THE TETRAMERIUM LINEAGE (ACANTHACEAE: JUSTICIEAE) DOES NOT SUPPORT THE PLEISTOCENE ARC HYPOTHESIS FOR SOUTH AMERICAN SEASONALLY DRY FORESTS1

Thomas  Daniel; Ana Luiza Cortes

A review of previously published palaeo-ecological data reveals a complex biogeographical history of seasonally dry tropical forests (SDTF) in South America over the Late Quaternary. Some dry forest communities (e.g. Colombian Andes) have undergone considerable re-assortment of species through the Holocene, whilst others (e.g. south-eastern Brazil) appear to have been more stable. In contrast

Much of the immense present day biological diversity of Neotropical rainforests originated from the Miocene onwards, a period of geological and ecological upheaval in South America. We assess the impact of the Andean orogeny, drainage of Lake Pebas and closure of the Panama isthmus on two clades of tropical trees (, 31 spp.; and , 14 spp.; both Annonaceae). Phylogenetic inference revealed similar patterns of geographically restricted clades and molecular dating showed diversifications in the different areas occurred in parallel, with timing consistent with Andean vicariance and Central American geodispersal. Ecological niche modelling approaches show phylogenetically conserved niche differentiation, particularly within . Niche similarity and recent common ancestry of Amazon and Guianan species contrast with dissimilar niches and more distant ancestry of Amazon, Venezuelan and Guianan species of , suggesting that this element of the similar patterns of disjunct distributions in the t...

The relationships among the morphoclimatic domains of South America have been a major biogeographical issue of recent years. Palynological, geological and phytogeographical data suggest that the Amazon Forest and the Atlantic Forest were connected during part of the Tertiary and Quaternary periods. This study uses amphibians as model organisms to investigate whether relict northeastern forests are a transition between the Amazon Forest and the Atlantic Forest. We compiled matrices of species composition for four different phytogeographic formations and “Brejos de Altitude,” and analyzed them using clustering methods and Cladistic Analysis of Distributions and Endemism. Our results indicate that the anurofauna of these northeastern forest relicts is most similar in composition to the areas of the Atlantic Forest included in this study, and most dissimilar to the Amazon Forest, which leads us to affirm that events of biotic exchange were more frequent within the Atlantic Forest areas.

AJB Advance Article published on June 15, 2015, as 10.3732/ajb.1400558. The latest version is at http://www.amjbot.org/cgi/doi/10.3732/ajb.1400558 A M E R I C A N J O U R N A L O F B OTA N Y RESEARCH ARTICLE THE TETRAMERIUM LINEAGE (ACANTHACEAE: JUSTICIEAE) DOES NOT SUPPORT THE PLEISTOCENE ARC HYPOTHESIS FOR SOUTH AMERICAN SEASONALLY DRY FORESTS1 ANA LUIZA A. CÔRTES2,4, ALESSANDRO RAPINI2, AND THOMAS F. DANIEL3 2Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, Av. Transnordestina s/n, Novo Horizonte 44036-900, Feira de Santana, Bahia, Brazil; and 3Department 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 relationships 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 terminals. 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 congruence among biogeographical events found for the Tetramerium lineage suggests that the dry forest centers currently dispersed 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. 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 Panama 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), favored the appearance of a unique biota. The formation of a land 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), Reflora (CAPES and CNPq), and AuxPe-PNADB (CAPES). It is also part of the first 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 bridge with North America and the Andes orogeny were geologic events that, together with climatic changes since the Neogene—particularly pronounced in the Pleistocene (Zachos et al., 2001)—appear to have significantly affected the history of South American vegetation and patterns of species distribution, resulting in a complex and extremely diversified biota (Antonelli et al., 2009; Rull, 2011; Hughes et al., 2013). Nevertheless, the sequence and relative importance of these events during the diversification of lineages is questionable and deserves attention. South American moist forests, including those in the Amazon region to the north and Atlantic region to the east [including open, mixed and closed evergreen, semideciduous and deciduous 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 continuous 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 American Journal of Botany 102(6): 1–16, 2015; http://www.amjbot.org/ © 2015 Botanical Society of America 1 Copyright 2015 by the Botanical Society of America 2 • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • A M E R I C A N J O U R N A L O F B O TA N Y Amazonian and Atlantic forest ‘blocks’ connected only by gallery forests and moist enclaves in Caatinga (Brejos) (OliveiraFilho 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 (Caatinga), along the watersheds of the Paraguay and Paraná 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 Pacific 