the Tetramerium lineage in the
Cerrado, and the presence of representatives of the Thyrsacan-
thus clade in dry forest remnants on the boundaries of the Cer-
rado and Atlantic forest domains. The divergence between the
lineages in these two blocks is relatively old and must have
occurred before the Pleistocene, like estimates in other dry for-
est genera ( Pennington et al., 2004 ; Särkinen et al., 2012 ), or
less likely at the beginning of the Pleistocene, agreeing with the
estimated coalescence age for geographically isolated popula-
tions of Handroanthus impetiginosus (Mart. ex DC.) Mattos
( Collevatti et al., 2012 ). Therefore, the Caatinga and Missiones
blocks of seasonally dry forests would not represent relicts of
the LGM (upper Pleistocene), as proposed by the dry forest
Pleistocene Arc hypothesis ( Prado and Gibbs, 1993 ).
Our results support the scenario suggested by Werneck et al.
(2011) , whereby lower temperatures and greater aridity would
have limited the distribution of dry forests during the glacial peri-
ods of Pleistocene and an eventual continuity between the two dry
forest blocks would have occurred at the beginning of the Pleisto-
cene or even in the Neogene. This scenario also agrees with May-
le’s (2006) suggestion that individual long-distance dispersals
might properly explain disjunct distribution of species in frag-
ments of dry forest. According to him, Anadenanthera colubrina
(Vell.) Brenan, a dominant species in several fragments of dry
forests in South America and considered a key taxon for the Pleis-
tocene Arc hypothesis ( Prado and Gibbs, 1993 ; Mogni et al.,
2015 ), probably dispersed to the Bolivian Chiquitano very re-
cently, since it was absent from this fragment during the LGM.
Prado and Gibbs (1993) argued that current disjunctions be-
tween isolated patches of seasonally dry forests represent a con-
sistent phytogeographic pattern, especially for legume species
( Mogni et al., 2015 ), and that a more parsimonious explanation
for this pattern would be a relatively recent fragmentation of a
continuous distribution. The postulated pattern, however, seems
to only represent selected examples and does not comprise the
distribution of a majority of dry forest species. Queiroz (2006) ,
for example, showed that most species of legumes from Caat-
inga are endemic, while only 11% of them occur disjunctly in
any other fragment of dry forest. Although a continuous dry
forest biome may have existed across the South America, its
fragmentation probably occurred much earlier than the LGM
and determined the current geographic structure observed in
most lineages. In this case, most species disjunctly distributed
in dry forest blocks got this pattern due to stochastic long-dis-
tance dispersal and their populations are not remnants of a con-
tinuous distributed ancestral.
Moist forests— Our data do not support a single lineage of
moist forest-taxa composed of the Pachystachys and Schaue-
ria - Fittonia clades, but this alternative (scenario B) cannot be
ruled out. The relationship between Fittonia albivenis and
Schaueria could suggest dispersal from Amazonia to Atlantic
forest, followed by vicariance. The segregation among lineages
of moist forest-taxa seems to coincide with the expansion of dry
forest lineages during the late Miocene, suggesting the forma-
tion of a diagonally shaped corridor of arid biomes in South
America during part of this epoch. This corridor, initially com-
posed of dry forests, but subsequently by savannas as well,
would have broken the continuity between moist forests in
South America. Presently, moist forest clades of the Tetramer-
ium lineage are concentrated in regions of greater climatic sta-
bility and high species richness, that are believed to be
ecological refuges, i.e., Fittonia and the Pachystachys clades in
western Amazonia at the base of the Andes occupies a region
little affected by the decrease in humidity during the colder pe-
riods of the Pleistocene ( Cheng et al., 2013 ), and the Schaueria
clade in the central and northern parts of the Atlantic forest,
from Bahia to Rio de Janeiro was less subject to Pleistocene
forest contractions ( Carnaval and Moritz, 2008 ).
The disjunction of the moist forest clades of the Tetramerium
lineage agrees with an older vicariance pattern, between the low-
lands of western Amazonia and the central region of the Atlantic
forest, stretching from Bahia to northern São Paulo, but reaching
the lowland of the southern region of Brazil. According to this
pattern of divergence, the connection between the two biomes
would have occurred via the Paran á River basin, south of the
Cerrado and through Mato Grosso, or through the Chaco and ar-
eas of transitional savannas in Paraguay and Bolivia, and would
have been interrupted at the end of the Neogene ( Batalha-Filho et
al., 2013 ). More recent connections between forests of Amazonia
and Atlantic remained via gallery forests in the Cerrado of central
Brazil and the Caatinga of northeastern Brazil, even if in a more
restricted way, allowing the transit of plants ( Oliveira-Filho and
Ratter, 1995 ) and animals ( Costa, 2003 ). This network of inter-
connections would have allowed the more recent dispersal of Pa-
chystachys spicata , for example.
Taxonomic implications — Schaueria clade— Traditionally,
Schau eria included approximately 20 species ( Nees, 1847 ; Clarke,
1900 ; Rusby, 1927 ; Daniel, 1990 ), however, phylogenetic analy-
ses reveal that six of its species are more closely related to other
lineages ( Fig. 2 ). According to our results, Schaueria should
comprise 14 species of herbs and shrubs, including three new spe-
cies and one species transferred from Justicia , J. paranaensis .
The genus is distinguished by its linear to lanceolate bracts (1–
19 × 0.5–2.5 mm), linear-triangular calyx (1.5–25 × 0.3–1 mm)
and usually a small, white corolla (1–2 cm long), but attaining up
to 5.5 cm in length when yellow ( S. calycotricha (Link & Otto)
Nees, S. sulfurea Nees, and the undescribed S. “ pyramidalis”).
The fl owers are nototribic with a shift to sternotribic in S. lophura
Nees. Based on shape, color and size of corolla, species are prob-
ably pollinated by bees and/or hummingbirds ( Nees, 1847 ; Côrtes,
personal observation). The lineage is restricted to the Atlantic for-
est, and occurs in rainforests, semideciduous forests and restin-
gas . The majority of species have restricted distributions that are
mostly confi ned to northeastern, southeastern, or southern regions
of Brazil (Côrtes et al., in prep.).
Pachystachys clade— Like Schaueria , this clade also has a
strong preference for South American moist forests; however, it
THE TETR AMERIUM LINEAGE—CÔRTES ET AL. • VOL. 102 , NO. 6 JUNE 2015 • 11
is geographically disjunct from the Schaueria clade. Pachys-
tachys clade consists of 18 species (Côrtes, et al., in prep.) dis-
tributed in the Amazon basin, with its center of diversity in Peru
( Wasshausen, 1986 ). Species of Streblacanthus (except for
Streblacanthus monospermus , type species of the genus) form a
basal grade from which Pachystachys s.s. is derived. Schaueria
azaleifl ora is sister to the rest of the species in the Pachystachys
clade and is the only species of the clade that occurs in the dry
forests of Bolivia ( Wasshausen and Wood, 2004 ).
Narrow bracts ( ≤ 1.5 mm wide) are characteristic of the basal
grade of the Pachystachys lineage ( Schaueria azaleifl ora , P .
“linearibracteata”, Streblacanthus dubiosus (Lindau) V.M.
