Phylogenetic Relationships in Bromeliaceae Subfamily Bromelioideae based on Chloroplast DNA Sequence Data

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1 Phylogenetic Relationships in Bromeliaceae Subfamily Bromelioideae based on Chloroplast DNA Sequence Data Author(s): Timothy M. Evans, Rachel S. Jabaily, Ana Paula Gelli de Faria, Leandro de Oliveira F. de Sousa, Tania Wendt, and Gregory K. Brown Source: Systematic Botany, 40(1): Published By: The American Society of Plant Taxonomists URL: BioOne ( is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

2 Systematic Botany (2015), 40(1): pp Copyright 2015 by the American Society of Plant Taxonomists DOI / X Date of publication February 12, 2015 Phylogenetic Relationships in Bromeliaceae Subfamily Bromelioideae based on Chloroplast DNA Sequence Data Timothy M. Evans, 1,7 Rachel S. Jabaily, 2 Ana Paula Gelli de Faria, 3 Leandro de Oliveira F. de Sousa, 4 Tania Wendt, 5 and Gregory K. Brown 6 1 Biology Department, 1 Campus Drive, Grand Valley State University, Allendale, Michigan 49401, U. S. A. 2 Department of Biology, Rhodes College, 2000 N. Parkway, Memphis, Tennessee 38112, U. S. A. 3,5 Departamento de Botânica, Universidade Federal de Juiz de Fora, ICB, Campus Universitário, , Juiz de Fora, Minas Gerais, Brazil. 4 Departamento de Ciências Vegetais, Universidade Federal Rural do Semi-Árido, , Mossoró, Rio Grande do Norte, Brazil. 5 Departamento de Botânica, Universidade Federal do Rio de Janeiro, IB, CCS, Ilha do Fundão, , Rio de Janeiro, Rio de Janeiro, Brazil. 6 Department of Botany, University of Wyoming, Laramie, Wyoming 82071, U. S. A. 7 Author for correspondence (evanstim@gvsu.edu) Communicating Editor: Andrea Weeks Abstract Of the eight subfamilies currently recognized in Bromeliaceae, Bromelioideae is perhaps the most poorly understood. Generic circumscriptions are unclear, and an exceptionally diverse morphology coupled with an unusually low rate of sequence divergence within Bromeliaceae has made it difficult to resolve phylogenetic relationships within the subfamily. Although recent molecular studies have begun elucidating relationships among species in Bromeliaceae, most have not sampled deeply and/or broadly across Bromelioideae. The purpose of this study was to conduct a phylogenetic analysis within subfamily Bromelioideae using three chloroplast DNA regions (matk, psba-trnh, and trnl-trnf), with the inclusion of multiple species from a broad sampling of bromelioid genera. Ochagavia, Deinacanthon, Fascicularia, Bromelia and Fernseea diverged relatively early in the history of the subfamily, with the remaining taxa being placed in a large and poorly resolved eubromelioid clade. Bromelia and Cryptanthus were found to be monophyletic, while 13 other genera were polyphyletic. Aechmea, the most morphologically diverse genus within the subfamily, was highly polyphyletic, with species distributed among 12 different lineages, with little support for subgeneric circumscriptions. Keywords matk, phylogeny, psba-trnh, trnl-trnf. Bromeliaceae, with 58 genera and approximately 3,200 species, is the sixth largest monocot family, and the largest plant family that is endemic to the New World (Smith 1934; Luther 2010). Bromelioideae, with 33 genera and roughly 800 species, is second largest of the eight subfamilies in Bromeliaceae (Givnish et al. 2007, 2011), and is the most poorly understood subfamily taxonomically (Benzing 2000; Brown and Leme 2000; Schulte et al. 2009; Sass and Specht 2010). Most genera in the subfamily are grouped by molecular data into a large eubromelioid clade, but relationships both within that clade and among the earliest-diverging lineages in the subfamily have not been well resolved into strongly supported clades (Fig. 1; Schulte et al. 2009; Sass and Specht 2010; Givnish et al. 2011; Silvestro et al. 2014). Generic circumscriptions in the subfamily are unclear, with many genera being defined by one or more highly labile characters that almost certainly do not reflect phylogenetic relationship. With considerable misgiving, Smith and Downs (1979) separated many Bromelioideae genera using presence/absence of petal appendages, and noted explicitly that their classification of the then recognized 27 genera was artificial. Rauh (1979) corroborated the poor understanding of Bromelioideae genera and noted that the delineation of individual genera is difficult, and in no way can be considered final. A review of the pre-smith and Downs (1979) Bromelioideae classification illustrates some of the chronic generic-level problems. Baker (1889) recognized 19 genera with 268 species in the tribe Bromelieae (= Bromelioideae). While there is substantial similarity between the generic names used by Baker (1889) and Smith and Downs (1979), their concepts of taxa differ dramatically. For example, Quesnelia from Baker (1889) includes species that Smith and Downs (1979) placed into Quesnelia, three subgenera of Aechmea, plus the genera Ronnbergia, Eduandrea, and Pitcairnia (subfamily Pitcairnioideae). Harms (1930) recognized 34 genera with approximately 440 species in Bromelioideae, and Mez ( ) presented a similar treatment. Notable differences between the Harms and Mez Bromelioideae treatments include expanded concepts for Bromelia and Streptocalyx in Mez, and differing concepts for Aregelia (now Neoregelia) and Nidularium. Finally, significant disagreement exists between the Bromelioideae treatments of Mez ( ) and Smith and Downs (1979), especially concerning the largest, most ambiguous genus in the subfamily, Aechmea. Smith and Downs (1979) rejected Mez s classification because there was not enough information in the pollen characters emphasized by Mez ( ) to make the classification workable. Aechmea, consisting of roughly 260 species in eight subgenera, represents nearly a third of the species in Bromelioideae, and embodies a substantial portion of the taxonomic difficulties within the subfamily. Smith and Downs (1979) acknowledged that the genus is likely polyphyletic, but the lack of reliable morphological characters within the group and among its segregate genera has impeded systematic progress. Recent morphological and molecular systematic studies have consistently demonstrated Aechmea to be highly polyphyletic (Faria et al. 2004; Horres et al. 2007; Sass and Specht 2010; Givnish et al. 2011; Silvestro et al. 2014). Much work remains in circumscribing a monophyletic Aechmea and related genera, and in discovering phylogenetically informative morphological characters with diagnostic utility. Bromeliaceae has been the focus of several molecular phylogenetic studies (Terry et al. 1997; Horres et al. 2000; Givnish et al. 2004, 2007, 2011; Crayn et al. 2004; Schulte et al. 2005, 2009; Schulte and Zizka 2008; Sass and Specht 2010; 116