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 conservatism and limited dispersal among centers, resulting in a significant spatial arrangement and diversification 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 evidence 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 forest biomes do not favor fossilization (Queiroz, 2006) and the available paleo-ecological data are insufficient to infer their distribution during the Last Glacial Maximum (Mayle, 2006). One of the most emblematic theories to explain Neotropical diversification, particularly in Amazonia, is the theory of Pleistocene refuges (Haffer, 1969; Pennington et al., 2000). According to this theory, moist forests would have been continuous during interglacial periods, but would retract into isolated centers, 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 diversification governed by climatic fluctuations during 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 forests constitute refuges of a more extensive and continuous formation in the past, and which would have formed a corridor during the colder and drier periods of the Quaternary, coinciding 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 heterogeneous pattern of diversity in Amazonia may simply reflect a sampling error, and the refuges would not necessarily be species-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 forests are resilient to drier climatic conditions and probably covered 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 diversification by geographic isolation, with divergences generally 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 Tetramerium 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 distributions estimated. The congruence of results from our phylogenetic 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 relevant 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 diversification of numerous groups of unrelated plants from seasonally dry forests in South America. The Tetramerium lineage—The Tetramerium lineage belongs to the tribe Justicieae of Acanthaceae, and consists of approximately 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 floral 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 five main genera: Pachystachys Nees, Schaueria Nees, Fittonia Coem., Streblacanthus Kuntze, and Thyrsacanthus Moric. (formerly species of Anisacanthus Nees from South America) comprising approximately 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 exclusively distributed in lowland regions of the Amazon basin [excluding Streblacanthus monospermus Kuntze, which diverges close to the root of the Tetramerium lineage and does not pertain to this group (Daniel et al., 2008), and Streblacanthus cordatus 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 (Goiás and São THE TETRAMERIUM LINEAGE—CÔRTES E T AL. • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • 3 species from the Tetramerium lineage were collected. We sequenced 31 silicagel dried specimens and eight herbarium specimens, representing almost the totality of known South American species in the lineage: Thyrsacanthus (5 species 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 five undescribed species (all ined.): Pachystachys “linearibracteata”, P. “gracilis”, Schaueria “hirta”, S. “pyramidalis”, and S. “thyrsiflora” (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 parviflora (Buckley) Wassh. clade, Carlowrightia clade, Mirandea clade, Tetramerium 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). Fig. 1. Representatives of genera in the Tetramerium lineage. (A) Fittonia albivenis. (B) Pachystachys lutea. (C) Pachystachys spicata. (D) Schaueria calycotricha. (E) Streblacanthus dubiosus. (F) Thyrsacanthus ramosissimus. Photos A, D, and E: T. F. Daniel; B, C, and F: A. L. A. Côrtes. 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 diversification 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 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 azaleiflora Rusby, S. hirsuta Nees, S. malifolia Nees, S. parviflora (Leonard) T.F. Daniel, and Pachystachys “gracilis”, we used the Qiagen Dneasy kit (Qiagen Sciences, Germantown, Maryland, USA. For amplifications, 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 introns 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 MgCl2, 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 Sciences) with 0.2 mM primer for each region and 1 µL of total DNA. PCR products were cleaned with PEG 20% (polyethylene glycol) and sequenced using the same primers as for amplification, except for trnL, trnL-trnF, which were amplified 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 Zimmer, 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 Terminator 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 (Appendix 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 congruence 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 significant when p < 0.01 (Cunningham, 1997). Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) analyses were conducted for the three datasets. Editing and viewing 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 bootstrap 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. 