Baum + P . gracilis , Streblacanthus roseus (Radlk.) B.L. Burtt,
and Streblacanthus cordatus Lindau), whereas broader and
more conspicuous bracts ( ≥ 4 mm wide) characterizes the Pa-
chystachys s.s. clade, with a reversal to narrow bracts in P . ba-
diospica Wassh. (c. 1.5 mm wide). Large fl owers, with a distally
expanded tube and a typically red corolla (white in P . lutea
Nees), are probably pollinated by hummingbirds. Such fl owers
represent a plesiomorphic state of the basal grade composed of
S. azaleifl ora and P . “linearibracteata”. The grade consisting of
species of Streblacanthus is distinguished by a narrow and long
fl oral tube and red, pink, or lavender corolla, and are possibly
pollinated by Lepidoptera. This fl oral diversity likely refl ects
adaptations to specifi c pollinators and suggests more than one
shift for hummingbird pollination ( Wasshausen, 1986 ; Daniel,
1993 , 1996 ; Smick, 2004 ; Daniel et al., 2008 ).
Thyrsacanthus clade— Thyrsacanthus comprises fi ve species
that are characterized by a shrubby habit, profuse branching,
inconspicuous bracts, and fl owers with a large and red corolla
having an expanded tube. The fl owers are probably pollinated
by hummingbirds ( Côrtes et al., 2010 ). The inclusion of Justi-
cia angustissima , Schaueria humulifl ora , Carlowrightia sul-
cata , Mirandea hyssopus , and Yeatesia mabryi renders the
Thyrsacanthus clade quite heterogeneous due to their different
fl owers, i. e., corolla generally small, white, purple or blue, and
with a narrow tube in J. angustissima , S. humulifl ora and Y.
mabryi , and infundibuliform corolla with bilabiate limb in C.
sulcata and M. hyssopus , refl ecting more than one change in
pollinator, that appears to vary from Lepidoptera and bees to
hummingbirds ( Hilsenbeck, 1989 ; Daniel, 2003 ; Daniel et al.,
2008 ; Côrtes, personal observation).
Justicia gonzalezii and J. zopilotensis , on the other hand,
share fl oral and pollen morphology with Thyrsacanthus ( Hen-
rickson and Hiriart, 1988 ; Daniel et al., 2008 ); both have large,
funnelform, reddish, and nototribic fl owers and 3-colporate,
6-pseudocolpate pollen. Justicia gonzalezii had been treated in
Anisacanthus ( Hagen, 1941 ), and both were treated in Justi-
cia ( Henrickson and Hiriart, 1988 ) for presenting nototribic
fl owers (vs. sternotribic in Anisacanthus ). Despite their het-
erogeneous morphology, a 366 bp deletion in the ndhF-rpl32
sequence (unpublished data) appears to represent a diagnostic
molecular synapomorphy for taxa of the Thyrsacanthus clade.
Taxa of the Thyrsacanthus clade occupy seasonally dry for-
est centers in South America and Mexico. This pattern is not
unexpected given the niche conservatism often associated with
this type of habitat among related species (e.g., Pennington
et al., 2004 , 2006 ; Särkinen et al., 2012 ; Cardoso et al., 2013 ).
Three species ( T. ramosissimus , T. microphyllus , and Justicia
angustissima ) occur in the Caatinga, the largest and most iso-
lated center of dry forests ( Pennington et al., 2000 ; Queiroz,
2006 ), and S. humulifl ora occurs in seasonal deciduous forests
of Bahia. Two species ( T. boliviensis (Nees) A. Côrtes & Rap-
ini and Carlowrightia sulcata ) are distributed in dry forests of
Bolivia (Piedmont centers), Chiquitano and Missiones centers,
whereas T. secundus is distributed in dry forests of northern
South America, with incursions into Amazon forests and in
northern restingas . The relationship with the Mexican clade ( J.
gonzalezii , J. zopilotensis , Yeatesia mabryi , and Mirandea hys-
sopus ) and the position of J. zopilotensis in this clade are here
confi rmed, as had been suggested by Daniel et al. (2008) .
CONCLUSIONS
Our results indicate a coincident chronology in events that
led to the ecological structuring of the Tetramerium lineage.
They suggest a diagonal corridor of dry biomes connecting the
Caatinga in the northeastern Brazil to the Missiones block of
seasonally dry forests in southwestern South America during
the Neogene. The dry forest lineage dispersed from North
America through the north coast of South America to northeast-
ern Brazil and, subsequently, to southwestern South America.
This corridor probably started to be fragmented in the Pliocene,
with the expansion of the Cerrado, suggesting a relatively old
isolation between these blocks.
The coincidence between the expansion of dry forests and
the vicariance between Amazonian and Atlantic moist forests
agrees with age estimates for older disjunctions associated with
the end of the moist forest corridors to the south of the Cerrado
( Batalha-Filho et al., 2013 ). The subsequent fragmentation of
dry forest centers is compatible with the pre-Pleistocene esti-
mates for divergences in plant genera typical of dry forests
( Pennington et al., 2004 ; Lavin, 2006 ; Särkinen et al., 2012 ),
and concur with the divergence age estimate between popula-
tions of dry forests that indicate a coalescence of isolated cen-
ters on a geographic scale for the lower Pleistocene ( Collevatti
et al., 2012 ). Our results are also in line with paleodistribution
simulations of dry forests in the LGM ( Mayle, 2004 ; Werneck
et al., 2011 ), and areas of climatic stability in the Atlantic forest
( Carnaval and Moritz, 2008 ) and in Amazonia ( Cheng et al.,
2013 ). The congruence between complementary biogeographi-
cal events in moist and dry forest clades and the consilience
among results obtained from phylogenetic, phylogeographic,
paleo-ecological, and paleodistribution modeling approaches
with different groups of organisms confer reliability to the age
estimated in this study for vicariance between dry forest blocks
from southwestern South America and northeastern Brazil. To-
gether, these results suggest the absence of a connection be-
tween Missiones block and Caatinga at least since the early
Pleistocene, clearly contrasting with a continuous dry forest
formation in South America during the Last Glacial Maximum,
as inferred by Prado and Gibbs (1993) in their classic Pleisto-
cene Arc hypothesis.
LITERATURE CITED
A LI , S. S. , Y. YU , M . P FOSER , AND W . W ETSCHNING . 2012 . Inferences of
biogeographical histories within subfamily Hyacinthoideae using
S-Diva and Bayesian binary MCMC analysis implemented in RASP
(Reconstruct Ancestral State in Phylogenies). Annals of Botany 109 :
95 – 107 .
A NTONELLI , A. , J. A. A. NYLANDER , C . P ERSON , AND I . S ANMARTÍN . 2009 .
Tracing the impact of the Andean uplift on Neotropical plant evo-
lution. Proceedings of the National Academy of Sciences, USA 106 :
9749 – 9754 .
12 • VOL. 102 , NO. 6 JUNE 2015 • AMERICAN JOURNAL OF BOTANY
B ATALHA-FILHO , H . , J . F JELDSA , F . P IERRE-HENRI , AND C. Y. MUYAKI . 2013 .
Connections between the Atlantic and Amazonia Forest avifaunas rep-
resent distinct historical events. Journal of Ornithology 154 : 41 – 50 .
B IGARELA , J. J. , D. ANDRADE-LIMA , AND P. J. RIEHS . 1975 . Considerações
a respeito das mudanças paleoambientais na distribuição de algumas
espécies vegetais e animais no Brasil. Anais da Academia Brasileira
de Ciências 47 : 411 – 464 .
B RUMMITT , R. K. 1978 . Proposal (447) to conserve the name Fittonia
Coemans over Adelaster Lindley ex Veitch (Acanthaceae). Taxon 27 :
307 – 309 .
C AETANO , S . , D . P RADO , R. T. PENNINGTON , S . B ECK , A . O LIVEIRA-FILHO ,
R . S PICHIGER , AND Y . N ACIRI . 2008 . The history of Seasonally Dry
Tropical Forests in eastern South America: Inferences from the ge-
netic structure of the tree Astronium urundeuva (Anacardiaceae).
Molecular Ecology 17 : 3147 – 3159 .