3 2015] EVANS ET AL.: BROMELIOIDEAE (BROMELIACEAE) PHYLOGENY 117 Fig. 1. Different basal relationships recovered by different molecular phylogenetic studies of Bromelioideae. Relationships from Silvestro et al. (2014) are similar to those of Schulte et al. (2009) except for the placement of Bromelia, which is sister to the Fernseea-Eubromelioid clade. Versieux et al. 2012; Silvestro et al. 2014), and many longstanding questions about the evolutionary and biogeographic history of the family are now being resolved with increasing precision and success. Fine-scale relationships among Bromelioideae species have not been the principal focus in most of these studies, however, and thus they have not resulted in a comprehensive, robustly resolved phylogeny for species and genera within the subfamily. They have generally included a relatively small number of Bromelioideae species as part of a larger family-wide survey (Terry et al. 1997; Givnish et al. 2004, 2007, 2011), they have focused primarily on dense sampling in a few Bromelioideae genera (Sass and Specht 2010), or they have included many bromelioid genera but with only one to a few species included as placeholders for most genera (Horres et al. 2000; Crayn et al. 2004; Schulte et al. 2005, 2009), precluding an examination of generic circumscriptions and monophyly. More recently, Silvestro et al. (2014) generated a larger-scale phylogeny for the subfamily, with greater taxon density than any previous study. Table 1 provides a comparison of taxa sampled for each of these Bromelioideae-focused studies (Schulte et al. 2005, 2009; Horres et al. 2007; Schulte and Zizka 2008; Sass and Specht 2010; Silvestro et al. 2014). This study and that of Silvestro et al. (2014) are the largest in terms of the numbers of species and genera sampled. This study includes, for the first time, representation from Eduandrea, as well as the largest samples to date for Billbergia, Canistrum, Neoregelia, Nidularium, Ronnbergia, andwittrockia. An additional impediment to the resolution of phylogenetic relationships is the unusually low rate of sequence divergence in Bromeliaceae (Smith and Donoghue 2008), and specifically in Bromelioideae (Horres et al. 2000, 2007; Crayn et al. 2004; Schulte et al. 2009), despite the tremendous amount of morphological variation within the group. In a broad survey of flowering plants, Smith and Donoghue (2008) note that Bromeliaceae exhibits substitutions per site per million years, a rate much lower even than the notably slowly-evolving palms. This low rate of molecular evolution has made it difficult to find enough sequence variation among bromeliads to yield robust phylogenies. The purpose of this study was to conduct a phylogenetic analysis of about 2,000 base pairs of plastid molecular data within subfamily Bromelioideae, with the inclusion of multiple species from a broad sampling of bromelioid genera. Specific goals were to: 1) examine relationships among the first diverging lineages of Bromelioideae (Bromelia, Deinacanthon, Fascicularia, Ochagavia); 2) examine boundaries of the remaining genera, with an emphasis on the problematic genus Aechmea and its eight subgenera; and 3) provide a broad

4 118 SYSTEMATIC BOTANY [Volume 40 Table 1. Taxon sampling comparison for the subfamily Bromelioideae-focused molecular phylogenetic studies. A zero (0) indicates that no species from that genus were included in the respective study. Nomenclature and species counts follow Luther (2010). Genus Number of species Schulte et al Horres et al Schulte and Zizka 2008 Schulte et al Sass and Specht 2010 Silvestro et al Acanthostachys Aechmea Ananas Androlepis Araeococcus Billbergia Bromelia Canistropsis Canistrum Cryptanthus Deinacanthon Disteganthus Edmundoa Eduandrea Fascicularia Fernseea Greigia Hohenbergia Hohenbergiopsis Lapanthus Lymania Neoglaziovia Neoregelia Nidularium Ochagavia Orthophytum Portea Pseudaechmea Pseudananas Quesnelia Ronnbergia Ursulaea Wittrockia This study (Evans et al.) total species no. genera represented % of 882 species sampled NA 6.6% 6.8% 6.5% 4.9% 16.3% 12.7% 16.9% % of genera represented NA 81.8% 81.8% 81.8% 66.7% 60.6% 94% 84.8% Average percent of species sampled per genus NA 26.6% 28.1% 26.6% 21.9% 29.8% 42.1% 41.2% framework upon which to develop a reliable, phylogenetically based taxonomy for the subfamily. Materials and Methods Taxon Sampling DNA sequences were generated from 149 species representing 28 genera of subfamily Bromelioideae (Appendix 1). Five species of Puya were used as outgroup taxa, based on previous studies that place the genus as sister to the subfamily (Givnish et al. 2007, 2011; Jabaily and Sytsma 2010). Samples were either collected from naturally occurring populations, or obtained from cultivated material from Marie Selby Botanical Gardens (SEL) or Refúgio do Gravatás, Teresópolis, Brazil (Appendix 1). Leaf tissue was dried in silica gel. Locations for herbarium voucher specimens are provided in Appendix 1. Nomenclature used here follows Luther (2010). DNA Extraction, Amplification and Sequencing Total DNA was isolated using a modification of the 2 +CTAB buffer method (Doyle and Doyle 1987; Smith et al. 1991) followed by purification with the QIAamp DNA mini kit (Qiagen, Valencia, California). The protocols for PCR amplification and sequencing of all three cpdna regions follow Johnson and Soltis (1995), with the addition of 5% DMSO. For PCR amplification of matk, primers two and five from Crayn et al. (2000) were used, and the psba-trnh and trnl-trnf primer sequences were from Sang et al. (1997). Sequencing reactions were performed using BigDye