4 • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • A M E R I C A N J O U R N A L O F B O TA N Y Two biomes were specified: (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 five main centers delimited as: (1) dry forests of North America (extending to Costa Rica); (2) dry forests of northern South America; (3) Caatinga (northeastern Brazil); (4) Piedmont dry forests; and (5) Chiquitano and Missiones (southwestern South America). Three additional areas representing the distribution of the Tetramerium lineage were specified, 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 ancestral 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 topologies, 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 distributions to four ancestral areas. And lastly, ancestral distribution reconstructions were plotted onto the chronogram. For the BI analyses, the best-fitting 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 (Ronquist 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, including 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 Miocene (11.62 Ma; Mautino, 2011). Due to its narrow stratigraphic range, the areoles 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 followed 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 five partitions, models of nucleotide substitution and clock were set to unlinked, models of sequence evolution were applied as in phylogenetic analysis, and base frequencies were estimated for all loci. The analysis was conducted with the Yule speciation model, a random starting tree, and uncorrelated lognormal distribution (UCLD; Mean = 1.6; Stdev = 1.5; Offset = 11.5, 5% quantile 11.5; 95% quantile 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 predefined 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 first 10% of saved trees (burn-in) (Drummond and Rambaut, 2007). 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 Tetramerium lineage (PP = 1/ BS_MP = 60% / BS_ML = 94%), but did not recover the Neotropical clade. A few clades were recovered 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 Carlowrightia parviflora 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 parviflora (−/−/69). Moreover, this method supported a single dry forest lineage ((Mirandea clade + core Tetramerium lineage) Thyrsacanthus clade) (−/−/65) (−/−/91), whereas the Pachystachys clade appeared segregated, partly as sister to Schaueria clade (−/−/76) forming two clades and, together with species of Streblacanthus, partly as a grade of the dry forest lineage clade (Table 1; Appendix S1 (see Supplemental Data with the online version of this article)). Ancestral distribution—Species distributions were obtained from herbarium collections and literature (Wasshausen, 1986; Hilsenbeck, 1989; Ezcurra, 1994; Smick, 2004; Côrtes et al., 2010). For a definition 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). TABLE 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 68 trnL+trnL-trnF 84 trnT-trnL 80 rps16 86 trnS-trnG ITS 98 Plastid Combined 99 (ITS + plastid 104 combined) Alinh 1086 934 1064 1017 866 5917 6783 PI L CI RI 1 2 3 4 5 6 7 8 9 10 11 155 395 0.8 0.9 79 86 95 71 — — — — — 112 306 0.86 0.89 — 54 — — — — — — — 135 352 0.8 0.86 50 — 69 — — — — — — 147 442 0.81 0.85 77 — — — — — — — — 284 1508 0.51 0.66 −/−/- 1/94/94 56/-/92 −/−/−/−/−/−/- 51/-/91 −/−/−/−/- 56/56/- −/−/652 1796 0.81 0.87 1/96/99 96/-/1 1/97/1 96/-/74 93/67/89 — −/−/- 57/-/91 91/-/73 — — 936 3341 0.67 0.77 1/1/1 1/1/99 1/99/99 1/91/94 100/-/93 95/−/− 98/−/− — — — −/−/22 THE TETRAMERIUM LINEAGE—CÔRTES E T AL. 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 resolved 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 genus 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 significant incongruence. However, topological conflicts 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 morphological affinities, whereas plastid data appear to be strongly influenced 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 angustissima A. Côrtes & Rapini and Schaueria humuliflora Nees (0.97/61/99), the first 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. microphyllus, 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 marginata 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 conflicts are also present in the relationships among the main clades of the Tetramerium lineage. The Pachystachys 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 resolved 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)). According to the SH test, the best tree recovered was that with the combined plastid and nuclear data (Fig. 3A). However, alternative trees, either with ITS or plastid dataset, were not rejected and alternative scenarios for ancestral distribution reconstructions were considered: one of the scenarios establishes an initial dichotomy between moist forest lineages (Pachystachys clade and Schaueria clade) and dry forest lineages (Thyrsacanthus 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 • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • 5 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 supported relationships. The BI is the only analysis that recovers relationships among the main clades of the Tetramerium lineage with significant support (PP = 0.95–1). Our results (Fig. 2) confirm 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 American genera are sustained in distinct clades. Schaueria parviflora and Fittonia albivenis collapsed into a basal polytomy of the Tetramerium lineage with ITS. However, S. parviflora 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). Schaueria forms a clade (1/100/99) if S. azaleiflora, S. hirsuta, S. humuliflora, S. malifolia, and S. parviflora 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 albivenis. 