C ARDOSO , D. , L. P. QUEIROZ , H. C. LIMA , E . S UGANUMA , C . VAN DEN BERG ,
AND M . L AVIN . 2013 . A molecular phylogeny of the Vatireoid le-
gumes underscores fl oral evolvability that is general to many early-
branching Papilionoid lineages. American Journal of Botany 100 :
403 – 421 .
C ARNAVAL , A. C. , AND C . M ORITZ . 2008 . Historical climate modeling pre-
dicts patterns of current biodiversity in the Brazilian Atlantic forest.
Journal of Biogeography 35 : 1187 – 1201 .
C ERLING , T. E. , J. M. HARRIS , B. J. MACFADDEN , M. G. LEAKEY , J . Q UADEK ,
V . E ISENMANN , AND J. R. EHLERINGER . 1997 . Global vegetation change
through the Miocene / Pliocene boundary. Nature 389 : 153 – 158 .
C HENG , H . , A . S INHA , F. W. CRUZ , X . W ANG , R. L. EDWARDS , F. M. D’HORTA ,
C. C. RIBAS , ET AL . 2013 . Climate change patterns in Amazonia and
biodiversity. Nature Communications 4 : 1411.
C LARKE , C. B. 1900 . Acanthaceae . In D. Oliver, W. T. T. Dyer, and A. W.
David [eds.], Flora of Tropical Africa, vol. 5, part 2, 1–242. L. Reeve,
London, UK.
C OATES , A. G. , L. S. COLLINS , M. O. AUBRY , AND A. N. D. W. A. BERGGREN .
2004 . The geology of the Darien, Panama, and the late Miocene-
Pliocene collision of the Panama arc with northwestern South
America. Geological Society of America Bulletin 116 : 1327 – 1344 .
C OHEN , K. M. , S. C. FINNEY , P. L. GIBBARD , AND J.-X. FAN . 2013 . (updated).
The ICS International Chronostratigraphic Chart. Episodes 36 : 199 – 204 .
C OLINVAUX , P. A. , AND P. E. DE O LIVEIRA . 2001 . Amazon plant diversity and
climate through the Cenozoic. Palaeogeography, Palaeoclimatology,
Palaeoecology 166 : 51 – 63 .
C OLLEVATTI , R. G. , L. C. TERRIBILE , M. S. LIMA-RIBEIRO , J. C. NABOUT , G .
O LIVEIRA , T. F. RANGEL , S. G. RABELO , AND J. A. F. DINIZ-FILHO . 2012 .
A coupled phylogeographical and species distribution modelling ap-
proach recovers the demographical history of a Neotropical season-
ally dry forest tree species. Molecular Ecology 21 : 5845 – 5863 .
C OLLEVATTI , R. G. , L. C. TERRIBILE , G . O LIVEIRA , M. S. LIMA-RIBEIRO , J. C.
N ABOUT , T. F. RANGEL , AND J. A. F. DINIZ-FILHO . 2013 . Drawbacks to
palaeodistribution modelling: The case of South American Seasonally
Dry Forests. Journal of Biogeography 40 : 345 – 358 .
C ÔRTES , A. L. A. , R. L. B. BORGES , AND A . R APINI . 2010 . Reinstatement of
Thyrsacanthus Moric. (Acanthaceae) and taxonomic novelties in the
genus. Taxon 59 : 965 – 972 .
C OSTA , L. P. 2003 . The historical bridge between the Amazon and the
Atlantic forest of Brazil: A study of molecular phylogeography with
small mammals. Journal of Biogeography 30 : 71 – 86 .
C UNNINGHAM , C. B. 1997 . Is incongruence between data partitions a
reliable predictor for phylogenetic accuracy? Empirically testing an
interactive procedure for choosing among phylogenetic methods.
Systematic Biology 46 : 464 – 478 .
D ANIEL , T. F. 1983a . Carlowrightia (Acanthaceae). Flora neotropica,
monograph 34. New York Botanical Garden, Bronx, New York, USA.
D ANIEL , T. F. 1983b . Systematics of Holographis (Acanthaceae). Journal
of the Arnold Arboretum 64 : 129 – 160 .
D ANIEL , T. F. 1985 . Artifi cial interspecifi c hybridization of three spe-
cies of Anisacanthus (Acanthaceae). Journal of the Arizona-Nevada
Academy of Science 19 : 85 – 88 .
D ANIEL , T. F. 1986 . Systematics of Tetramerium (Acanthaceae).
Systematic Botany Monographs , vol. 12. American Society of Plant
Taxonomists, Ann Arbor, Michigan, USA.
D ANIEL , T. F. 1990 . New and reconsidered Mexican Acanthaceae IV.
Proceedings of the California Academy of Sciences 4 : 279 – 287 .
D ANIEL , T. F. 1993 . Taxonomic and geographic notes on Central
American Acanthaceae. Proceedings of the California Academy of
Sciences 4 : 119 – 130 .
D ANIEL , T. F. 1996 . Sciaphyllum amoenum (Acanthaceae) is a Peruvian
Streblacanthus. Novon 6 : 147 – 149 .
D ANIEL , T. F. 2003 . A new combination in Mirandea (Acanthaceae). Acta
Botanica Mexicana 62 : 9 – 13 .
D ANIEL , T. F. , L. A. MCDADE , M . M ANKTELOW , AND C. A. KIEL . 2008 . The
“ Tetramerium Lineage” (Acanthaceae: Acanthoideae: Justicieae):
Delimitation and intra-lineage relationships based on cp and nrITS
sequence data. Systematic Botany 33 : 416 – 436 .
D ESFEUX , C . , S . M AURICE , J. P. HENRY , B . L EJEUNE , AND P. H. GOUYON . 1996 .
The evolution of reproductive system in the genus Silene . Proceedings
of the Royal Society of London, B: Biological Sciences 263 : 409 – 414 .
D ORADO , O. , L. H. RIESEBERG , AND D. M. ARIAS . 1992 . Chloroplast
DNA introgression in southern California sunfl owers. Evolution 46 :
566 – 572 .
D OYLE , J. J. , AND J. L. DOYLE . 1987 . A rapid DNA isolation procedure for
small amounts of fresh leaf tissue. Phytochemical Bulletin 19 : 11 – 15 .
D RUMMOND , A. J. , AND A . R AMBAUT . 2007 . BEAST: Bayesian evolution-
ary analysis by sampling trees. BMC Evolutionary Biology 7 : 214 .
E ZCURRA , C. 1994 . Carlowrightia sulcata (Acanthaceae), una especie de
Sudamerica Austral tratada previamente en Siphonoglossa. Novon 4 :
221 – 223 .
F ARRIS , J. S. , M. KÄLLERSJÖ , A. G. KLUGE , AND C . B ULT . 1994 . Testing
signifi cance of incongruence. Cladistics 10 : 315 – 319 .
F EHRER , J . , B . G EMEINHOLZER , J . C HRTEK J R . , AND S . B RÄUTIGAM . 2007 .
Incongruent plastid and nuclear DNA phylogenies reveal ancient
intergeneric hybridization in Pilosella hawkweeds ( Hieracium ,
Cichorieae, Asteraceae). Molecular Phylogenetics and Evolution 42 :
347 – 361 .
G UINDON , S. , J. F. DUFAYARD , V . L EFORT , M . A NISIMOVA , W . H ORDIJK , AND
O . G ASCUEL . 2010 . New algorithms and methods to estimate maxi-
mum-likelihood phylogenies: Assessing the performance of PhyML
3.0. Systematic Biology 59 : 307 – 321 .
H AFFER , J. 1969 . Speciation in Amazonian forest birds. Science 165 :
131 – 137 .