Terminator Reaction Mix version 3.1 (Life Technologies), and sequences were obtained on an ABI 310 automated sequencer. Forward and reverse strands were assembled using Autoassembler version 2.0 (ABI Prism) or DNA Baser (HeracleSoftware). Almost all DNA sequences were originally generated for this study and are available from GenBank (Appendix 1). Data Analysis All matrices for this study were deposited in TreeBASE (study number 14,406). DNA sequences for each region were aligned manually using the program Se-Al v2.0a11 (Rambaut 2003), employing the similarity criterion of Simmons (2004), and subsequently combined into a single data matrix. Gap characters were coded using the simple indel coding method described by Simmons and Ochoterena (2000) and were used in the parsimony analysis. Missing data and gaps resulted in 9.7% of the nucleotide positions across all taxa being scored as missing. Maximum parsimony analyses were performed using PAUP* version 4.b10 (Swofford 2003). Heuristic searches were conducted using a multiple islands search, with an initial search using the TBR branch swapping algorithm, MULTREES off, and 10,000 random addition replicates. Trees obtained from this search were used as starting trees, with TBR branch swapping, MULTREES on, and MAXTREES set to 100,000. All characters were unordered and assigned equal weight. Bootstrap (BS) values were determined to evaluate support for each node, again with MAXTREES set to 100,000. For the bootstrap analysis, one hundred replicate searches were performed using TBR, with random addition of 100 replicates and 100 trees saved from each replicate. Bayesian analyses were conducted using MrBayes version (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) on the CIPRES Portal teragrid (Miller et al. 2010). Appropriate models of evolution were selected for the matk region (HKY) and the combined non-coding cpdna regions ( JC + G) using a hierarchical ratio test as implemented in jmodeltest v (Guindon and Gascuel 2003; Posada 2008). The temperature setting in MrBayes was reduced from the default

5 2015] EVANS ET AL.: BROMELIOIDEAE (BROMELIACEAE) PHYLOGENY 119 setting of to create appropriate levels of chain swapping and increase the likelihood of convergence. The analysis consisted of four chains each in two parallel runs with 10,000,000 generations and samples taken every 1,000 generations. The split frequency diagnostic between the two runs was used to assess convergence; runs with average standard deviations of < 0.01 were considered converged. Additionally, the sample parameter traces were visualized using Tracer version 1.6 (Rambaut and Drummond 2013). The runs converged at approximately 100,000 generations. As a conservative approach, trees from the first 2,500 samples were discarded (i.e. the first 25% of the generations were the burn-in stage). Gap data were treated as restriction site data as recommended in the MrBayes manual (Ronquist and Huelsenbeck 2003) and initially included in Bayesian analyses. Even with the reduced temperature, however, analyses containing gap data failed to run to a point at which the chains showed convergence (based on the split frequency diagnostic), so gaps were subsequently omitted from these analyses. The Shimodaira-Hasegawa (S-H) test (Shimodaira and Hasegawa 1999) was employed using ML to compare alternative topologies to an unconstrained baseline ML phylogeny, with the same models of molecular evolution that were used in the Bayesian analyses. Specifically, individual tests were conducted in which each genus: Acanthostachys, Araeococcus, Billbergia, Hohenbergia, Lymania, Neoglaziovia, Neoregelia, and Portea was constrained to be monophyletic, while no constraints were placed on the remaining genera. Additionally, alternative relationships among basal lineages within Bromelioideae that have been recovered in other studies (Fig. 1; Schulte et al. 2009; Jabaily and Sytsma 2010; Givnish et al. 2011) were tested. Maximum-likelihood analyses were conducted using GARLI (Zwickl 2006) on the CIPRES Portal (Miller et al. 2010), and the S-H test was implemented in PAUP* version 4b.10 (Shimodaira and Hasegawa 1999) using 1,000 bootstrap replicates. Results Aligned sequences yielded a concatenated data matrix of 1,979 characters (matk = 902 characters, trnl-trnf = 433 characters, and psba-trnh = 644 characters; Table 2). Gap coding resulted in the addition of 26 characters to the data matrix for parsimony analysis, characters which were omitted from the Bayesian analysis. Parsimony analysis yielded 100,000 most parsimonious trees (Fig. 2; 510 steps, CI = 0.65, RI = 0.79), with consistently short branch lengths among the early-diverging Bromelioideae lineages. Parsimony and Bayesian (Fig. 3) analyses produced consensus trees that are largely congruent with each other, each with relatively poor resolution, particularly near the base of the eubromelioid clade. In each analysis, subfamily Bromelioideae is monophyletic, with four genera (Bromelia, Deinacanthon, Fascicularia, and Ochagavia) placed near the base of the rest of the subfamily. Bromelia is monophyletic, whereas Ochagavia is not, and is part of a moderately supported clade (BS: 57%, PP: 0.99) that contains Deinacanthon and Fascicularia (Figs. 2, 3). A single deletion in psba-trnh of four nucleotides was found in every species in the analysis except for the outgroup (Puya), the earliest-diverging clade in Bromelioideae (Deinacanthon, Ochagavia, andfascicularia), and Hohenbergia catingae (presumed independent gain; positions 1,559 1,562 in the data matrix; Fig. 2). Table 2. Summary statistics for three chloroplast DNA regions for phylogenetic analysis of Bromelioideae. matk psba-trnh trnl-trnf All regions No. of characters in matrix ,979 No. of variable characters No. of parsimony uninformative characters No. of parsimony informative characters Each analysis recovered a weakly supported eubromelioid clade with numerous individual species placed in an unresolved polytomy (Figs. 2, 3). All members of this clade, with the exception of Fernseea itatiaiae, contain a five-nucleotide deletion in the trnl-trnf region that is not found outside the clade (positions 1,247 1,251 in data matrix; Fig. 