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 azaleiflora 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); includes a lineage from Mexico (Yeatesia mabryi Hilsenb., Mirandea hyssopus (Nees) T.F. Daniel, Justicia gonzalenzii, and J. zopilotensis); and otherwise consists of species of Thyrsacanthus, Carlowrightia sulcata, Justicia angustissima, and Schaueria humuliflora (Table 1; Fig. 2). Unlike other species of South American Anisacanthus that are now placed in Thyrsacanthus, the taxonomic affinities 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 maximum credibility clades is presented in Appendix S3 (see Supplemental 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 Tetramerium 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 Miocene/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 incongruent positions between plastid and nuclear results. Bayesian posterior probability (PP) support values are reported above branches; parsimony (BS_MP) and likelihood 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 • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • A M E R I C A N J O U R N A L O F B O TA N Y THE TETRAMERIUM LINEAGE—CÔRTES E T AL. Ancestral reconstruction— The Tetramerium lineage originated in the Old World (A, Fig. 3A, node 1) with one dispersal event to the New World. The arrival of the ancestor in the Neotropical 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 Pachystachys diversified in their respective current areas of distribution: 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, represented by Schaueria azaleiflora (Appendix S4, node 10’), a species that appears to be confined to more humid mountainous areas. Figure 3A summarizes the reconstruction of ancestral distributions through time; the complete reconstruction, including Fittonia albivenis, whose ancestral distribution is also shown in this figure (node 9’) is present in Appendix S4 (see Supplemental Data with the online version of this article). Considering the alternative reconstructions, in the first 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 forest 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 appears 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, indicating 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 • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • 7 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), Pachystachys (sensu Wasshausen, 1986), and Thyrsacanthus (sensu Côrtes et al., 2010). These lineages are morphologically distinct and geographically structured between dry and moist forest biomes. 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 Tetramerium lineage). An alternative tree (Fig. 3B), with moist forest 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 molecular 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 sufficient to recover relatively ancient relationships resulted from rapid radiation. This is probably the main reason for uncertainties regarding relationships among the main clades of the Tetramerium lineage, hindering the establishment of the sequence of diversification for the group during the end of the Miocene, when the lineage seems to have undergone an ecological restructuring, with the formation of clades predominantly restricted either to dry or moist forests. Recent rapid diversifications, on the other hand, may often result in incomplete lineage sorting, and this is possibly an explanation for incongruences in the Tetramerium 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 Appendix S4 (see Supplemental Data with the online version of this article)). Species of Thyrsacanthus clade show variation in floral morphology that appears to conform to different pollination syndromes (e.g., butterflies in S. humuliflora and hummingbirds 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, flies, butterflies, and hummingbirds), and some of these have been shown to be at least partially interfertile in Carlowrightia (Daniel, 1983a), Anisacanthus (Daniel, 1985), and Tetramerium (Daniel, 1986). Although hybrids were not identified in natural populations of Schaueria and Thyrsacanthus species, older interspecific → Fig. 3. (A) Chronogram (in million years) based on the combined data (for confidence 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 • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • A M E R I C A N J O U R N A L O F B O TA N Y THE TETRAMERIUM LINEAGE—CÔRTES E T AL. • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • 9 TABLE 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 first clade. Alternative topologies (Mirandea (Thyrsacanthus + core Tetramerium lineage)) Pachystachys + Thyrsacanthus Schaueria + Pachystachys Fittonia + Pachystachys Schaueria (sensu Nees, 1847) Streblacanthus Thyrsacanthus (sensu Côrtes et al., 2010) Not reject Not reject Not reject reject reject reject reject hybridization events could be involved in the diversification 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 