H AGEN , S. H. 1941 . A revision of the North American species of the genus
Anisacanthus. Annals of the Missouri Botanical Garden 28 : 385 – 408 .
H AMILTON , M. B. 1999 . Four primer pairs for the amplifi cation of chlo-
roplast intergenic regions with intraspecifi c variation. Molecular
Ecology 8 : 521 – 523 .
H ENRICKSON , J . , AND P . H IRIART . 1988 . New species and transfers into
Justicia (Acanthaceae). Aliso 12 : 45 – 58 .
H ILSENBECK , R. A. 1989 . Taxonomy of Yeatesia (Acanthaceae). Systematic
Botany 14 : 427 – 438 .
H OORN , C. , F. P. WESSELINGH , H . TER STEEGE , M. A. BERMUDEZ , A . M ORA , J .
S EVINK , I . S ANMARTÍN , ET AL . 2010 . Amazonia through time: Andean
uplift, climate change, landscape evolution, and biodiversity. Science
330 : 927 – 931 .
H UGHES , C. E. , R. T. PENNINGTON , AND A . A NTONELLI . 2013 . Neotropical
plant evolution: Assembling the big picture. Botanical Journal of the
Linnean Society. Linnean Society of London 171 : 1 – 18 .
IBGE . 2012 . Manual técnico da vegetação brasileira. Instituto Brasileiro
de Geografi a e Estatística, Rio de Janeiro, Brazil.
I TURRALDE-VINENT , M. A. , AND R. D. E. MACPHEE . 1999 . Palaeogeography
of the Caribbean region: Implications for Cenozoic biogeography.
Bulletin of the American Museum of Natural History 238 : 1 – 95 .
J ABAILY , R. S. , AND K. J. SYTSMA . 2013 . Historical biogeography and life
history evolution of Andean Puya (Bromeliaceae). Botanical Journal
of the Linnean Society. Linnean Society of London 171 : 201 – 224 .
K IEL , C. A. , L. A. MCDADE , T. F. DANIEL , AND D . C HAMPLUVIER . 2006 .
Phylogenetic delimitation of Isoglossinae (Acanthaceae: Justiceae)
and relationships among constituent genera. Taxon 55 : 683 – 694 .
K IKUCHI , R . , P . J AE-HONG , H . T AKAHASHI , AND M . M AKI . 2010 . Disjunct
distribution of chloroplast DNA haplotypes in the understory peren-
nial Veratrum album ssp. oxysepalum (Melanthiaceae) in Japan as a
result of ancient introgression. New Phytologist 188 : 879 – 891 .
THE TETR AMERIUM LINEAGE—CÔRTES ET AL. • VOL. 102 , NO. 6 JUNE 2015 • 13
L AMBECK , K. , T. M. ESAT , AND E.-K. POTTER . 2002 . Links between cli-
mate and sea levels for the past three million years. Nature 419 :
199 – 206 .
L AVIN , M. 2006 . Floristic and geographical stability of discontinuous
Seasonally Dry Forests explains patterns of plant phylogeny and en-
demism. In R. T. Pennington, G. P. Lewis, and J. A. Ratter [eds.],
Neotropical savannas and dry forests: Plant diversity, biogeography
and conservation, 433–447. CRC Press, Taylor and Francis Group,
Boca Raton, Florida, USA.
L ÓPEZ , R. P. , D. L. ALC ÁZAR , AND M. J. MACÍA . 2006 . The arid and dry
plant formations of South America and their fl oristic connections:
New data, new interpretation? Darwiniana 44 : 18 – 31 .
M ADDISON , W. P. , AND D. R. MADDISON . 2010 . Mesquite: A modular
system for evolutionary analysis , version 2.74. Website http://mes-
quiteproject.org . [accessed 5 January 2013].
M ARTINS , F. M. , A. R. TEMPLETON , A. C. O. PAVAN , B. C. KOHLBACH , AND
J. S. MORGANTE . 2009 . Phylogeography of the common vampire
bat ( Desmodus rotundus ): Marked population structure, Neotropical
Pleistocene vicariance and incongruence between nuclear and mtDNA
markers. BMC Evolutionary Biology 9 : 294 – 307 .
M AUTINO , I. R. 2011 . Nuevas especies de palinomorfos de las formacio-
nes San José y Chiquimil (Mioceno Medio y Superior), noroeste de
Argentina. Revista Brasileira de Paleontologia 14 : 279 – 290 .
M AYLE , F. E. 2004 . Assessment of the Neotropical dry forest refugia hy-
pothesis in the light of palaeoecological data and vegetation model
simulations. Journal of Quaternary Science 19 : 713 – 720 .
M AYLE , F. E. 2006 . The late Quarternary biogeographical history of South
American Seasonally Dry Tropical Forests: Insights from Palaeo-
ecological data . In R. T. Pennington, G. P. Lewis, and J. A. Ratter
[eds.], Neotropical savannas and dry forests: Plant diversity, bioge-
ography and conservation, 395–416. CRC Press, Taylor and Francis
Group, Boca Raton, Florida, USA.
M AYLE , F. E. , AND M. J. POWER . 2008 . Impact of a drier Early-
Mid-Holocene climate upon Amazonian forests. Philosophical
Transactions of the Royal Society of London, B, Biological Sciences
363 : 1829 – 1838 .
M CDADE , L. A. , T. F. DANIEL , S. E. MASTA , AND K. M. RILEY . 2000 .
Phylogenetic relationships within the tribe Justicieae (Acanthaceae):
Evidence from molecular sequences, morphology and cytology.
Annals of the Missouri Botanical Garden 87 : 435 – 458 .
M OGNI , V. Y. , L. J. OAKLEY , AND D. E. PRADO . 2015 . The distribution of
woody legumes in neotropical dry forests: The Pleistocene arc theory
20 years on. Edinburgh Journal of Botany 72 : 35 – 60 .
M ONTES , C . , A . C ARDONA , R . M CFADDEN , S. E. MORÓN , C. A. SILVA , R. D.
A. RESTREPO-MORENO , N . H OYOS , ET AL . 2012 . Evidence for middle
Eocene and younger land emergence in central Panama: Implications
for Isthmus closure. Geological Society of America Bulletin 124 :
780 – 799 .
M ORLEY , R. J. 2000 . Origin and evolution of tropical rain forests . Wiley,
Chichester, UK.
M YERS , N. , R. A. MITTEMEIER , C. G. MITTERMEIER , G. A. B. FONSECA , AND J .
K ENT . 2000 . Biodiversity hotspots for conservation priorities. Nature
403 : 853 – 858 .
N EES , C. G. 1847 . Acanthaceae . In A. Candolle [ed.], Prodromus
Systematis Naturalis Regni Vegetabilis, vol. 11, 46–519. Victor
Masson, Paris, France.
N ELSON , B. W. , C. A. C. FERREIRA , M. F. SILVA , AND M. L. KAWASAKI .
1990 . Endemism centres, refugia and botanical collection density in
Brazilian Amazonia. Nature 345 : 714 – 716 .
N YLANDER , J. A. A. 2008 . MrModeltest 2.3. Program distributed by the author.
Evolutionary Biology Centre. Uppsala University, Uppsala, Sweden.
Website: https://www.abc.se/~nylander/mrmodeltest2/mrmodeltest2.
html/ [accessed 6 June 2015].
O KUYAMA , Y . , N . F UJII , M . W AKABAYASHI , A . K AWAKITA , M . I TO , M .
W ATANABE , N . M URAKAMI , AND M . K ATO . 2005 . Nonuniform convert
evolution and chloroplast capture: Heterogeneity of observed intro-
gression patterns in three molecular data partition phylogenies of
Asian Mitella (Saxifragaceae). Molecular Biology and Evolution 22 :
285 – 296 .