2; Aechmea bocainensis lacked sequence data for this region, so it was not scored for the indel). Accessions from the highly problematic Aechmea provide a substantial portion of the large polytomy at the base of the eubromelioid clade (12 of 26 unresolved species for parsimony analysis; 10 of 22 for the Bayesian analysis; Figs. 2, 3). Members of this polyphyletic genus, excluding Aechmea bocainensis, are found in a minimum of 11 separate lineages throughout the phylogenies from either analysis. Both parsimony and Bayesian analyses recovered two monophyletic genera, Bromelia (stated above) and Cryptanthus. Monophyly of Bromelia is supported by a 140-nucleotide deletion in trnl-trnf (positions 1,051 1,190 in data matrix), and Cryptanthus is supported by a five-nucleotide deletion in psba-trnh (positions 1,410 1,414 in data matrix; also independently gained in Hohenbergia catingae; Fig. 2). Every other genus in the subfamily for which multiple species were represented was polyphyletic or, in the case of Lymania, unresolved (Figs. 2, 3). Monophyly of several genera was not tested either because they were represented by only one species (Disteganthus, Fernseea, Lapanthus), or because they were absent from this analysis (Androlepis, Greigia, Pseudaechmea, Pseudananas, Ursulaea). Deinacanthon, Eduandrea, Fascicularia, andhohenbergiopsis are each monotypic. The S-H tests for alternative topologies failed to reject a monophyletic Acanthostachys, Araeococcus, Lymania, Neoglaziovia, Neoregelia, or Portea (Table 3). Monophyly for Billbergia and Hohenbergia were each rejected, with p = 0.04 and p < 0.001, respectively (Table 3). The S-H tests failed to reject any of the alternative basal relationships recovered by Schulte et al. (2009), Jabaily and Sytsma (2010), or Givnish et al. (2011). Discussion Early Lineages within Bromelioideae Several morphological characters unite Bromelioideae, including the presence of spines on the leaf margins, inferior or nearly inferior ovaries, and baccate and indehiscent fruits (Smith and Downs 1979), but morphology alone has proved unreliable for resolving relationships within the subfamily. Molecular data have consistently yielded a monophyletic Bromelioideae, with Puya sister to the subfamily (Terry et al. 1997; Horres et al. 2000; Givnish et al. 2004, 2007, 2011; Crayn et al. 2004; Schulte et al. 2005, 2009; Schulte and Zizka 2008; Jabaily and Sytsma 2010; a notable exception to this, however, is found in Jabaily and Sytsma 2010, in which the nuclear region PHYC places Ochagavia, Greigia, anddeinacanthon in a clade that is sister to Puya, resulting in a non-monophyletic Bromelioideae). Within Bromelioideae, the current study, as well as recent molecular studies (Schulte et al. 2009; Sass and Specht 2010; Givnish et al. 2011; Silvestro et al. 2014), have recovered a large albeit often weakly supported eubromelioid clade (sensu Schulte et al. 2009) that contains the majority of bromelioid taxa (BS < 50%, PP < 0.50; Figs. 2, 3). Parsimony analysis places a clade containing Ochagavia, Deinacanthon, andfascicularia (BS = 57%) as the earliest diverging lineage within Bromelioideae, followed by Bromelia (BS = 100%), with Fernseea and the remaining genera in a polytomy (Fig. 2), while Bayesian analysis yielded

6 120 SYSTEMATIC BOTANY [Volume 40 Fig. 2. A single representative of 100,000 trees recovered from parsimony analysis of chloroplast DNA sequence data for Bromelioideae. Branch length is proportional to the number of changes along that branch. Branches shaded gray are supported in majority-rule consensus but collapse in the strict consensus; branches in red collapse in majority-rule consensus. Numbers above branches represent bootstrap values; branches with no number are supported by < 50% bootstrap value. Aechmea subgenera are indicated in bold. Deletions that support specific clades are indicated.

7 2015] EVANS ET AL.: BROMELIOIDEAE (BROMELIACEAE) PHYLOGENY 121 Fig % majority-rule consensus tree from Bayesian analysis of chloroplast DNA sequence data for Bromelioideae. Numbers above branches indicate Bayesian posterior probability values. Aechmea subgenera are indicated in bold. Deletions that support specific clades are indicated.

8 122 SYSTEMATIC BOTANY [Volume 40 Table 3. Statistics from S-H tests of alternative topologies. *Unconstrained phylogeny -ln L = Significant p values are highlighted in bold. Group for which monophyly was tested -ln L Difference from unconstrained phylogeny* Acanthostachys 6, Araeococcus 6, Billbergia 6, Hohenbergia 6, < Lymania 6, Neoglaziovia 6, Neoregelia 6, Portea 6, Schulte et al , Jabaily and Sytsma 6, (Total Data) Jabaily and Sytsma 6, (PHYC) Givnish et al , the same clades of Fascicularia/Ochagavia/Deinacanthon (PP = 0.99), Bromelia (PP = 1.00) and the remaining Bromelioideae species (PP < 0.50), but placed them in a basal polytomy (Fig. 3). The placement of these taxa near the base of the subfamily is largely in agreement with previous molecular studies, but relationships among the early-diverging taxa vary among studies (Fig. 1). Combined chloroplast and nuclear data (4,281 characters; Schulte et al. 2009) placed Bromelia as the earliestdiverging lineage in the subfamily with low support, followed by a clade containing Ochagavia, Deinacanthon, Fascicularia,and Greigia (not represented in this study). Chloroplast data (9,341 characters; Givnish et al. 2011) yielded a single poorly supported clade containing Fascicularia, Ochagavia, Deinacanthon, Bromelia, and Greigia as sister to the eubromelioid clade. Fernseea was not included in that study. Jabaily and Sytsma (2010) recovered a topology in which a single species of Ochagavia (O. lindleyana), a monophyletic Bromelia, and the remaining members of the subfamily form a nested grade based on three chloroplast regions (trns-trng, matk, and rps16; 2,694 characters). Nuclear data (PHYC; 1,048 characters) from the same study placed a clade containing Ochagavia elegans, Greigia, and Deinacanthon sister to Puya, resulting in a non-monophyletic Bromelioideae. Each of these studies places the same genera as sister to the eubromelioids with relatively strong support, with the exception of the PHYC data of Jabaily