uncertain. The Schaueria-Fittonia clade seems to be one of the first to diverge in South America. Both Schaueria and Fittonia have flowers with small, yellow or white corollas, potentially pollinated by bees/flies, 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 diversification mainly concentrated in seasonally dry forests of North America. The Thyrsacanthus clade has nototribic flowers (androecium dehiscing toward the inferior lip of the corolla), a symplesiomorphy of the lineage, whereas sternotribic flowers (androecium dehiscing toward the superior lip of the corolla) emerged as a synapomorphy of the TABLE 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 1 3 4 5 6 7 8 9 10 11 12 13 14 16 17 Average (My) 95% HPD (My) 14.04 11.4 9.11 8.25 7.91 7.41 5.15 3.29 3.41 5.16 6.53 4.11 4.28 3.28 1.99 7.3-22.92 6.08-18.72 4.88-14.87 4.35-13.39 4.15-12.78 3.96-12.01 1.9-9.89 1.38-6.43 1.38-6.53 2.54-8.82 3.45-10.64 1.84-7.31 1.94-7.98 1.41-5.99 0.55-4.13 -lnL -lnL alternative topology Difference p 30530.60362 30530.60362 30530.60362 30530.60362 30530.60362 30530.60362 30530.60362 30541.80358 30538.43570 30535.49404 30551.75331 31600.32591 30577.12348 30612.08106 11.19996 7.83208 4.89042 21.14968 1069.7229 4651985 81.47744 0.21 0.22 0.29 0.02 0.00 0.02 0.01 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 sufficient to establish the initial sequence of colonization in the New World. During the Late Miocene, the lineage would already be possibly occupying the Amazon basin in South America, and occurring in North and Central America, probably in both moist and dry environments. 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 Fittonia clades were confined 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 fire regime in the central region of South America (Simon et al., 2009; Simon and Pennington, 2012), and where taxa of the Tetramerium lineage do not occur, may have represented an insurmountable 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 regions 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 decrease 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 restingas 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 facilitated by the expansion of dry forests, starting from the Caatinga 10 • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • A M E R I C A N J O U R N A L O F B O TA N Y 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 dispersal between discontinuous blocks of dry forest would have similar reconstruction (node 11), but then we would expect current 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 Tetramerium lineage. Vicariance between dry forests in the Caatinga and Missiones blocks may have been caused by a subsequent expansion of fire-adapted savannas (3–8 Ma; Cerling et al., 1997; Simon et al., 2009). Dry forest lineages, sensitive to fires (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 Thyrsacanthus clade in dry forest remnants on the boundaries of the Cerrado 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 forest 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 populations 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 periods of Pleistocene and an eventual continuity between the two dry forest blocks would have occurred at the beginning of the Pleistocene or even in the Neogene. This scenario also agrees with Mayle’s (2006) suggestion that individual long-distance dispersals might properly explain disjunct distribution of species in fragments 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 Pleistocene Arc hypothesis (Prado and Gibbs, 1993; Mogni et al., 2015), probably dispersed to the Bolivian Chiquitano very recently, since it was absent from this fragment during the LGM. Prado and Gibbs (1993) argued that current disjunctions between isolated patches of seasonally dry forests represent a consistent 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 Caatinga 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-distance dispersal and their populations are not remnants of a continuous distributed ancestral. Moist forests—Our data do not support a single lineage of moist forest-taxa composed of the Pachystachys and Schaueria-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 formation of a diagonally shaped corridor of arid biomes in South America during part of this epoch. This corridor, initially composed 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 Tetramerium lineage are concentrated in regions of greater climatic stability 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 periods 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 lowlands 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 Paraná River basin, south of the Cerrado and through Mato Grosso, or through the Chaco and areas 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 interconnections would have allowed the more recent dispersal of Pachystachys spicata, for example. Taxonomic implications— Schaueria clade—Traditionally, Schaueria included approximately 20 species (Nees, 1847; Clarke, 1900; Rusby, 1927; Daniel, 1990), however, phylogenetic analyses 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 species 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 flowers are nototribic with a shift to sternotribic in S. lophura Nees. Based on shape, color