O LIVEIRA , P . E . DE , A . M . F . B ARRETO , AND K . S UGUIO . 1999 . Late
Pleistocene/Holocene climatic and vegetational history of the
Brazilian caatinga: The fossil dunes of the middle São Francisco
river. Palaeogeography, Palaeoclimatology, Palaeoecology 152 :
319 – 337 .
O LIVEIRA-FILHO , A. T. , AND J. A. RATTER . 1995 . A study of the origin of
the central Brazilian forests by analysis of plant species distribution.
Edinburgh Journal of Botany 52 : 141 – 194 .
O LSON , D . , E . D INERSTEIN , E . W IKRAMANAYAKE , N . B URGESS , G . P OWELL , E .
U NDERWOOD , J . D ’ AMICO , ET AL . 2001 . Terrestrial ecoregions of the
world—a new map of life on Earth. Bioscience 51 : 933 – 938 .
O XELMAN , B . , M . L INDEN , AND D . B ERGLUND . 1997 . Chloroplast rps16
intron phylogeny of the tribe Sileneae (Caryophyllaceae). Plant
Systematics and Evolution 206 : 393 – 410 .
P ALMA-SILVA , C . , T . W ENDT , F . P INHEIRO , T . B ARBARA , M. F. FAY , S .
C OZZOLINO , AND C . L EXERS . 2011 . Sympatric bromeliad species
( Pitcairnia spp.) facilitate tests of mechanisms involved in species co-
hesion and reproductive isolation in Neotropical lineages. Molecular
Ecology 20 : 3185 – 3201 .
P ENNINGTON , R. T. , M. LAVIN , AND A . O LIVEIRA-FILHO . 2009 . Woody plant
diversity, evolution, and ecology in the tropics: Perspectives from
Seasonally Dry Tropical Forests. Annual Review of Ecology Evolution
and Systematics 40 : 437 – 457 .
P ENNINGTON , R . T . , M . L AVIN , D. E. PRADO , C. A. PENDRY , S. K. PELL ,
AND C. A. BUTTERWORTH . 2004 . Historical climate change and
speciation: Neotropical seasonally dry forest plants show patterns
of both Tertiary and Quaternary diversifi cation. Philosophical
Transactions of the Royal Society of London, B, Biological
Sciences 359 : 515 – 537 .
P ENNINGTON , R. T. , D. E. PRADO , AND C. A. PENDRY . 2000 . Neotropical
seasonally dry forests and Quaternary vegetation changes. Journal of
Biogeography 27 : 261 – 273 .
P ENNINGTON , R. T. , J. E. RICHARDSON , AND M . L AVIN . 2006 . Insights into
the historical construction of species-rich biomes from dated plant
phylogenies, neutral ecological theory and phylogenetic community
structure. New Phytologist 172 : 605 – 616 .
P RADO , D. E. , AND P. E. GIBBS . 1993 . Patterns of species distributions in
the dry seasonal forests of South America. Annals of the Missouri
Botanical Garden 80 : 902 – 927 .
Q UEIROZ , L. P. 2006 . The Brazilian Caatinga: Phytogeographical patterns
inferred from distribution data of the Leguminosae . In R. T. Pennington,
G. P. Lewis, and J. A. Ratter [eds.], Neotropical savannas and dry for-
ests: Plant diversity, biogeography and conservation, 121–157. CRC
Press, Taylor and Francis Group, Boca Raton, Florida, USA.
R AMBAUT , A. 2009 . FigTree: Tree fi gure drawing tool, version 1.3.1. Website
http://tree.bio.ed.ac.uk/software/fi gtree . [accessed 5 January 2013].
R AMBAUT , A . , AND A . D RUMMOND . 2007 . Tracer: A program for analyzing
results from Bayesian MCMC programs, version 1.4. Website http://
evolve.-zoo.ox.ac.uk/software [accessed 5 January 2013].
R AVEN , P. H. , AND D. I. AXELROD . 1974 . Angiosperm biogeography
and past continental movements. Annals of the Missouri Botanical
Garden 61 : 539 – 673 .
R IESEBERG , L. H. , AND J. F. WENDEL . 1993 . Introgression and its consequences
in plants. In R. Harrison [ed.], Hybrid zones and the evolutionary process,
70–109. Oxford University Press, New York, New York, USA.
R OKAS , A. , B. L. WILLIANS , N . K ING , AND S. B. CARROLL . 2003 . Genome-
scale approaches to resolving incongruence in molecular phylogenies.
Nature 425 : 798 – 804 .
R ONQUIST , F . , AND J. P. HUELSENBECK . 2003 . MrBayes 3.1.2: Bayesiana
phylogenetic inference under mixed models. Bioinformatics (Oxford,
England) 19 : 1572 – 1574 .
R ULL , V. 2008 . Speciation timing and Neotropical biodiversity: The
Tertiary-Quaternary debate in the light of molecular phylogenetic evi-
dence. Molecular Ecology 17 : 2722 – 2729 .
R ULL , V. 2011 . Neotropical biodiversity: Timing and potential drivers.
Trends in Ecology & Evolution 26 : 508 – 513 .
R USBY , H. H. 1927 . Descriptions of new species of plants collected on
the Mulford Biological Exploration of the Amazon Valley, 1921–22.
Memoirs of the New York Botanical Garden 7 : 205 – 387 .
14 • VOL. 102 , NO. 6 JUNE 2015 • AMERICAN JOURNAL OF BOTANY
S ANTOS , A. M. M. , D. R. CAVALCANTI , J. M. C. SILVA , AND M . T ABARELLI .
2007 . Biogeographical relationships among tropical forests in North-
Eastern Brazil. Journal of Biogeography 34 : 437 – 446 .
S ÄRKINEN , T. , R. T. PENNINGTON , M . L AVIN , M. F. SIMON , AND C. E. HUGHES .
2012 . Evolutionary islands in the Andes: Persistence and isolation
explain high endemism in Andean dry tropical forests. Journal of
Biogeography 39 : 884 – 900 .
S HIMODAIRA , H . , AND M . H ASEGAWA . 1999 . Multiple comparisons of log-
likelihoods with applications to phylogenetic inference. Molecular
Biology and Evolution 16 : 1114 – 1116 .
S ILVA , S. M. 1999 . Diagnóstico das restingas do Brasil. In Fundação Bio
Rio [ed.], Workshop Avaliação e Ações Priorit árias para a Conservação
da Biodiversidade da Zona Costeira, Ilhéus, Bahia, Brasil.
S IMON , M. F. , R. GRETHER , L. P. QUEIROZ , C . S KEMA , R. T. PENNINGTON , AND C .
E. HUGHES . 2009 . Recent assembly of the Cerrado, a Neotropical plant
diversity hotspot, by in situ evolution of adaptations to fi re. Proceedings
of the National Academy of Sciences, USA 106 : 20359 – 20364 .
S IMON , M. F. , AND T . P ENNINGTON . 2012 . Evidence for adaptation to fi re re-
gimes in the tropical savannas of the Brazilian Cerrado. International
Journal of Plant Sciences 173 : 711 – 723 .
S MALL , R. L. , R. C. CRONN , AND J. F. WENDEL . 2004 . Use of nuclear genes
for phylogeny reconstruction in plants. Australian Systematic Botany
17 : 145 – 170 .
S MICK , G. A. 2004 . Monograph of Streblacanthus Kuntze (Acanthaceae):
Taxonomy and phylogenetics. M.S. thesis. San Francisco State
University, San Francisco, California, USA.
S ONG , S . , L . L IU , S. V. EDWARDS , AND S . W U . 2012 . Resolving confl ict
in eutherian mammal phylogeny using phylogenomics and the mul-
tispecies coalescent model. Proceedings of the National Academy of
Sciences, USA 109 : 14942 – 14947 .