and Sytsma (2010), but the relationships among the basal lineages tend to be more poorly supported and differ slightly among the studies. Our results are similar to those of Schulte et al. (2009), Givnish et al. (2011), and Silvestro et al. (2014) in uniting Ochagavia, Fascicularia, and Deinacanthon, but each of these studies differ in the placement of Bromelia. Likewise, our results are similar to the PHYC results of Jabaily and Sytsma (2010) in the placement of Bromelia sister to the eubromelioid clade, but these results differ in the placement of Deinacanthon, Greigia, and Ochagavia. The placement of Bromelia sister to the eubromelioid clade is supported in the analysis by the presence of a single deletion in the psba-trnh region that is lacking in the remaining early-diverging genera (Fig. 2). Interestingly, Fernseea is the only member of the eubromelioid clade that lacks a five-base pair deletion in the trnl-trnf region, suggesting either an early divergence from the rest of the clade, which is supported by Silvestro et al. (2014) or, less likely, an independent gain of those five nucleotides. The S-H tests failed to reject any of the alternative hypotheses for early relationships within Bromelioideae, including p the non-monophyletic Bromelioideae recovered by PHYC data of Jabaily and Sytsma (2010; Table 3). The short branch lengths found in the phylogeny reflect the relatively low phylogenetic signal that has plagued Bromelioideae molecular systematics, making it difficult to select from alternative phylogenetic hypotheses with confidence. Thus, while this data set provides weak support for a particular topology, including monophyly of the subfamily, the resolving power at the deep branches is not strong enough to confidently reject any of the topologies of the other recent molecular studies of Bromelioideae. It is clear that the subfamily underwent an early radiation resulting in short branches near the base of the phylogeny, and early relationships within the subfamily are going to remain elusive until all genera, with multiple included species, are included along with a relatively large number of gene sequences. Givnish et al. (2011) hypothesized that the lineages leading to Puya and Bromelioideae diverged approximately 10 Mya, with Puya diversifying along the Andes and Bromelioideae diversifying elsewhere, possibly in northern South America. Subsequently, Givnish et al. (2011) hypothesized a divergence between Puya and Bromelioideae in and around the southern Andes, if not in central Chile (Jabaily and Sytsma 2010), based on biogeography and phylogenetic relationships among the early lineages of Bromelioideae. Jabaily and Sytsma (2010) hypothesized a central Chilean origin for Bromelioideae based on the Chilean distribution of both basal Bromelioideae and the earliest-diverging clades in Puya. Of the early-diverging lineages in Bromelioideae, Bromelia (58 species) is widespread, with areas of high diversity in Central America/northern South America, and the Brazilian Shield. Ochagavia (four species) and Fascicularia (one species) are exclusively Chilean, and the monotypic Deinacanthon is found in Paraguay and northern Argentina (Smith and Downs 1979). Although the basal relationships found here differ in some details from those of Givnish et al. (2011) and Jabaily and Sytsma (2010), the basal position of Fascicularia, Ochagavia and Deinacanthon in our phylogeny lends strong support to their hypotheses of an early Andean diversification for Bromelioideae. Specifically, the Andean distribution of Puya, along with the basal position of the southern Andean Fascicularia and Ochagavia in the parsimony analysis (compared to the basal position of the widespread, but non-andean Bromelia found by Schulte et al. 2009; Fig. 2), suggest an Andean origin for Bromelioideae, followed by a subsequent diversification of the subfamily in eastern Brazil. Generic Boundaries within Bromelioideae Generic-level systematics in subfamily Bromelioideae is poorly understood at best. Smith and Downs (1979) state that in going from subfamily Pitcairnioideae to Tillandsioideae to Bromelioideae, the determination of genus is progressively more difficult. The Bromelioideae are a step worse [than Tillandsioideae] in that the genera are so poorly defined that even with complete information, it is difficult to assign some species to genera. Recent molecular studies have shed light upon many of the systematic problems within the family; they have completely redefined Pitcairnioideae and subfamilial classification in Bromeliaceae (Givnish et al. 2007), illuminated relationships among the subfamilies (Givnish et al. 2011), resolved major lineages and generic circumscriptions within Tillandsioideae (Horres et al. 2000; Barfuss et al. 2005; Givnish et al. 2011; Versieux et al. 2012), and revealed a possible relationship between geographic co-occurrence and relationship within

9 2015] EVANS ET AL.: BROMELIOIDEAE (BROMELIACEAE) PHYLOGENY 123 the family (Sass and Specht 2010). Additionally, significant progress has been made in recent years to elucidate relationships within Bromelioideae (Givnish et al. 2011; Horres et al. 2007; Jabaily and Sytsma 2010; Sass and Specht 2010; Schulte et al. 2009; Schulte and Zizka 2008; Silvestro et al. 2014), although homoplasy, relatively low or uneven taxon coverage (Table 1), and an exceptionally low rate of molecular evolution within the subfamily have made the task particularly difficult. In this study, 28 of the 33 genera in Bromelioideae (sensu Luther 2010) are represented from a broad geographical range, with most non-monotypic genera represented by multiple species. This broad taxon coverage can provide some insights into generic circumscriptions and relationships that are not possible with less inclusive coverage. Of the 28 Bromelioideae genera sampled here, Bromelia and Cryptanthus were found to be monophyletic, and Ananas was monophyletic in the parsimony analysis (BS < 50%) and unresolved in the Bayesian analysis (Figs. 2, 3). Both Bromelia and Ananas have been supported as monophyletic in previous molecular studies (Schulte et al. 2005, 2009; Horres et al. 2007; Schulte and Zizka 2008), although Sass and Specht (2010) and Silvestro et al. (2014) each recovered a paraphyletic Ananas. Silvestro et al. (2014) recovered a polyphyletic Cryptanthus, with members of the genus being found in three different lineages and some being closely related to Orthophytum. Unfortunately, only two of the nine species that were collectively included in the two studies were common to both, making a direct comparison difficult. The genus was either absent (Sass and Specht 2010), represented by a single accession (Schulte et al. 2009), or unresolved (Schulte et al. 2005; Horres et al. 2007; Schulte and Zizka 2008) in other molecular studies. Monophyly for Acanthostachys, Araeococcus,andNeoglaziovia are neither supported nor refuted due to lack of resolution. The S-H tests fail to reject monophyly for any of these genera (Table 3), however, indicating that a phylogeny in which any of these genera are constrained to be monophyletic is not significantly less likely than the phylogeny that was recovered here. Additional sequence data will be needed to provide insight into the circumscriptions of these genera. Hohenbergia and Neoregelia are each nearly monophyletic, having a single species that is found in a separate lineage from the rest of their respective congenerics. Disteganthus, Fernseea, and Lapanthus are each represented by a single species, and Deinacanthon, Eduandrea, Fascicularia, and Hohenbergiopsis are monotypic. The remaining 13 genera in this study are polyphyletic. Aechmea is the most taxonomically problematic genus in Bromelioideae. Of the roughly 33 genera and 880 species within Bromelioideae, about 30% (260) of the species are placed within Aechmea (sensu Smith and Read 1976). In commentary after the description of the genus, Smith and Downs (1979) noted that Aechmea includes some very discordant elements and is very likely of polyphyletic origin. Further research is likely to divide it with some parts becoming independent genera and others merging with genera at present considered distinct. Circumscription of Aechmea has been problematic, with numerous genera differing from it by only one or two characters, and those characters are often highly homoplasious. Members of Aechmea are found along at least 12 different lineages in this study. In addition to its significant contribution to a large polytomy within Bromelioideae, Aechmea species are found in clades containing elements of numerous other genera, including Billbergia, Canistrum, Hohenbergia, Quesnelia, and Ronnbergia. These results strongly support other recent studies (Faria et al. 2004; Horres et al. 2007; Sass and Specht 2010; Givnish et al. 2011; Silvestro et al. 2014) in indicating that Aechmea, as currently defined, is highly polyphyletic and will need to be extensively redefined. Smith and Downs (1979) divided Aechmea into eight subgenera based largely on floral and inflorescence characters. Multiple species from every subgenus except Podaechmea, which was not sampled, were included in this study to test the monophyly of the subgenera. Chloroplast DNA data do not support the monophyly of any of the seven Aechmea subgenera that were sampled. Indeed, species from most of the subgenera are placed into numerous clades representing numerous lineages. Aechmea vallerandii and A. floribunda represent two species that were previously placed into the genus Streptocalyx (S. poeppigii and S. floribundus, respectively, sensu Smith and Downs 1979). Streptocalyx was distinguished from Aechmea based solely on the absence of petal appendages, a character that has since been shown to be unreliable at the generic level (Brown and Terry 1992), prompting Smith and Spencer (1992) to subsume the genus into Aechmea. Parsimony analysis placed both species in a large polytomy at the base of the eubromelioids, whereas Bayesian analysis placed A. floribunda in a medium-supported clade (PP = 0.66) containing Canistrum triangulare and four species of Orthophytum plus A. vallerandii, which was part of the large polytomy at the base of the core bromelioids (Figs. 2, 3). These results, which lack sufficient evidence to unite A. vallerandii and A. floribunda, are in agreement with morphological data (Faria et al. 2004), which also lacked evidence of a monophyletic Streptocalyx. Four of the nine species of Araeococcus were included in this study: A. goeldianus, A. micranthus, A. parviflorus, and A. chlorocarpus. Bayesian analyses placed A. goeldianus and A. parviflorus into a clade with high support (PP = 0.98; Fig. 3), whereas parsimony analyses failed to resolve any of the species, placing all of them into the basal polytomy. Araeococcus was divided into two subgenera based on the presence of pedicellate versus sessile flowers (Mez ; Smith and Downs 1979). Siqueira-Filho and Leme (2007) noted that the subgenera were artificial and redefined them based on several morphological characters, such as plant height when flowering, leaf texture, and the presence of spines on leaf blades and floral bracts. With the revised classification, subgenus Araeococcus species are restricted to the Amazonian region and subgenus Pseudaraeococcus species are found in the Atlantic Forest region of northeastern Brazil. The grouping of the pedicellate Amazonian A. goeldianus (subgenus Araeococcus) with the sessile-flowered Atlantic Forest species A. parviflorus (subgenus Pseudaraeococcus) inthe Bayesian analysis is curious, particularly since it is strongly supported (PP = 0.99) and the parsimony analysis failed to unite any of the species in the genus. Results from Schulte et al. (2005, 2009), Schulte and Zizka (2008) and Silvestro et al. (2014) suggest that Araeococcus, based on A. goeldianus and A. flagellifolius, is monophyletic. Sass and Specht (2010) included five species of Araeococcus, four from subg. Araeococcus, and one, A. parviflorus, from subg. Pseudaraeococcus. They found the genus to be polyphyletic, with A. parviflorus placed in a separate clade from subgenus Araeococcus. Additional rapidly evolving markers will be needed to resolve relationships in this genus and to test the unexpected placement of A. goeldianus and A. parviflorus as well as the circumscription of the genus sensu Siqueira-Filho and Leme (2007).