and size of corolla, species are probably pollinated by bees and/or hummingbirds (Nees, 1847; Côrtes, personal observation). The lineage is restricted to the Atlantic forest, and occurs in rainforests, semideciduous forests and restingas. The majority of species have restricted distributions that are mostly confined 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 TETRAMERIUM LINEAGE—CÔRTES E T AL. is geographically disjunct from the Schaueria clade. Pachystachys clade consists of 18 species (Côrtes, et al., in prep.) distributed 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 azaleiflora 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 azaleiflora, 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 Pachystachys s.s. clade, with a reversal to narrow bracts in P. badiospica Wassh. (c. 1.5 mm wide). Large flowers, with a distally expanded tube and a typically red corolla (white in P. lutea Nees), are probably pollinated by hummingbirds. Such flowers represent a plesiomorphic state of the basal grade composed of S. azaleiflora and P. “linearibracteata”. The grade consisting of species of Streblacanthus is distinguished by a narrow and long floral tube and red, pink, or lavender corolla, and are possibly pollinated by Lepidoptera. This floral diversity likely reflects adaptations to specific 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 five species that are characterized by a shrubby habit, profuse branching, inconspicuous bracts, and flowers with a large and red corolla having an expanded tube. The flowers are probably pollinated by hummingbirds (Côrtes et al., 2010). The inclusion of Justicia angustissima, Schaueria humuliflora, Carlowrightia sulcata, Mirandea hyssopus, and Yeatesia mabryi renders the Thyrsacanthus clade quite heterogeneous due to their different flowers, i. e., corolla generally small, white, purple or blue, and with a narrow tube in J. angustissima, S. humuliflora and Y. mabryi, and infundibuliform corolla with bilabiate limb in C. sulcata and M. hyssopus, reflecting 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 floral and pollen morphology with Thyrsacanthus (Henrickson and Hiriart, 1988; Daniel et al., 2008); both have large, funnelform, reddish, and nototribic flowers and 3-colporate, 6-pseudocolpate pollen. Justicia gonzalezii had been treated in Anisacanthus (Hagen, 1941), and both were treated in Justicia (Henrickson and Hiriart, 1988) for presenting nototribic flowers (vs. sternotribic in Anisacanthus). Despite their heterogeneous 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 forest 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 isolated center of dry forests (Pennington et al., 2000; Queiroz, 2006), and S. humuliflora occurs in seasonal deciduous forests • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • 11 of Bahia. Two species (T. boliviensis (Nees) A. Côrtes & Rapini 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 hyssopus) and the position of J. zopilotensis in this clade are here confirmed, 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 northeastern 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 estimates 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 populations of dry forests that indicate a coalescence of isolated centers 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). 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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. WERNECK, F. P. 2011. The diversification of eastern South American open vegetation biomes: Historical biogeography and perspectives. Quaternary Science Reviews 30: 1630–1648. WERNECK, F. P., G. C. COSTA, G. R. COLLI, D. E. PRADO, AND J. W. SITES JR. 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. WHITE, T. J., T. BRUNS, S. LEE, AND J. TAYLOR. 1990. Amplification 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. YU, Y., A. J. HARRIS, AND X. HE. 2010. S-DIVA (statistical dispersalvicariance analysis): A tool for inferring biogeographic histories. Molecular Phylogenetics and Evolution 56: 848–850. ZACHOS, J., M. PAGANI, L. SLOAN, E. THOMAS, AND K. BILLUPS. 2001. Trends, rhythms, and aberrations in global climate 65 Ma to present. Science 292: 686–693. THE TETRAMERIUM LINEAGE—CÔRTES E T AL. • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • 15 APPENDIX 1. Taxa, Genbank accession numbers (rps16, 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 quadrifidus 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 parviflora (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 albiflora 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 grandiflora 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 floribundum 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 azaleiflora Rusby; KJ535224; —, —, KJ535288, KJ535187; J.R.I. Wood 12593 (CAS). Schaueria azaleiflora 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 humuliflora Nees; KJ535235, 16 • V O L . 1 0 2 , N O. 6 J U N E 2 0 1 5 • A M E R I C A N J O U R N A L O F B O TA N Y 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 parviflora (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 thyrsiflora 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 931457 (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).

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