S TADEN , R. , D. P. JUDGE , AND J. K. BONFIELD . 2003 . Analysing sequences
using the Staden Package and EMBOSS . In S. A. Krawetz and D.
Womble [eds.], Introduction to bioinformatics. A theoretical and
practical approach. Human Press, Totawa, New Jersey, USA.
S UN , Y. , D. Z. SKINNER , G. H. LIANG , AND S. H. HULBERT . 1994 . Phylogenetic
analysis of Sorghum and related taxa using internal transcribed spacers
of nuclear ribosomal DNA. Theoretical and Applied Genetics 89 : 26 – 32 .
S WOFFORD , D. L. 2000 . PAUP: Phylogenetic analysis using parsimony,
version 4.0b10 . Sinauer, Sunderland, Massachusetts, USA.
T ABERLET , P . , L . G IELLY , G . P AUTOU , AND J . B OUVET . 1991 . Universal
primers for amplifi cation of three non-coding regions of chloroplast
DNA. Plant Molecular Biology 17 : 1105 – 1109 .
T HIERS , B. 2013 (continuously updated) . Index Herbariorum: A global
directory of public herbaria and associated staff. New York Botanical
Garden's Virtual Herbarium. Website http://sweetgum.nybg.org/ih/
[accessed 5 January 2013].
T RIPP , E. A. , T. F. FATIMAH , I . D ARBYSHIRE , AND L. A. MCDADE . 2013 .
Origin of African Physacanthus (Acanthaceae) via wide hybridiza-
tion. PLoS ONE 8 : e55677 .
T RIPP , E. A. , AND L. A. MCDADE . 2014 . A rich fossil record yields cali-
brated phylogeny for Acanthaceae (Lamiales) and evidence for
marked biases in timing and directionality of intercontinental disjunc-
tions. Systematic Biology 10.1093/sysbio/syu029 .
W ASSHAUSEN , D. C. 1986 . The systematics of the genus Pachystachys
(Acanthaceae). Proceedings of the Biological Society of Washington
99 : 160 – 185 .
W ASSHAUSEN , D. C. , AND J. R. I. WOOD . 2004 . Acanthaceae of Bolivia.
Contributions from the United States National Herbarium, vol.
49 . Department of Botany, National Museum of Natural History,
Washington, District of Columbia, USA.
W EN , J . , AND E. A. ZIMMER . 1996 . Phylogeny and biogeography of Panax
L. (the ginseng genus, Araliaceae): Inferences from ITS sequences of
nuclear ribosomal DNA. Molecular Phylogenetics and Evolution 6 :
167 – 177 .
W ERNECK , F. P. 2011 . The diversifi cation of eastern South American
open vegetation biomes: Historical biogeography and perspectives.
Quaternary Science Reviews 30 : 1630 – 1648 .
W ERNECK , F . P . , G . C . C OSTA , G. R. COLLI , D. E. PRADO , AND J . W . S ITES
J R . 2011 . Revisiting the historical distribution of Seasonally Dry
Tropical Forests: New insights based on palaeodistribution modeling
and palynological evidence. Global Ecology and Biogeography 20 :
272 – 288 .
W HITE , T. J. , T. BRUNS , S . L EE , AND J . T AYLOR . 1990 . Amplifi cation and
direct sequencing of fungal ribosomal genes for phylogenetics. In M.
A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White [eds.], PCR
protocols: A guide to methods and applications, 315–322. Academic
Press, Orlando, Florida, USA.
Y U , Y. , A. J. HARRIS , AND X . H E . 2010 . S-DIVA (statistical dispersal-
vicariance analysis): A tool for inferring biogeographic histories.
Molecular Phylogenetics and Evolution 56 : 848 – 850 .
Z ACHOS , J . , M . P AGANI , L . S LOAN , E . T HOMAS , AND K . B ILLUPS . 2001 .
Trends, rhythms, and aberrations in global climate 65 Ma to present.
Science 292 : 686 – 693 .
THE TETR AMERIUM LINEAGE—CÔRTES ET AL. • VOL. 102 , NO. 6 JUNE 2015 • 15
A PPENDIX 1. Taxa, Genbank accession numbers ( rps 16, trnL-F , trnT-L , trnS- G, ITS; — indicate unavailable sequences), voucher specimens for DNA sequences
used in this study: Collector and number (herbarium acronym, according to Thiers, 2013 ). GenBank accession numbers ( McDade et al., 2000 ; Daniel et al.,
2008 ; Tripp et al., 2013 ).
Anisacanthus junceus Hemsl.; —,—, EU081110, EU0810421, EU0874301; M.
Manktelow 720 (UPS). Anisacanthus linearis (S.H. Hagen) Henrickson
& E.J. Lott; EU0874821, EU0875381, EU0811111, EU0874311; Louie
s.n. (CAS). Anisacanthus puberulus (Torr.) Henrickson & E.J. Lott; —, —,
EU0811121, EU0810441, AF2897781; L. McDade 1179 (ARIZ).
Anisacanthus quadrifi dus var. wrightii (Torr.) Henrickson; —, —, —, —,
EU0874321; M. Manktelow 688 (UPS). Anisacanthus tetracaulis
Leonard; EU0874991, —, EU0811331, EU0810451, EU0874491; J.M.
Tucker 629 (CAS). Anisacanthus trilobus Lindau; KJ535244, KJ535276,
KJ535174, —, KJ535209, A.L.A. Côrtes et al. 97 (HUEFS). Aphanosperma
sinaloensis (Leonard & Gentry) T.F. Daniel; EU0875031, EU0875501,
EU0811381, EU0810721, EU0874541; T.F. Daniel 4060cv (CAS).
Carlowrightia arizonica A. Gray; EU0874851, AF0631231, EU0811151,
EU0810481, EU0874341; C.E. Jenkins 8924 (ARIZ). Carlowrightia
hapalocarpa B.L. Rob. & Greem.; EU0875001, EU0875481, EU0811341,
EU0810671, EU0874501; M. Manktelow 715 (UPS). Carlowrightia
huicholiana T.F. Daniel; EU0875011, —, EU0811351, EU0810661,
EU0874511; J.A. Bauml & G.Voss 1896 (CAS). Carlowrightia linearifolia
(Torr.) A. Gray; EU0874881, EU0875421, EU0811191, EU0810501, —;
M. Manktelow 722 (UPS). Carlowrightia mcvaughii T.F. Daniel;—,
EU0875491, EU0811361, EU0810681, EU0874521; T.F. Daniel 5262
(CAS). Carlowrightia myriantha (Standl.) Standl.; EU0875041,—,
EU0811391, EU0810731, EU0874551; T.F. Daniel 8267 (CAS).
Carlowrightia neesiana (Schauer ex Nees) T.F. Daniel; EU0874871,
EU0875411, EU0811181, EU0810491, EU0874361; M. Manktelow 708
(UPS). Carlowrightia parvifl ora (Buckley) Wassh.; EU0875021,—,
EU0811371, EU0810691, EU0874531; M. Manktelow 704 (UPS).
Carlowrightia serpyllifolia A. Gray; EU0874891, —, EU0811201,
EU0810521, EU0874371; M. Manktelow 694 (UPS). Carlowrightia
sulcata (Nees) C. Ezcurra; —, —, KJ535171, KJ535303, KJ535206; A.
Krapovickas 17381 (CAS). Carlowrightia torreyana Wassh.;
EU0874911,—, EU0811221, EU0810541, EU0874391; M. Manktelow
690 (UPS). Chalarothyrsus amplexicaulis Lindau; EU0875051,
AF2897401, EU0811401, EU0810741, AF2897801; T.F. Daniel & B.
Bartholomew 4842cv (CAS). Chlamydocardia buettneri Lindau;
EU0875351; EU0875691; EU0811741; EU0811071; EU0874801; A.
Krapovickas 17381 (CAS). Chlamydocardia buettneri Lindau;
EU0875351, EU0875691, EU0811741, EU0811071, EU0874801;
cultivated, National Botannic Garden of Belgium (native to Cameroun,
Gabon, Ivory Coast and Nigeria), accession No. 95-0034-44 (BR).
Clinacanthus siamensis Bremek.; EU0875341, EU0875681, EU0811731,
EU0811061, EU0874791; cultivated, National Botannic Garden of
Belgium (native to Thailand), accession No. 1979-0344 (BR). Dicliptera
sp. ( Daniel 9194 ); —, AF2897231, —, —, AF2897641; T.F. Daniel 9194
(CAS). Dicliptera suberecta (André) Bremek.; —, AF2897221, —, —,
AF2897631; L. McDade 1176 (ARIZ). Dyschoriste albifl ora Lindau;
KC420528, KC420612, KC118466, KC420586, KC420544; B. Luwiika
et al. 580 (MO). Ecbolium madagascariense Vollesen; EU3157901, —,
EU0811681, EU0811011, —; T.F. Daniel et al. 10412 (PH). Ecbolium
tanzaniense
Vollesen; EU0875301, —, EU0811691, EU0811021,
EU0874751; G.S. Bidgood et al. 567 (CAS). Ecbolium viride (Forssk.)
Alston; EU0875311,—, EU0811701, EU0811031, EU0874761; Ib. Friis
& K. Vollesen 5050 (CAS). Fittonia albivenis (Lindl. ex Veitch) Brummitt;
KJ535213, KJ535246, —, KJ535277, KJ535176; A.L.A. Côrtes 235
(HUEFS). Harpochilus nessianus Mart. ex Nees;—, AF2897211,—,—,
AF2897621; Souza et al. 5413 (CAS). Harpochilus phaeocarpus Nees;—
,—,—,—, KJ535210; L.P. Queiroz 13899 (HUEFS). Henrya insularis
Nees; EU0875071, AF0631251, EU0811421, EU0810711, AF1698431;
C.E. Jenkins 89-432 (ARIZ). Herpetacanthus stenophyllus Gómez-Laur.
& Grayum;—,—,—,—, AF2897951; J. Herrera 3855 (ARIZ). Hoverdenia
speciosa Nees; EU0875191, AF2897381, EU0811571, EU0810891,
AF2897771; T.F. Daniel & M. Baker 3739 (CAS). Hygrophila corymbosa
Lindau; EU529024, AF063120, EU529090, EU528961, AF169836;
897223 (MO). Isoglossa grandifl ora C.B. Clarke; —, AF289745,
DQ3724451, DQ3724901, AF2897881; T.F. Daniel s.n. (CAS). Isoglossa
sp. ( Daniel 9106 ); —, AF2897461, —, —, AF2897891; T.F. Daniel 9106
(CAS). Justicia adhatoda L.; DQ0592141, AF289734, EU0811761,
DQ0592961, AF2897731; G.W. Barr 60-393 (ARIZ). Justicia
angustissima A. Côrtes & Rapini; KJ535241, KJ535273, KJ535170,
KJ535302, KJ535205; E. Melo et al. 4642 (HUEFS). Justicia betonica L.;
—, AF2897311,—,—, AF2897701; T.F. Daniel 9369 (CAS). Justicia
brandegeeana Wassh. & L.B. Sm.;—,—,—,—, AF2897591; C. Starr 32
(ARIZ). Justicia caudata A. Gray; EU5290281, AF0631341, EU5290931,
EU5289641, AF1698371; A. Faivre 64 (ARIZ). Justicia comata (L.)
Lam.;—,—,—,—, AF2897601; A. Faivre 59 (ARIZ). Justicia gonzalezii
(Greenm.) Henrickson & P. Hiriart; KJ535242, KJ535274, KJ535172,
KJ535304, KJ535207; B.Cruz 1093 (CAS). Justicia medranoi Henrickson
& P. Hiriart; EU0922551, —, EU0811561, EU0810881, EU0874651; T.F.
Daniel & M. Baker 3742 (CAS). Justicia paranaensis (Rizzini) Wassh. &
L.B. Sm.; KJ535233, KJ535265, KJ535163, KJ535294, KJ535198;
A.L.A. Côrtes et al. 266 (HUEFS). Justicia zopilotensis Henrickson & P.
Hiriart; KJ535243, KJ535275, KJ535173, KJ535305, KJ535208; T.F.
Daniel 5351 (CAS). Metarungia galpinii (Baden) Baden; EU5290461,—,
EU5289841,—, AF2897761; T.F. Daniel 9322 (CAS). Mexacanthus
mcvaughii T.F. Daniel; EU0874841, EU0875391, EU0811141,
EU0810471, EU0874331; T.R.. van Devender 94-23 (CAS). Mirandea
grisea Rzed.; EU0875221,—, EU0811611, EU0810951, AF2897831; T.F.
Daniel & M. Baker 3717 (CAS). Mirandea huastecensis T.F. Daniel;
EU0875231, EU0875601, EU0811621, EU0810961, EU0874691; M.
Manktelow 706 (UPS). Mirandea hyssopus (Nees) T.F. Daniel;
EU0875121, EU0875551, EU0811471, EU0810941, EU0874591; B.
Diaz & E. Carranza 7498 (CAS). Mirandea nutans (Nees) T.F. Daniel;
EU0875201, —, EU0811581, EU0810901, EU0874661; G.C. Rzedowski
53366 (IEB). Pachystachys badiospica Wassh.; KJ535214, KJ535247,
KJ535145, KJ535278, less 200 pb; P. Nuñez et al. 34040A (HUEFS).
Pachystachys coccinea (Aubl.) Nees;—, EU0875571, EU0811521,
EU0810831, EU0874621; R. Gustafsson 330 (NY). Pachystachys killipii
Wassh.; KJ535215, KJ535248, KJ535146, KJ535279, KJ535177; P.
Nuñez et al. 34053 (HUEFS). Pachystachys lutea Nees; KJ535216,
KJ535249, KJ535147, KJ535280, KJ535178; A.L.A.Côrtes et al. 162
(HUEFS). Pachystachys lutea Nees; EU0875161, AF0631281,
EU0811511, EU0810821, AF1698441; L. McDade 1181 (DUKE).
Pachystachys ossolae Wassh.; KJ535217, KJ535250, KJ535148,
KJ535281, KJ535179; P. Nuñez et al. 34023 (HUEFS). Pachystachys
puberula Wassh.; KJ535218, KJ535251, KJ535149, KJ535282,
KJ535180; P. Nuñez et al. 34042 (HUEFS). Pachystachys rosea Wassh.;
KJ535219, KJ535252, KJ535150, KJ535283, KJ535181; P. Nuñez et al.
34002 (HUEFS). Pachystachys linearibracteata sp. ined.; KJ535222,
KJ535255, KJ535153, KJ535286, KJ535184; P. Nuñez et al. 34047
(HUEFS). Pachystachys spicata (Ruiz & Pav.) Wassh.; KJ535221,
KJ535254, KJ535152, KJ535285, KJ535183; P. Nuñez et al. 34052
(HUEFS). Pachystachys spicata (Ruiz & Pav.) Wassh.; KJ535220,
KJ535253, KJ535151, KJ535284, KJ535182; A.L.A. Côrtes & A.C. Mota
119 (HUEFS). Poikilacanthus macranthus Lindau; EU5290541,
AF0670661, EU5291211, EU5289941, AF1698381; W. Haber 707 (MO).
Populina richardii Baill.; EU0875321, EU0875661, EU0811711,
EU0811041, EU0874771; M. Keraudren 1671 (P). Pseuderanthemum
atropurpureum (W. Bull.) Radlk.;—,—,—,—, JF3461661.
Pseuderanthemum fl oribundum T.F. Daniel;—,—,—, DQ3725071,
DQ3724791; T.F. Daniel 5381cv (CAS). Rhinacanthus gracilis Klotzsch.;
EU5290571,—, EU5289951,—, AF2897661; T.F. Daniel s.n. (CAS).
Ruellia humilis Nutt; AF482604, AF482604, KC11850, EU431038, —; E.
Tripp 14 (PH). Schaueria azaleifl ora Rusby; KJ535224; —, —, KJ535288,
KJ535187; J.R.I. Wood 12593 (CAS). Schaueria azaleifl ora Rusby;
EU0875151, —, EU0811501, EU0810811, EU0874611; J.R.I. Wood
12593 (CAS). Schaueria calycotricha (Link & Otto) Nees; KJ535225,
KJ535257, KJ535155, KJ535289, KJ535188; A.L.A. Côrtes & A.C. Mota
160 (HUEFS). Schaueria capitata Nees; KJ535226, KJ535258,
KJ535156, KJ535290, KJ535191; A.L.A. Côrtes et al. 200 (HUEFS).
Schaueria capitata Nees;—,—,—,—, KJ535189; A.L.A. Côrtes et al. 187
(HUEFS). Schaueria capitata Nees;—,—,—,—, KJ535190; A.L.A.
Côrtes et al. 198 (HUEFS). Schaueria gonystachya Nees; KJ535227,
KJ535259, KJ535157, KJ535291,—; A.L.A. Côrtes & R.L.B. Borges 239
(HUEFS). Schaueria gonystachya Nees;—,—,—,—, KJ535192; A.L.A.
Côrtes 237 (HUEFS). Schaueria hirsuta Nees; KJ535245;—, KJ535175,
KJ535306; KJ535211; M.N.S. Stapf et al. 349 (HUEFS). Schaueria hirta
sp. ined.; KJ535228, KJ535260, KJ535158, —, KJ535193; A.L.A. Côrtes
& R.L.B. Borges 253 (HUEFS). Schaueria humulifl ora Nees; KJ535235,
16 • VOL. 102 , NO. 6 JUNE 2015 • AMERICAN JOURNAL OF BOTANY
KJ535267, KJ535165, KJ535296, KJ535201; A.L.A. Côrtes et al. 31
(HUEFS). Schaueria lachynostachya Nees; KJ535230, KJ535262,
KJ535160, —, KJ535195; A.L.A. Côrtes & A.C. Mota 147 (HUEFS).
Schaueria lophura Nees; KJ535231, KJ535263, KJ535161, KJ535293,
KJ535196; A.L.A. Côrtes et al. 193 (HUEFS). Schaueria malifolia
Nees;—,—,—,—, KJ535212; C.A.L. Oliveira 1917 (GUA). Schaueria
marginata Nees; KJ535232, KJ535264, KJ535162, —, KJ535197; A.L.A.
Côrtes et al. 231 (HUEFS). Schaueria parvifl ora (Leonard) T.F.
Daniel;—,—,—,—, KJ535200; J.I. Calzada 1773 (CAS). Schaueria
pyramidalis sp. ined.; KJ535229, KJ535261, KJ535159, KJ535292,
KJ535194; R.P. Oliveira et al. 747 (HUEFS). Schaueria thyrsifl ora sp.
ined.; KJ535234, KJ535266, KJ535164, KJ535295, KJ535199; D.M. Braz
& A.H.N. Souza 333 (HUEFS). Stenostephanus chiapensis T.F. Daniel;—,
AF2897471, DQ3724611, DQ3725061, AF2897921; D.E. Breedlove & C.
Burns 72688cv (CAS). Stenostephanus lobeliiformis Nees;—,—,
DQ3724601, DQ3725051, DQ3724781; D. Wasshausen 2350 (US).
Streblacanthus cordatus Lindau; EU0875171, AF2897421, EU0811531,
EU0810841, AF2897841; T.F. Daniel et al. 8203 (CAS). Streblacanthus
dubiosus (Lindau) V.M. Baum; KJ535223, KJ535256, KJ535154,
KJ535287, KJ535185; P. Nuñez et al. 34008 (HUEFS). Streblacanthus
dubiosus (Lindau) V.M. Baum; EU0875181, EU0875581,—, EU0810851,
EU0874631; T.F. Daniel 10174 (CAS). Streblacanthus gracilis sp. ined.;—
,—,—,—, KJ535186; J.M. Silva 4977. Streblacanthus monospermus
Kuntze;—,—, EU081155, EU081087, EU087464; T.F. Daniel et al. 6230
(CAS). Streblacanthus roseus (Radlk.) B.L. Burtt;—,—, EU0811541,
EU0810861, AF2897851; T.F. Daniel s.n. (CAS). Tetramerium abditum
(Brandegee) T.F. Daniel; EU0874921,—, EU0811231, EU0810551,
EU0874401; M. Manktelow 727 (UPS). Tetramerium glandulosum Oerst.;—,
EU0875441, EU0811241, EU0810561, EU0874411; T.R. van Devender 93-
1457 (ARIZ). Tetramerium nervosum Nees; EU0874931, EU0875451(AS),
EU0811261, EU0810591(AS), AF1698471; M. Jenkins 1154 (ARIZ).
Tetramerium ochoterenae (Miranda) T.F. Daniel; EU0874941,—,
EU0811271, EU0810601, EU0874421; Q. Gonzales 3631 (DS). Tetramerium
tenuissimum Rose; EU0874911,—, EU0811301, EU0810631, EU0874431;
M. Manktelow 730 (UPS). Thyrsacanthus boliviensis (Nees) A. Côrtes &
Rapini; KJ535236, KJ535268, KJ535166, KJ535297, —; A.L.A. Côrtes et al.
264 (HUEFS). Thyrsacanthus boliviensis (Nees) A.Côrtes & Rapini;
EU0875081, EU0875511, EU0811431, EU0810751, EU0874561; J.R.I.
Wood & M. Serrano 14841 (CAS). Thyrsacanthus microphyllus A. Côrtes;
KJ535237, KJ535269, KJ535167, KJ535298, KJ535202; A.L.A. Côrtes &
R.L.B. Borges 175A (HUEFS). Thyrsacanthus ramosissimus Moric.;
EU0875091, EU0875521, EU0811441, EU0810761, EU0874571; L.A. Silva
2333(US). Thyrsacanthus ramosissimus Moric.; KJ535238, KJ535270,—,
KJ535299, —; A.L.A. Côrtes et al. 108 (HUEFS). Thyrsacanthus ramosus
(Nees) A. Côrtes & Rapini; KJ535239, KJ535271, KJ535168, KJ535300,
KJ535203; G.P. Hamilton 122 (CEN). Thyrsacanthus secundus (Leonard) A.
Côrtes & Rapini; KJ535240, KJ535272, KJ535169, KJ535301, KJ535204;
A.L.A.Côrtes & M.L.S. Carvalho 218 (HUEFS). Yeatesia mabryi Hilsenb.;
EU0875111, EU0875541, EU0811461, EU0810781, EU0874601; T.F Daniel
& M. Baker 3698 (CAS). Yeatesia platystegia (Torr.) Hilsenb.; EU0875211,
EU0875591, EU0811591, EU0810911, EU0874671; L. McDade 1187
(ARIZ).