10 124 SYSTEMATIC BOTANY [Volume 40 Neither parsimony nor Bayesian analysis yielded a monophyletic Lymania, although the S-H test failed to reject monophyly (Table 3). Of the six species that were included here, three (L. azurea, L. alvimii, and L. spiculata) were placed in a strongly supported clade (BS = 89%, PP = 1.00), with the remaining three (L. corallina, L. globosa, and L. smithii) being part of the large polytomy near the base of the tree. Our taxon sample for Lymania is identical to that of Sass and Specht (2010). Both studies obtain the well supported L. azurea, L. alvimii, and L. spiculata clade, but Sass and Specht found a paraphyletic Lymania whereas the genus as a whole is unresolved in our phylogeny. Sousa et al. (2007) recovered a monophyletic Lymania based on combined morphological and molecular data, albeit with only weak support (BS = 52%, PP = 0.51). Additionally, the combined molecular/morphological analysis fully resolved the relationships among Lymania species, placing species into two moderately supported clades, one of which includes the three species that are grouped together in this study plus L. smithii (Sousa et al. 2007). Each clade is supported by morphological characters that are unambiguous within Lymania but homoplasious within Bromelioideae as a whole. The clade recovered here, for example, with the inclusion of L. smithii, is supported by a furrowed ovary and scape bracts that disintegrate (Sousa et al. 2007). Furrowed ovaries are found in species of Billbergia and Fernseea, however, and scape bracts that disintegrate can be found in members of Cryptanthus and Orthophytum. This situation illustrates both the potential utility of carefully examined morphological characters for phylogenetic reconstruction in Bromelioideae at the infrageneric level, as well as the difficulties of applying morphological characters broadly across the subfamily. The genus Hohenbergia is divided into two subgenera, Hohenbergia and Wittmackiopsis, based on ovule characteristics (ovules mostly apiculate to caudate in subg. Hohenbergia, vs. obtuse in subg. Wittmackiopsis) and flower color (flowers yellow, green, to lilac blue in subg. Hohenbergia, vs. mostly white in subg. Wittmackiopsis; Smith and Downs 1979; Siqueira- Filho and Leme 2007). Additionally, the two subgenera exhibit different biogeographic patterns, with subgenus Hohenbergia mostly restricted to Brazil and subgenus Wittmackiopsis found mainly in the Greater Antilles (Smith and Downs 1979; Siqueira-Filho and Leme 2007). Of the six species of Hohenbergia included in this study, the five species of subgenus Hohenbergia were all placed into a single strongly supported clade (BS = 82%, PP = 1.00), and the single species representing subgenus Wittmackiopsis (H. attenuata) was placed into a separate strongly supported clade with Aechmea patentissima, A. turbinocalyx and Ronnbergia brasiliensis (BS = 80%, PP = 1.00). The finding of a non-monophyletic Hohenbergia corroborates Sass and Specht (2010) and Silvestro et al. (2014), with the addition of many more taxa from subg. Hohenbergia of Brazil. Sass and Specht (2010) and Silvestro et al. (2014) each sampled extensively within subgenus Wittmackiopsis and found this subgenus to be monophyletic. In Sass and Specht (2010), subgenus Wittmackiopsis is nested within a clade containing A. lingulata and many affiliate Aechmea, as well as two species of Ronnbergia. Several of these affiliate Aechmea (A. fraseri, A. involucrata) are contrastingly placed in the current study within the broader Hohenbergia-Orthophytum clade containing subg. Hohenbergia. While the non-monophyly of the genus is not unexpected given the morphological and biogeographic disjunctions of the subgenera, the placement of H. attenuata among the group of Aechmea species was not anticipated based on morphology. Aechmea patentissima, which is sister to H. attenuata (BS = 52%, PP = 1.00), is part of the A. lingulata species complex, for which specific circumscriptions and relationships have historically been poorly defined. Siqueira-Filho and Leme (2007) have recently offered a relatively narrow circumscription of A. lingulata, with members of the species being restricted to the Lesser and Greater Antilles, the Bahamas, and the Amazon region. The other 15 species in the A. lingulata complex, including A. patentissima represented in this study, have a more eastern and southern distribution in the Brazilian coastal states of Bahia, Alagoas, Pernambuco, Espírito Santo, and Rio de Janeiro, and the inland states of Ceará and Minas Gerais. Thus, biogeographic distribution provides one potential explanation for the apparent shared history of H. attenuata and the Aechmea lingulata complex. Orthophytum, a genus of 55 species, is polyphyletic in this analysis, with species being found in two separate lineages. Schulte and Zizka (2008) and Schulte et al. (2009) found a close relationship between Orthophytum and Cryptanthus,with Cryptanthus glaziovii the single species of the genus represented in their analyses being nested within Orthophytum, albeit with relatively low support. Silvestro et al. (2014) found a polyphyletic Orthophytum, with species of Cryptanthus and Lapanthus being associated with the genus. Neither clade containing Orthophytum species in this analysis is placed near Cryptanthus. In the parsimony analysis, one Orthophytum clade is nested within a larger clade containing Hohenbergia subg. Hohenbergia,severalAechmea species, and a single species each of Lapanthus (formerly Orthophytum supthutii), Hohenbergiopsis, Canistrum, Billbergia, and Ronnbergia (supported only in 50% majority-rule tree). The remaining Orthophytum species are placed in a well-supported clade (BS = 76%) sister to Canistrum triangulare (BS = 54%). Bayesian analyses recovered similar relationships, except the latter clade also included two additional Billbergia species, B. kuhlmannii and B. morelii. Orthophytum has been divided into two informal groups based on the inflorescence being either scapose or sessile (Leme 2004; Louzada and Wanderley 2010; Louzada and Versieux 2010). Two species, O. supthutii, O. itambense, have been segregated to establish the new genus Lapanthus (Louzada and Versieux 2010) based on petal, stamen and molecular data. In this analysis, the two clades containing Orthophytum species represent each of these two groups, with two species bearing sessile inflorescences (O. humile, O. navioides,) being placed into one clade and four scapose species (O. benzingii, O. disjunctum, O. glabrum,ando. fosterianum) in another. Except for Sass and Specht (2010), Orthphytum supthutii, nowlapanthus duartei, has been included in all of the Bromelioideae-focused molecular studies. However, in Schulte et al. (2005), Horres et al. (2007), and Schulte and Zizka (2008), this was the only included Orthophytum. In Schulte et al. (2005) and Horres et al. (2007) Lapanthus (as O. supthutii) was unresolved in a large polytomy. In Schulte and Zizka (2008) Lapanthus came out in a wellsupported unresolved clade with the only two species of Cryptanthus included. In Schulte et al. (2009) Lapanthus, again as O. supthutii, came out as sister-taxon to a clade containing one Cryptanthus species and O. disjunctum and O. maracasense. This ((Cryptanthus Orthophytum) Lapanthus) clade is in a weakly supported clade with a monophyletic Ananas. Our results concur with Schulte et al. (2009) in regards to a Lapanthus Orthophytum relationship, but not Cryptanthus. These results support the statement by Siqueira-Filho and

11 2015] EVANS ET AL.: BROMELIOIDEAE (BROMELIACEAE) PHYLOGENY 125 Leme (2007) that this is a poorly studied group for which we lack much basic knowledge of species circumscriptions and ecology, and inclusion of a greater number of taxa in future phylogenetic studies would certainly aid in resolving questions about the monophyly of the group. Quesnelia is polyphyletic, with the two species included in this study being placed into two separate lineages. Other molecular studies (Schulte et al. 2005; Horres et al. 2007) as well as morphological (Faria et al. 2004; Almeida et al. 2009) phylogenetic analyses have also failed to yield a monophyletic Quesnelia. Faria et al. (2004) and Almeida et al. (2009) each recovered a monophyletic subgenus Quesnelia and a polyphyletic subgenus Billbergiopsis based on analysis of morphological characters. Although the inclusion of a single species from either subgenus in this study does not permit a test of the monophyly of either subgenus here, the placement of Q. arvensis is notable in that its placement is similar to that found in morphological studies. Almeida et al. (2009) placed Q. arvensis in a clade with Q. quesneliana that is sister to Aechmea nudicaulis, while Faria et al. (2004) found Q. arvensis to be in a clade containing Q. quesneila and A. vanhoutteana as well as two other species of Quesnelia. In this study, Q. arvensis is a member of a clade containing both A. nudicaulis and A. vanhoutteana, as well as A. distichantha (BS = 58%, PP = 1.00), lending support to the relationship between members of Quesnelia and A. nudicaulis and A. vanhoutteana. Both the parsimony and Bayesian analyses produced a large, weakly supported Billbergia-nidularioid clade (supported in parsimony majority-rule consensus, PP = 0.67) containing all included species of Canistropsis, Edmundoa, Nidularium, Wittrockia, Eduandrea, and Neoregelia, and the majority of sampled Billbergia, as well as several Aechmea from several subgenera. Sixteen species of Billbergia within this broader clade form a weakly to well-supported monophyletic lineage (supported in parsimony majority-rule consensus, PP = 0.86), but Bayesian analysis placed three species of Billbergia within the Hohenbergia-Orthophytum clade. Both analyses placed B. chlorantha outside of these two major clades, with weak support as a close relative of a clade of Aechmea (supported in parsimony majority-rule consensus, PP = 0.55). The two Billbergia subgenera Billbergia and Helicodea of Smith and Downs (1979) are not supported by the molecular data presented here. All of Neoregelia form a well-supported clade (BS = 78%, PP = 0.92), with the exception of N. carolinae, which is found in the basal polytomy of the clade. All nine species of Canistropsis are united in a single clade (BS < 50%, PP = 0.87), but the genus is not monophyletic due to the inclusion of Wittrockia paulistana, Edmundoa, and two species of Nidularium. Sass and Specht (2010) recovered a similar clade containing Nidularium, Wittrockia, and Edmundoa. In their study both Edmundoa and Nidularium were polyphyletic, in agreement with the topology presented here, but this clade was placed with low support as sister to a well-supported clade of Lymania and some species of Aechmea subg. Lamprococcus, a group not here included within the Billbergia-nidularioid clade. Schulte et al. (2009) recovered a similar grouping with species of Quesnelia being placed into three different lineages, also in agreement with the recovered polyphyletic Quesnelia. The relatively high level of phylogenetic resolution within the Billbergia-nidularioid clade suggests that this might be a group of taxa most ready for further taxonomic and molecular sampling ahead of generic revisions. Nidularioid genera have undergone a significant amount of taxonomic and nomenclatural revision in recent years (i.e. Pereira and Leme 1986; Leme 1997, 1998, 2000), and additional resolution of this clade using molecular systematic methods is needed. Prospects for a Phylogenetically Based Taxonomy within Bromelioideae Bromelioid systematics has been an active field of research in recent years (e.g. Horres et al. 2007; Schulte and Zizka 2008; Schulte et al. 2009; Sass and Specht 2010; Silvestro et al. 2014), yet the subfamily has resisted efforts to produce a robust and well-resolved phylogeny with high taxon-density. The low nucleotide substitution rate has made it necessary to sample multiple genes to obtain even modest resolution among species (e.g. Schulte and Zizka 2008; Sass and Specht 2010; Silvestro et al. 2014), while morphological characters are plagued by a high degree of homoplasy (e.g. Faria et al. 2004), particularly at deeper branches within the subfamily, making interpretation of homology exceedingly difficult and the phylogenetic utility of those characters problematic at deeper levels of the subfamily. Additionally, molecular studies in Bromelioideae tend to focus on a limited number of genera, often including only one or two placeholder species for many genera, making it difficult to examine generic boundaries across the subfamily. This study includes multiple species for a majority of genera in the subfamily, providing an opportunity to examine the monophyly of genera across the Bromelioideae. While relationships among numerous lineages within the core Bromelioideae remain largely unresolved, a framework for examining generic boundaries and relationships in the subfamily is emerging. For example, molecular studies have consistently placed Bromelia, Ochagavia, Deinacanthon, Fascicularia, and Fernseea near the base of the bromelioid phylogeny (Horres et al. 2007; Schulte and Zizka 2008; Schulte et al. 2009). Likewise, there is growing support for a Billbergia-nidularioid group, albeit with slight variations of the exact composition of this group. Aechmea is consistently found to be highly polyphyletic, whereas Cryptanthus and Bromelia appear to be monophyletic. As a general consensus begins to emerge regarding major clades within Bromelioideae, however, the problem of constructing a taxonomy that reflects the phylogenetic relationships remains. Characters that have traditionally been used to define groups are now known to be highly homoplasious, and many groups (e.g. the Hohenbergia- Orthophytum clade) that are recovered in the molecular phylogenies are difficult to define based on currently understood morphological grounds. There is little doubt that we are edging closer to the goal of producing a phylogenetically based taxonomy, however, as we begin to produce a reliable phylogeny for the subfamily. Acknowledgments. The authors wish to thank the Brazilian Forestry Service (IBAMA) and Brazilian Research Council (CNPq) for the field collection permits, and the colleagues E. M. C. Leme, Refúgio do Gravatás, Teresópolis, RJ, Brazil, and the late H. Luther, Marie Selby Botanical Gardens, Sarasota, FL for providing access to living materials. Two anonymous reviewers greatly improved this manuscript. Stephanie Kortering and John Drake provided assistance in data collection. This work was funded by National Science Foundation grants to TME (DEB ) and GKB (DEB ); and CNPq for productive grant to TW. Literature Cited Almeida, V. R., A. F. da Costa, A. Mantovani, V. Gonçalves-Esteves, R. de Oliveira, and R. C. Forzza Morphological phylogenetics of Quesnelia (Bromeliaceae, Bromelioideae). Systematic Botany 34: