PHYLOGENETIC RELATIONSHIPS IN SUBFAMILY TILLANDSIOIDEAE (BROMELIACEAE) BASED ON DNA

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1 American Journal of Botany 92(2): PHYLOGENETIC RELATIONSHIPS IN SUBFAMILY TILLANDSIOIDEAE (BROMELIACEAE) BASED ON DNA SEQUENCE DATA FROM SEVEN PLASTID REGIONS 1 MICHAEL H. J. BARFUSS, 2,4 ROSABELLE SAMUEL, 2 WALTER TILL, 2,3 AND TOD F. STUESSY 2 2 Department of Higher Plant Systematics and Evolution, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria; and 3 Herbarium, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria Molecular phylogenetic studies of seven plastid DNA regions were used to resolve circumscriptions at generic and infrageneric levels in subfamily Tillandsioideae of Bromeliaceae. One hundred and ten tillandsioid samples were analyzed, encompassing 10 genera, 104 species, and two cultivars. Two species of Bromelioideae, eight species of the polymorphic Pitcairnioideae, and two species of Rapateaceae were selected as outgroups. Parsimony analysis was based on sequence variation of five noncoding (partial 5 and 3 trnk intron, rps16 intron, trnl intron, trnl-trnf intergenic spacer, atpb-rbcl intergenic spacer) and two coding plastid regions (rbcl and matk). Phylogenetic analyses of individual regions produced congruent, but mostly weakly supported or unresolved clades. Results of the combined data set, however, clearly show that subfamily Tillandsioideae is monophyletic. The earliest divergence separates a lineage comprised of Glomeropitcairnia and Catopsis from the core tillandsioids. In their present circumscriptions, genera Vriesea and Tillandsia, and their subgenera or sections, as well as Guzmania and Mezobromelia, are poly- and/or paraphyletic. Genera Alcantarea, Werauhia, Racinaea, and Viridantha appear monophyletic, but separation of these from Vriesea and Tillandsia makes the remainder paraphyletic. Nevertheless, Tillandsioideae separates into four main clades, which are proposed as tribes, viz., Catopsideae, Glomeropitcairnieae, Vrieseeae, and Tillandsieae. Key words: atpb-rbcl; Bromeliaceae; coding and noncoding plastid DNA; molecular phylogeny; rps16 intron; Tillandsioideae; trnk-matk; trnl-trnf. The Neotropical pineapple family (Bromeliaceae) is one of the most conspicuous and characteristic monocot angiosperm families. Because of their unique appearance, bromeliads have become very important horticultural plants, and numerous popular publications exist for the family (e.g., Rauh, 1990; Röth and Weber, 1991). Bromeliaceae are well known for their potential for adaptive radiation, comprising terrestrial to tankimpounding and extreme atmospheric epiphytic life forms (Benzing, 1990, 2000; Givnish et al., 1997). They are found in very different habitats ranging from sand dunes of coastal deserts and wet savannas to tropical montane cloud forests. The Bromeliaceae is a well-defined, generally accepted monocot family, its monophyly being supported by several phylogenetic and morphological studies (Chase et al., 1995a, 2000; Nandi et al., 1998; Hilu et al., 2003). Close relationships have been suggested to Velloziaceae based on cladistic analyses of morphological data (Gilmartin and Brown, 1987), as well as to Rapateaceae and Mayacaceae based on recent molecular studies (Clark et al., 1993; Duvall et al., 1993; Chase et al., 1995b, 2000; Linder and Kellogg, 1995; APG, 1998, 2003; Givnish et al., 1999). The current taxonomy of Bromeliaceae, however, is strongly in flux. As new living material becomes available, new floral 1 Manuscript received 29 December 2003; revision accepted 19 October The authors thank H. and L. Hromadnik (Austria), F. Hase (Germany), E. M. C. Leme (Brazil), the Botanical Garden of the University of Heidelberg (Germany), and the Marie Selby Botanical Gardens (US) for providing plant material; H. Voglmayr, A. N. Müllner, M. Toth and E. Grasserbauer for technical assistance; and three anonymous reviewers for their critical comments. Financial support for this study was provided by the Austrian Science Foundation (FWF) to Walter Till (grant no. P13690-BIO). 4 Author for correspondence ( michael.h.j.barfuss@univie.ac.at; a @unet.univie.ac.at). 337 morphological details result in numerous changes and additions at the generic level. Newer morphological studies of seeds (Gross, 1988), septal nectaries (Böhme, 1988), stigmas (Brown and Gilmartin, 1984, 1989), pollen (Halbritter, 1988, 1992) and petal appendages (Brown and Terry, 1992) all are in conflict with the generic concepts of Bromeliaceae in the last comprehensive monograph of the family by Smith and Downs (1974, 1977, 1979; see also Smith and Till, 1998). Evidence is growing that morphological characters traditionally used to circumscribe genera within the family fail to delimit natural groups (Brown and Terry, 1992; Grant, 1993b, 1995a). The first DNA sequencing studies to incorporate Bromeliaceae examined interfamilial relationships among monocots (Chase et al., 1993, 1995b; Duvall et al., 1993). In these investigations, Bromeliaceae were a well-supported, monophyletic group, but taxon sampling within the family was very limited. Restriction site variation in chloroplast DNA (Givnish et al., 1990; Ranker et al., 1990), as well as comparative sequencing of plastid rbcl (Clark and Clegg, 1990; Clark et al., 1993), ndhf (Terry and Brown, 1996; Terry et al., 1997a, b), trnl intron sequences (Horres et al., 2000), matk (Crayn et al., 2000, 2004), and rps16 intron sequences (Crayn et al., 2004) have been used to test the existing morphologically based classification of Bromeliaceae and its subfamilies or the evolution of CAM photosynthesis and the epiphytic habit. Three subfamilies of Bromeliaceae traditionally have been recognized, Bromelioideae, Pitcairnioideae and Tillandsioideae (Mez, ; Smith and Downs, 1974, 1977, 1979; Smith and Till, 1998). According to the current classification by Smith and Till (1998), Bromelioideae encompasses 31 genera with 724 species and Pitcairnioideae 16 genera with 946 species. Subfamily Tillandsioideae is the largest (approximate-

2 338 AMERICAN JOURNAL OF BOTANY [Vol. 92 ly 1100 species) and in the last taxonomic treatment for the whole Bromeliaceae (Smith and Till, 1998) is divided into nine genera (species numbers updated): Alcantarea (16 spp.), Catopsis (21 spp.), Glomeropitcairnia (2 spp.), Guzmania (176 spp.), Mezobromelia (9 spp.), Racinaea (56 spp.), Tillandsia (551 spp.), Vriesea (188 spp.) and Werauhia (73 spp.). Generic limits and relationships in Tillandsioideae are currently undergoing change. Catopsis and Glomeropitcairnia are delimited most clearly, being defined by several distinctive features (e.g., fruit, seed, and pollen morphology, septal nectaries). Their placement in Bromeliaceae, however, has been controversial, but since Harms (1930), they have been recognized as belonging to Tillandsioideae (Mez, ; Hutchinson, 1973; Smith and Downs, 1977; Smith and Till, 1998; Till, 2000a). Inclusion of both Catopsis and Glomeropitcairnia into Tillandsioideae is also supported by cladistic analyses of morphological characters (Gilmartin et al., 1989), plastid ndhf (Terry et al., 1997a, b), and trnl intron sequences (Horres et al., 2000). In contrast, circumscriptions for most of the remaining Tillandsioideae are not clear, because morphological characters used for separation are of questionable diagnostic utility (e.g., spiral vs. distichous arrangement of the flowers, various connations of the corolla tube, inclusion vs. exsertion of stamens and styles, and, especially, presence vs. absence of petal appendages). Recent studies on flower gross morphologies, stamens and anthers, pollen, stigmata, ovaries, septal nectaries, ovules, fruits, and seeds (Brown and Gilmartin, 1984, 1989; Böhme, 1988; Gross, 1988; Gortan, 1991; Halbritter, 1992; W. Till, unpublished data) indicate that the four most heterogeneous genera, Guzmania, Mezobromelia, Tillandsia, and Vriesea, are all paraphyletic, having at least two lineages in Mezobromelia, three lineages in Guzmania, 12 lineages in Tillandsia, and five lineages in Vriesea (W. Till, unpublished data). In addition, the transfer of species from Guzmania to Mezobromelia (Utley and Luther, 1991), from Tillandsia to Guzmania (Rauh, 1991), to Mezobromelia (Weber and Smith, 1983), and to Racinaea (Grant, 1993a, 1994b; Spencer and Smith, 1993) as well as from Vriesea to Alcantarea (Grant, 1995a), to Guzmania (Utley, 1978), to Mezobromelia (Utley and Luther, 1991; Grant, 1993a), to Werauhia (Grant, 1995a, b), and to Tillandsia (Grant, 1993b, 1994a, 1995b), demonstrate the unreliability of the current morphologically based taxonomy. The shifting of xeric members of Vriesea with exserted stamens and styles to Tillandsia subgen. Tillandsia (Grant, 1993b), based on overall similarity (despite distinguishing petal appendages), the segregation of Viridantha (Espejo-Serna, 2002), and the resurrection of Sodiroa (Betancur and Miranda-Esquivel, 1999) has not been universally accepted (Luther and Sieff, 1997, 1998; Luther, 2000, 2002). The most comprehensive molecular analyses of Tillandsioideae so far are the studies of Terry et al. (1997b) using ndhf for 25 species and Horres et al. (2000), comparing trnl intron for 24 tillandsioid species. Results from these studies support the monophyly of Tillandsioideae as circumscribed by Smith and Downs (1977) and the isolated positions of Catopsis and Glomeropitcairnia, but resolution of the core Tillandsioideae genera Alcantarea, Vriesea, Werauhia, Guzmania, Mezobromelia, Racinaea, and Tillandsia (including Viridantha) was poor. Molecular data, however, indicate that the splitting of Tillandsia into six subgenera (T. subgen. Allardtia, T. subgen. Anoplophytum, T. subgen. Diaphoranthema, T. subgen. Phytarrhiza, T. subgen. Pseudalcantarea, and T. subgen. Tillandsia) and the segregation of the two genera Racinaea (previously treated as T. subgen. Pseudo-Catopsis) and Viridantha (formerly included in T. subgen. Allardtia) seem to circumscribe only partly natural groups. The same is valid for the genera Massangea and Sodiroa, which were segregated from Guzmania in the past, for the two sections of Vriesea (Vr. sect. Vriesea and Vr. sect. Xiphion), and the genera Alcantarea and Werauhia. In the present study, we assess phylogenetic relationships in Tillandsioideae using nucleotide sequences from seven regions of the chloroplast genome, viz. rps16 intron, trnl intron, trnltrnf intergenic spacer, atpb-rbcl intergenic spacer, rbcl with a small part of the rbcl-accd intergenic spacer, and matk with parts of the flanking trnk intron. The utility of these noncoding and coding sequences for resolving phylogenetic relationships at generic and subgeneric levels has been highlighted by several authors (e.g., trnk intron: Nyffeler, 2002; rps16 intron: Oxelman et al., 1997; Crayn et al., 2004; trnl intron: Gielly and Taberlet, 1996; Porter et al., 2000; trnl-trnf intergenic spacer: Baker et al., 2000; Bayer et al., 2000; atpb-rbcl intergenic spacer: Natali et al., 1996; Hoot and Douglas, 1998; rbcl: Chase et al., 1999; Muellner et al., 2003; matk: Soltis et al., 2001; Samuel et al., 2003). Our specific aims are to (1) examine relationships of Tillandsioideae to the other two subfamilies; (2) assess phylogenetic relationships within subfamily Tillandsioideae; (3) test the monophyly of individual genera, subgenera, or sections; (4) resolve taxonomic positions of problematic genera and species; and (5) examine the usefulness of morphological characters used in previous classifications in the context of the molecular results. MATERIALS AND METHODS Taxon sampling All major groups of Bromeliaceae were sampled based on the work of Smith and Till (1998) and Till (2000a, b). One hundred and ten samples (104 species and two cultivars) represented all 10 tillandsioid genera (Alcantarea, Catopsis, Glomeropitcairnia, Guzmania, Mezobromelia, Racinaea, Tillandsia, Vriesea, Viridantha, and Werauhia) and all their recognized subgenera, sections, or species groups. In most cases, one individual was sampled per species, only in a few, two (Tillandsia bergeri, T. usneoides, and T. utriculata) or three (T. guatemalensis) individuals from different accessions have been taken. A total of 10 species of the other two subfamilies of bromeliads (two species of Bromelioideae, eight species representing all three tribes of Pitcairnioideae), and two species of the genus Stegolepis (Rapateaceae) were used as outgroups. Samples were taken from the living collection of the Botanical Garden of the University of Vienna, Austria, some from the Herbarium of the Institute of Botany of Vienna (WU), Austria, as well as from other botanical gardens and private living collections (see Acknowledgments). All DNA sequences were originally produced for this study and are available from GenBank ( accession numbers AY to AY614500). The references for all these taxa, including origin, specimen voucher, place of deposition of herbarium, and GenBank number of the sequences can be found in Appendix 1 (see Supplemental Data accompanying online version of this article). DNA extraction, amplification, and sequencing Total genomic DNA was isolated from fresh, or in a few cases from silica-gel-dried leaf or herbarium material, using the 2 CTAB procedure described by Doyle and Doyle (1987). Extracted DNA was electrophoresed on 1% agarose gels with ethidium bromide for testing the quantity and quality of the isolated product. Polymerase chain reaction (PCR) amplifications of the selected regions used 100- L reactions containing 0.3 L of 5 units/ L Taq DNA polymerase (Promega, Mannheim, Germany), 10 L 10 Mg-free DNA polymerase buffer (Promega), 8 L 25 mmol/l MgCl 2,2 L 10 mmol/l each dntp, 4 L 0.4% bovine serum albumin (BSA), 2 L each primer (20 mmol/l), 65.7 L ddh 2 O, and 1 L template DNA. Alternatively, 50 L reactions were carried

3 February 2005] BARFUSS ET AL. PHYLOGENETICS IN TILLANDSIOIDEAE (BROMELIACEAE) 339 out with 45 L 1.1 ReddyMix PCR Master Mix (Advanced Biotechnologies, ABgene House, UK), including 1.25 units Taq DNA polymerase, 75 mmol/l Tris-HCL (ph 8.8 at 25 C), 20 mmol/l ammonium sulfate, 2.5 mmol/ L MgCl 2, 0.01% Tween 20, and 0.2 mmol/l each dntp, to which were added 1 L each primer (20 mmol/l), 2 L 0.4% BSA, and 1 L template DNA. The trnl intron and the trnl-trnf intergenic spacer were amplified as a single PCR fragment using the two universal primers c and f of Taberlet et al. (1991). The atpb-rbcl intergenic spacer and the rps16 intron were also amplified using universal primers ( Oligo 2 and Oligo 5, Manen et al., 1994; rpsf and rpsr2, Oxelman et al., 1997). In the case of rbcl, the two conserved flanking primers 1F (positioned at the beginning of rbcl) and 1460R (positioned in the rbcl-accd intergenic spacer; Fay et al., 1998) were used. The matk-trnk region was amplified in two overlapping fragments using primers -19F (positioned in the 5 trnk intron; Molvray et al., 2000; Gravendeel et al., 2001), 390F and 1326R (Cuénoud et al., 2002), and one newly designed reverse primer 1710R (5 -GCT TGC ATT TTT CAT TGC ACA CG-3 ) positioned in the 3 trnk intron. The PCR profile consisted of an initial 3 min premelt at 94 C and 36 cycles of 1 min denaturation at 94 C, 1 min annealing at 48 C, and 1 min (or 2 min, depending on the length of the amplified fragment) extension at 72 C, followed by a final extension of 9 (or 8) min at 72 C. PCR products were purified with a QIAquick Gel Extraction kit (Qiagen, Margaritella, Vienna, Austria) according to the manufacturer s instructions. Cycle sequencing was performed using the ABI PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystems, ABI, Vienna, Austria) using the same primers that were used for PCR. Additional internal primers for rbcl ( 2F, forward, 5 -CTT TCC AAG GCC CGC CTC ATG GC-3 ; and 2R, reverse, 5 -GTA GTA CGG AAT CAT CTC CAA AG-3 ) and for the atpbrbcl intergenic spacer ( RS3, reverse, 5 -TAC TGA GAA AAA TTC CCT CT-3 ) were designed to get clear, unambiguous sequences. Cycle sequencing products were precipitated with ethanol/sodium acetate consistent with manufacturer s instructions. Sequences were edited, assembled and a consensus of both forward and reverse DNA strands for each taxon generated using AutoAssembler (ABI). A detailed list for all primers used for PCR amplification and cycle sequencing has been archived (see Appendix 2 in Supplemental Data accompanying online version of this article). Sequence alignment and indel coding Multiple sequence alignments were performed using ClustalX (Thompson et al., 1997), leaving default parameters unchanged, and were visually adjusted as necessary. The limits of coding regions (matk and rbcl) were determined by comparison to published sequences of related monocots. All indels were coded as binary (presence/ absence) characters with the simple indel coding method of Simmons and Ochoterena (2000) and appended to the combined sequence matrix. The aligned matrices are available from the first author upon request. Phylogenetic analysis Fitch parsimony analyses (Fitch, 1971) were done using PAUP* 4.0b10 (Swofford, 2002). Eleven data sets were analyzed (see Table 1): (1) partial 5 and 3 trnk intron sequences, (2) rps16 intron sequences, (3) trnl intron sequences, (4) trnl-trnf intergenic spacer sequences, (5) atpb-rbcl intergenic spacer sequences, (6) rbcl sequences, (7) matk sequences, (8) combined noncoding plastid sequences, (9) combined coding plastid sequences, (10) all plastid sequences combined excluding indels, and (11) all plastid sequences combined including indels. Gap positions resulting from introduction of indels were treated as missing data, but indels (coded as binary characters, as explained earlier) were added to the combined coding/ noncoding data matrix. For each data set, heuristic searches were conducted using 1000 random addition sequence replicates, tree bisection-reconnection (TBR) branch swapping holding only 10 trees at each step to reduce time spent swapping on non-optimal trees. Only 10 trees were saved per replicate. All trees from the 1000 replicates were collected and an attempt made to swap them to completion without restrictions. However, due to the large number of trees yielded in most cases (the computer ran out of memory after trees), only were saved, but the tree search was allowed to continue to check for shorter trees. Bootstrap analysis (Felsenstein, 1985; Felsenstein and Kashino, 1993; Hillis and Bull, 1993) was performed on each data set, comprising 1000 replicates and using equal weighting, TBR branch swapping, simple sequence addition, with 10 trees held at each step and saved from each replicate. An independent, model-based estimate from combined sequence data (excluding coded gaps) was generated using Bayesian inference (Larget and Simon, 1999; Huelsenbeck et al., 2001; Lewis, 2001) with the method implemented in MrBayes 2.01 (Huelsenbeck and Ronquist, 2001). The model of sequence evolution chosen was the general time-reversible model (Rodríguez et al., 1990) with a proportion of invariable sites and gamma distribution (GTR I G). This model best fit our combined data set according to a hierarchical likelihood ratio test conducted in Modeltest version 3.06 (Posada and Crandall, 1998, 2001). Parameters set were nst 6 and rate invgamma, others were used as default. Four Markov chains starting with a random tree were run simultaneously for generations, sampling from the trees every 10 th generation. Stationarity was reached at around generation ; thus, the first generations ( trees) were discarded as the burnin, and inference about relationships was based only on the remaining generations ( trees). RESULTS The attributes of the aligned matrices, together with the details of the shortest trees found from the parsimony analyses (including the number, length, consistency index [CI], and retention index [RI]) for each of the data sets constructed are given in Table 1. The main results of the analyses are given later. Frequently used abbreviations are BS for bootstrap support and PP for posterior probability. Figures shown are bootstrap consensus trees of the rps16 intron (Fig. 1A), the trnl intron (Fig. 1B), the atpb-rbcl intergenic spacer (Fig. 1C), the matk (Fig. 1D), the combined noncoding sequences (Fig. 2A), and the combined coding sequences (Fig. 2B); for the analysis of all sequences combined a bootstrap consensus tree (Fig. 3A), a strict consensus tree (Fig. 3B), a Bayesian majorityrule probability tree with posterior probability as well as bootstrap support for the same branches (Fig. 4), and a phylogram of one shortest tree (out of ) randomly chosen from the parsimony analysis (Fig. 5). Clades (A R) mentioned in the individual results of each region are only shown in the trees of the combined analyses (Figs. 2 4). Partial 5 and 3 trnk intron analysis (excluding matk) Monophyly for Bromeliaceae is well supported (Bootstrap support, BS 100), but because of a limited number of sequenced base pairs, the bootstrap consensus tree does not provide much resolution within the family, and therefore is not shown. Only some clades, which are also present in the analysis of all sequences combined, can be observed in a large polytomy, and these have only weak or moderate bootstrap support: A (BS 57), B (BS 84), K (BS 53), L (BS 61), N (BS 70), and R (BS 84). rps16 intron analysis The bootstrap consensus tree (Fig. 1A) shows monophyly of the whole Bromeliaceae with a BS of 100. The intrafamilial relationships are not well resolved. Subfamily Tillandsioideae is also not supported as a monophyletic group. Catopsis (clade B, BS 100) and Glomeropitcairnia (clade A, BS 100) are placed in a polytomy with other members of Bromeliaceae. Only the core tillandsioids form one clade (BS 67), which is divided into two different lineages, although support for each is very weak (BS 54, respectively BS 58). Other clades present also in the analysis of all sequences combined are: E (BS 52), I (BS 75), L (BS 95), O (BS 85), and R (BS 54).

4 340 AMERICAN JOURNAL OF BOTANY [Vol. 92 TABLE 1. Results obtained from parsimony analyses of the 11 different data sets of 110 Tillandsioideae and 12 outgroup taxa. All combined including indels All combined excluding indels Combined coding Combined noncoding trnl intron trnl-trnf atpb-rbcl rbcl matk rps16 intron Partial trnk intron Phylogenetic information Length of sequences (bp) Length of alignment (no. of characters) No. of constant characters (% of characters) (67.8%) (79.5%) (81.5%) (82.7%) (77%) (89.2%) (75.9%) (78.8%) (82.3%) (80.4%) (78.3%) No. of variable characters (% of characters) (32.2%) (20.5%) (18.5%) (17.3%) (23%) (10.8%) (24.1%) (21.2%) (17.7%) (19.6%) (21.7%) No. of informative characters (% of characters) (21.6%) (14.1%) (11.7%) (12.3%) (12.8%) (7.3%) (17.2%) (13.4%) (12.4%) (13%) (14%) Number of gaps Number of shortest trees Length of shortest trees ( steps ) CI RI No. of clades with 85 BS No. of clades with 50 BS Note: bp base pairs; CI consistency index; RI retention index; BS bootstrap support. trnl intron analysis The bootstrap consensus tree is given in Fig. 1B. Monophyly of Bromeliaceae is well supported (BS 100). Brocchinia is resolved as monophyletic (BS 90) and placed sister to the remaining Bromeliaceae, but support for this relationship is low (BS 55). Subfamily Tillandsioideae is supported as monophyletic (BS 62), having some internal clades present also in the analysis of all sequences combined: A (BS 85), B (BS 86), D (BS 62), L (BS 63), and Q (BS 57). trnl-trnf intergenic spacer analysis The bootstrap consensus tree (not shown) displayed very poor resolution of phylogenetic relationships in Bromeliaceae; only a few clades in a large polytomy were observed, e.g., A (BS 99), B (BS 61). atpb-rbcl intergenic spacer analysis Figure 1C is the bootstrap consensus tree. Monophyly of the Bromeliaceae is well supported (BS 100). Brocchinia clearly comes out as the most ancient bromeliad genus, supported by a BS of 99, with all other Bromeliaceae forming a monophyletic sister group (BS 78). In this second clade Lindmania appears sister to the rest, but bootstrap support is very low (BS 54) for the latter. Going up the tree, there is a polytomy of four lineages: (1) Pitcairnia; (2) Hechtia, Aechmea, Bromelia, and Puya (BS 73); (3) Catopsis and Glomeropitcairnia (BS 66); and (4) the core tillandsioids (BS 57). Catopsis (clade B, BS 100) and Glomeropitcairnia (clade A, BS 78) are clearly resolved as separate genera, but their affinities to other Tillandsioideae are uncertain. Relationships among the core tillandsioids are not well resolved. Only some taxa like Racinaea (BS 79) and a number of Tillandsia species (e.g., clade Q, BS 67) form identifiable clades within a large polytomy. rbcl analysis (excluding rbcl-accd intergenic spacer) Consistent with the other regions, monophyly of family Bromeliaceae is supported with a BS of 100, but intrafamilial relationships are not resolved or only with very low bootstrap support. Therefore, the bootstrap consensus tree is not shown. Clades present among others are A (BS 90), B (BS 98), M (BS 60), and O (BS 69). matk analysis (excluding trnk intron) The matk bootstrap is well resolved at inter- and intrasubfamilial levels (Fig. 1D). Bromeliaceae are monophyletic (BS 100) with Brocchinia monophyletic (BS 99) and placed sister to the rest (BS 82). Relationships of the remaining Bromeliaceae are not well resolved, but Tillandsioideae is monophyletic (BS 73), with Catopsis and Glomeropitcairnia in a clade (BS 84) in sister position to the core tillandsioids (BS 99). Both genera have maximal or nearly maximal bootstrap support (Catopsis: clade B, BS 99; Glomeropitcairnia: clade A, BS 100). The remaining Tillandsioideae are split into two different lineages. The first one (BS 89) comprises the genera Alcantarea, Vriesea, Werauhia (including Werauhia insignis), and Tillandsia singularis, and the second one (BS 56) includes most of the Tillandsia species, and all taxa from Guzmania, Mezobromelia, Racinaea, Viridantha, and Vriesea appenii. Further clades present in the analysis of all sequences combined are C (BS 59), D (BS 94), E (BS 84), F (BS 90), G (BS 66), I (BS 60), K (BS 98), L (BS 93), M (BS 56), O (BS 94), P (BS 79), Q (BS 59), and R (BS 82). Analysis of combined noncoding plastid sequences The bootstrap consensus tree is given in Fig. 2A. Monophyly of

5 February 2005] BARFUSS ET AL. PHYLOGENETICS IN TILLANDSIOIDEAE (BROMELIACEAE) 341 Fig. 1. Bootstrap consensus trees of 110 Tillandsioideae and 12 outgroup taxa obtained from individual analyses of rps16 intron (A), trnl intron (B), atpbrbcl intergenic spacer (C), and matk (D) data. Bootstrap support ( 50) is given above the branches. Numbers right beside Tillandsia guatemalensis, T. usneoides, and T. utriculata refer to different accessions of these taxa (see Appendix 1 in Supplemental Data accompanying online version of this article). the family is well supported (BS 100). Catopsis and Glomeropitcairnia are grouped with a BS of 88. Alcantarea, Werauhia, T. singularis, and all Vriesea species except Vr. appenii form a well-supported clade (BS 98) sister to a clade comprising Mezobromelia, Guzmania, Tillandsia, Viridantha, Racinaea, and Vr. appenii. Analysis of the combined noncoding plastid sequences provided much better resolution compared to individual analysis of trnk intron, rps16 intron, trnl intron, trnl-trnf intergenic spacer, and atpb-rbcl intergenic spacer. Analysis of combined coding plastid sequences The bootstrap consensus tree (Fig. 2B) of the coding sequences shows a nearly identical pattern as the noncoding one. Analysis of all plastid sequences combined A total of 6687 characters (including 176 characters of coded gaps) was analyzed. Figures shown are the bootstrap consensus tree (Fig. 3A), the strict consensus tree (Fig. 3B), the Bayesian majorityrule probability tree (Fig. 4), and a phylogram (Fig. 5). Excluding coded gaps from the parsimony analyses of the combined data set resulted in nearly congruent trees of slightly different proportions (and therefore are not shown). As demonstrated by individual region analyses, Bromeliaceae and its probable sister group, Rapateaceae (RA), are separated by a very long, well-supported branch (BS 100/posterior probability, PP 1, Fig. 4). Within Bromeliaceae, Brocchinia is monophyletic (BS 100/PP 1) and placed sister to a clade containing all other bromeliads (BS 96/PP 0.99); within this clade Lindmania is sister to the rest (BS 89/PP 1). Pitcairnioideae (PI) is not monophyletic; Brocchinia and Lindmania do not group with Hechtia, Pitcairnia, and Puya. These last three taxa form a clade (BS 87/PP 0.98) with the two representatives of sub-

6 342 AMERICAN JOURNAL OF BOTANY [Vol. 92 Fig. 2. Bootstrap consensus trees of 110 Tillandsioideae and 12 outgroup taxa obtained from individual analyses of the combined noncoding (A), and coding (B) data. Bootstrap support ( 50) is given above the branches. Bars and letters indicate resolved groups mentioned in the text. Numbers right beside Tillandsia guatemalensis, T. usneoides, and T. utriculata refer to different accessions of these taxa (see Appendix 1 in Supplemental Data accompanying online version of this article). family Bromelioideae (BR; which are grouped, BS 76/PP 0.78), and this larger clade is sister to Tillandsioideae (BS 99/ PP 1). Puya laxa is the closest relative of Bromelioideae (BS 99/PP 1). Monophyly of Tillandsioideae (including Glomeropitcairnia and Catopsis) and its separation from other Bromeliaceae are well supported (BS 99/PP 1). Within subfamily Tillandsioideae, the combined data identify two strongly supported clades. The first of these clades (BS 93/PP 0.99) consists of two lineages, one of Catopsis (clade B, BS 100/PP 1) and a second of Glomeropitcairnia (clade A, BS 100/PP 1), which are well separated from each other by long branches and maximal BS. The second clade (BS 100/PP 1) includes the core Tillandsioideae consisting of the genera Alcantarea, Vriesea, Werauhia, Mezobromelia, Guzmania, Tillandsia, Racinaea, and Viridantha. The core tillandsioids are split into two different lineages: (1) a strongly supported clade (BS 100/PP 1, Vriesea s.l. in the sense of Smith and Downs, 1977) consisting of the genera Alcantarea, Werauhia, and Vriesea, including W. insignis (syn. T. insignis) and T. singularis (T. subgen. Allardtia); and (2) a clade (BS 94/PP 1) of Mezobromelia, Guzmania, Tillandsia, Racinaea, and Viridantha, including T. barclayana (syn. Vr. barclayana), T. werneriana (syn. Vr. rauhii), and Vr. appenii (Vr. sect. Xiphion). In Vriesea s.l., at least four clades can be recognized. The first one consists of the genus Alcantarea (clade C, BS 69/PP 0.78), the second one of Vriesea species from eastern Brazil (clade D, BS 100/PP 1), the third of Andean members of Vriesea sect. Xiphion, including T. singularis (clade E, BS 98/PP 1), and the fourth of the genus Werauhia, with Vr. monstrum (Vr. sect. Xiphion)

7 February 2005] BARFUSS ET AL. PHYLOGENETICS IN TILLANDSIOIDEAE (BROMELIACEAE) 343 Fig. 3. Consensus trees of 110 Tillandsioideae and 12 outgroup taxa obtained from analysis of combined partial trnk intron, rps16 intron, trnl intron, trnltrnf intergenic spacer, atpb-rbcl intergenic spacer, rbcl, partial rbcl-accd intergenic spacer, matk, and indel data. Bars and letters indicate resolved groups mentioned in the text. Numbers right beside Tillandsia guatemalensis, T. usneoides, and T. utriculata refer to different accessions of these taxa (see Appendix 1 in Supplemental Data accompanying online version of this article). (A) Bootstrap consensus tree. Bootstrap support ( 50) is given above the branches. (B) Strict consensus tree of the trees saved during the final round of parsimony analysis. and Vr. splendens (Vr. sect. Vriesea) in basal positions (clade F, BS 82/PP 1). Mezobromelia hutchisonii is sister to a clade (BS 89/PP 1) comprising M. pleiosticha and all species of Guzmania, Tillandsia (except T. singularis), Racinaea, and Viridantha. Guzmania is monophyletic (clade G, BS 87/PP 1), but only by inclusion of M. pleiosticha. Presently, Tillandsia s.l. (including Racinaea and Viridantha) remains a weakly resolved assemblage of several distinct lineages, which may turn out to be well-supported clades when more data are collected. Tillandsia viridiflora (type species of T. subgen. Pseudalcantarea) is sister to the rest of Tillandsia/Racinaea/Viridantha, but this relationship has only weak bootstrap support (BS 53/ PP 1). Bootstrap analysis strongly groups T. cacticola and T. marconae (both T. subgen. Phytarrhiza, clade L, BS 100/PP 1), but the position of this clade, as well as that of T. disticha (T. subgen. Allardtia) and T. wagneriana (T. subgen. Phytarrhiza) are not yet well resolved. In particular, the position of T. disticha is very unstable in different analyses and currently not interpretable. In Bayesian analysis, however, T. wagneriana is sister to clade H, T. disticha is sister to clade K plus T. paniculata (T. subgen. Pseudalcantarea), and T. cacticola plus T. marconae form a polytomy together with clade M and clades N R. Viridantha plumosa and Vi. tortilis form a strongly supported clade (BS 100/PP 1) with T. tectorum (T. subgen. Allardtia) as its sister (clade M, BS 94/PP 1). Former xeric members of Vriesea (BS 72/PP 0.59) form a moderately supported clade with T. barthlottii (T. subgen. Allardtia), but the precise position of this group (clade H, BS 83/PP 1) within Tillandsia s.l. remains uncertain. Racinaea (syn. T. subgen. Pseudo-Catopsis) forms a well-supported clade (BS 99/PP 1)

8 344 AMERICAN JOURNAL OF BOTANY [Vol. 92 Fig. 4. Bayesian majority-rule probability tree of 110 Tillandsioideae and 12 outgroup taxa obtained from analysis of combined partial trnk intron, rps16 intron, trnl intron, trnl-trnf intergenic spacer, atpb-rbcl intergenic spacer, rbcl, partial rbcl-accd intergenic spacer, and matk data. Bootstrap support ( 50) and posterior probability ( 0.5) are given above the branches. Left bars and letters (indicating resolved groups mentioned in the text; A R Tillandsioideae, BR Bromelioideae, PI Pitcairnioideae, and outgroup family RA Rapateaceae) compare relationships in this study with the generic and subgeneric classification (right bars and text) in the monographs of Smith and Downs (Pitcairnioideae, 1974; Tillandsioideae, 1977; and Bromelioideae, 1979). Gray bars indicate taxa, which were classified as Tillandsia species by Smith and Downs. Asterisks (*) mark species that were described after publication of the monographs. Numbers right beside Tillandsia guatemalensis, T. usneoides, and T. utriculata refer to different accessions of these taxa (see Appendix 1 in Supplemental Data accompanying online version of this article). with T. venusta and T. lindenii (both T. subgen. Phytarrhiza) at its base (clade J, BS 83/PP 1). The sister relationship of T. dodsonii plus T. narthecioides (both T. subgen. Phytarrhiza, clade I, BS 98/PP 1) to clade J has only weak bootstrap support (BS 50/PP 0.97). Tillandsia subgen. Tillandsia forms a strongly supported clade (clade K, BS 100/PP 1), which includes T. guatemalensis (the type species of T. subgen. Allardtia) and T. remota (also T. subgen. Allardtia), with T. paniculata as its sister (BS 81/PP 1). Members of T. subgen. Allardtia, T. subgen. Anoplophytum, xeric T. subgen. Phytarrhiza, and T. subgen. Diaphoranthema form the largest (but internally partly unresolved) clade (BS 86/PP 1) within Tillandsia s.l. However, three strongly supported clades comprised of members of T. subgen. Anoplophytum can be recognized: (1) an eastern Brazilian clade (clade O, BS 100/PP 1), (2) an Andean clade (clade Q, BS 100/PP 1), and (3) a southeastern Brazilian clade (clade R, BS 100/PP 1). Further analyses will doubtless split this large group into several better-supported clades. DISCUSSION The present study includes 122 samples (110 ingroup taxa, 12 outgroup taxa) and seven plastid regions. In general, no region alone completely resolves intrafamilial relationships. The best single resolution is provided by matk, but better res-

9 February 2005] BARFUSS ET AL. PHYLOGENETICS IN TILLANDSIOIDEAE (BROMELIACEAE) 345 Fig. 4. Continued. olution of all important clades and their affinities to each other are provided by combined data from all seven plastid regions. Rates of evolution in the bromeliad chloroplast genome appear very conservative in comparison to other families for which variability in the same loci has been assessed (see also Terry et al., 1997a, b). This lack of variation in Bromeliaceae is also remarkable in view of the broad morphological diversification in the family. Results from Terry et al. (1997b) and Horres et al. (2000) regarding monophyly of some groups (Tillandsioideae as a whole, monophyly for the core Tillandsioideae) are consistent with our investigations. Relationships that were only weakly supported in these studies have now been corroborated, and a greater degree of phylogenetic resolution has been obtained. Following are discussions of the results in the context of the phytogeographic, evolutionary and taxonomic implications.

10 346 AMERICAN JOURNAL OF BOTANY [Vol. 92 Fig. 5. Phylogram of one most parsimonious tree (randomly chosen from shortest trees) of 110 Tillandsioideae and 12 outgroup taxa obtained from analysis of combined partial trnk intron, rps16 intron, trnl intron, trnl-trnf intergenic spacer, atpb-rbcl intergenic spacer, rbcl, partial rbcl-accd intergenic spacer, matk, and indel data. Branch length is given above the branches. Gray bars indicate the tribal classification of Tillandsioideae based on the present study (viz., Glomeropitcairnieae, Catopsideae, Vrieseeae, and Tillandsieae), black ones the other subfamilies (Bromelioideae and Pitcairnioideae) and outgroup family (Rapateaceae). Numbers right beside Tillandsia guatemalensis, T. usneoides, and T. utriculata refer to different accessions of these taxa (see Appendix 1 in Supplemental Data accompanying online version of this article).

11 February 2005] BARFUSS ET AL. PHYLOGENETICS IN TILLANDSIOIDEAE (BROMELIACEAE) 347 Phytogeography and evolution Our molecular data clearly demonstrate a pattern of geographic radiation during evolution of the bromeliad family. Both the super-outgroup (Stevenson et al., 1998; Givnish et al., 2000) and the outgroup (Holst, 1997) are centered in northern South America, and there is little doubt that the family, as well as Tillandsioideae, have evolved in this geologically very old and stable environment (Benzing et al., 2000a). From there early lineages migrated into eastern Brazil (Vriesea s.l.) and the Caribbean (Catopsis and Glomeropitcairnia). During the rather recent uplift of the Andes, Guzmania/Mezobromelia and Racinaea/Tillandsia/Viridantha displayed an explosive adaptive radiation perhaps due to the high number of new ecological niches. Along the Andes, these genera appear to have migrated northward to Mexico and southward to Chile. Tillandsia subgen. Allardtia has its center of distribution in the central and northern Andes, whereas T. subgen. Tillandsia has developed a secondary center of diversity in Mexico and Central America. Tillandsia subgen. Diaphoranthema also has developed a secondary center at the southern end of the range of the genus under very arid environments; only a few tillandsias have reached the eastern rim of the continent. All taxa of Tillandsia seem to be phylogenetically young, as detected by the low genetic divergence, i.e., weak resolution in our molecular cladograms. It is remarkable that the rapid evolution of the Andean groups (Guzmania, Tillandsia s.l.) has led to a loss of petal appendages, and that this loss appears to have been compensated by filament plication (e.g., T. subgen. Anoplophytum; Evans and Brown, 1989) or petal constriction just above the ovary (T. subgen. Tillandsia). Loss of petal appendages is very rare in Vriesea s.l. This interpretation of petal appendage evolution is more parsimonious than the assumption that these principally very similar structures had evolved five times independently. In the early diverging lineages of Tillandsioideae, mesophytic and phytotelm-forming terrestrial taxa greatly outnumber xerophytes (Alcantarea, Vriesea). In Tillandsia, which is in a terminal position in our trees, epiphytic xerophytes are common and by far outnumber the phytotelms (Till, 2000b). This is mirrored by the lack or rare occurrence of CAM photosynthesis in Catopsis, Glomeropitcairnia, Guzmania, and Vriesea s.l. and the numerous records for Tillandsia (Martin, 1994; Crayn, 2004). The prevailing pollinators are hummingbirds, but insects also are the regular pollinators in Catopsis and Tillandsia (Gardner, 1986; Benzing et al., 2000b). Bat pollination has been reported for Werauhia, Alcantarea, and for east Brazilian Vriesea (Vogel, 1969; Utley, 1983; Martinelli, 1994). Self-pollination is common in Andean Tillandsias (Till, 2000b), and cleistogamy is known only from T. subgen. Diaphoranthema (Gilmartin and Brown, 1985). Although some tendencies are evident, pollination syndromes are less correlated with the subfamily s phylogeny than is vegetative morphology and ecology, but obviously play a major role in species diversity. Taxonomy Subfamilial classification in Bromeliaceae traditionally uses fruit and seed types, and the position of the ovaries in relation to the perianth, which varies from completely superior (Tillandsioideae: Catopsis) to extremely inferior (most Bromelioideae). However, varying degrees of inferior are the rule. In general, Tillandsioideae have the most superior ovaries, Bromelioideae the most inferior, and with Pitcairnioideae in between. Pitcairnioideae Paraphyly of this subfamily in earlier studies (Terry et al., 1997a; Horres et al., 2000) is further corroborated by this study as is the sister position of Brocchinia to the rest of the family and that of Puya to Bromelioideae (Terry et al., 1997a; Horres et al., 2000). Fruits are capsular and open with septicidal and loculicidal splitting. Seeds are unappendaged or bear variously shaped (but never pseudo-pappiformously split) appendages. Leaves may have entire or serrate margins as in Bromelioideae, and the foliar trichomes have irregular cellular patterns and are nonabsorbing. Roots are responsible for nutrient and water uptake. Bromelioideae Our limited sampling corroborates the monophyly of this subfamily (Terry et al., 1997a). Fruits are berries, seeds are unappendaged or rarely bear sticky appendages, and the outer seed coat usually is slimy. Leaves vary in armature as in Pitcairnioideae, and foliar trichomes are not regularly constructed and are usually nonabsorbing. Roots are usually responsible for nutrient and water uptake but may function as holdfasts in epiphytic xerophytes. Tillandsioideae Our results support monophyly of Tillandsioideae (Figs. 2 5) as suggested by Terry et al. (1997a, b) and Horres et al. (2000). Fruits are septicidal capsules. The unique seed morphology with a pseudo-pappus for wind dispersal, the always entire leaf margins, and the absorptive trichomes with their regular cellular pattern further corroborate its monophyly. Roots function as holdfasts in most cases. Catopsis is unique in its apical seed appendage, which is formed of separate rows of multicellular hairs (in contrast, all other members of Tillandsioideae have seed appendages of cell rows derived from longitudinal splitting of the outer integument, which strongly elongates at the base during maturation of the seeds; the apical portion of the ovule rarely elongates and is not split into hairs ). This genus has a unique pollen type (Halbritter, 1992), characterized by sharply cut aperture margins. Glomeropitcairnia has semi-inferior ovaries and fruits. Long seed appendages are produced at both ends but are split only at the base, and the cell rows are distally divergent and form a hairy tuft. This seed morphology is very distinctive and resembles only that of Alcantarea, in which, the basal cell rows are united by a stomium and diverge proximally. Pollen, however, strongly resembles that of certain Guzmania species (as does stigma morphology; Till et al., 1997). The molecular divergence between Catopsis, Glomeropitcairnia, and the remaining Tillandsioideae (as evidenced by the long branches and high bootstrap support, Figs. 2 5) is interesting in the light of the morphological autapomorphies for each as mentioned earlier. From a morphological point of view, the quite isolated positions of both genera have never been a matter of debate. They appear to have diverged early in the evolution of the subfamily and have developed divergent autapomorphies. Vriesea, Alcantarea, and Werauhia have mostly distichous flower arrangements and a pair of petal appendages; petals are mostly yellow (often with green tips) or white. The convoluteblade stigma type (Brown and Gilmartin, 1984, 1989) is characteristic of Alcantarea (Fig. 4, clade C) and East Brazilian Vriesea (clade D) (but found also in Glomeropitcairnia and Guzmania), whereas the cupulate stigma type is distinctive for Werauhia (clade F in part). The Andean clade (E) clearly differs in its simple-erect or conduplicate-spiral stigma type from

12 348 AMERICAN JOURNAL OF BOTANY [Vol. 92 the east Brazilian (D) one. Pollen of Andean Vriesea is mainly of the diffuse-sulcus type (Halbritter, 1992), and similar pollen is found in Guzmania and Tillandsia, whereas that of the east Brazilian Vriesea species and Werauhia is of the insulae type. Alcantarea has a unique pollen type. These morphological characters confirm our four clades in Vriesea s.l. (Fig. 4). The close relationship of Alcantarea, Vriesea, and Werauhia has never been questioned, but the separation of Alcantarea and Werauhia has not been generally accepted. Our data show that Vriesea s.l. in the sense of Smith and Downs (1977; i.e., including Alcantarea and Werauhia) is a well-supported group, but only when some species previously treated here are excluded and others treated as Tillandsia are herein transferred. Taxa that should be excluded are the xeric Andean members, e.g., T. barclayana (syn. Vr. barclayana), T. werneriana (syn. Vr. rauhii), and Vr. appenii; those to be incorporated are, e.g., T. singularis and W. insignis (syn. T. insignis). In contrast, the results show convincingly that the separation of Alcantarea and Werauhia makes the remainder of Vriesea paraphyletic. The relationships of Werauhia to Vr. monstrum and Vr. splendens remain unclear. Mezobromelia and Guzmania have a mostly polystichous flower arrangement. Petals in the two genera are nearly exclusively white or yellow, rarely green, the former genus also bearing two appendages. Neither of these genera have conformity in stigma architecture. Pollen is of the diffuse sulcus type or inaperturate. Tillandsia, Racinaea, and Viridantha have a mainly distichous flower arrangement. Petals are mostly violet or purple and lack appendages, but exceptions are the xeric species formerly placed in Vriesea. Additional petal colors are white, green, yellow, orange, and red. Stigma architecture is variable, mostly of the conduplicate-spiral or simple-erect type. Some members of T. subgen. Phytarrhiza possess the unique coralliform stigma type (Brown and Gilmartin, 1989). The clustering of Racinaea together with different members of mesic T. subgen. Phytarrhiza (clade I, Fig. 4) supports the interpretation of the stigma morphology of Racinaea as a simplification of the coralliform stigma type. Pollen is most variable in Tillandsia (Halbritter, 1992). The diffuse-sulcus type prevails; in addition, the insulae type, operculum type, and Vriesea (Alcantarea) imperialis type also occur. Stigma and pollen morphology reflect well the heterogeneity of this genus as demonstrated by molecular data. Seeds, nectaries and ovules also provide useful comparative data within Tillandsioideae. Gross (1988) has studied the micromorphology of bromeliad seeds. Her proposals for infrageneric classifications in Tillandsioideae are rather well supported by our molecular data, especially in Guzmania and Vriesea s.l. Böhme (1988) analyzed septal nectary morphology and concluded that Catopsis is basal with Guzmania/Mezobromelia/Tillandsia/Vriesea constituting a derived group (corresponding to the molecular core group!). Ovule morphology has hitherto been neglected, but preliminary observations suggest that it may provide another useful set of characters (W. Till, unpublished data). Development of the chalazal appendage as well as ovule shape vary considerably. Therefore more extensive morphological data sets have to be generated first before generic and subgeneric grouping can be made to parallel clades created by molecular analyses. This work is in progress and will be presented in a separate article. Based on the results of the present study, a four-tribe classification of Tillandsioideae is proposed (Fig. 5) here. Genera provisionally accepted are mentioned after each tribe. Catopsideae: Ovary superior, capsule septicidal; petal appendages lacking; stigma of the simple-erect type; pollen with sharply cut aperture margins, exine reticulate (Catopsis type). Catopsis. Glomeropitcairnieae: Ovary half-inferior, capsule only partly septicidal; petal appendages present; stigma of the convolute-blade type; pollen with a diffuse aperture, exine reticulate. Glomeropitcairnia. Vrieseeae W. Till & Barfuss, tribus nov.: Ovario partialiter inferiore, capsula septicida, petalis plerumque appendiculatis, stigmate plerumque typo laminae-convolutae vel cupulari, rariter simplici-erecto et polline plerumque insulae typo vel typo Vrieseae-imperialis, rariter aperture diffusa recedit. Typus: Vriesea Lindl. (1843), nom. conserv. Ovary partly inferior, capsule septicidal; petal appendages usually present; stigma mainly of the convolute-blade and cupulate types, rarely of the simple-erect type (Brown and Gilmartin, 1989, reported conduplicate-spiral, probably a misinterpretation); pollen mainly of the insulae type or the Vriesea imperialis type, less often with a diffuse aperture. Alcantarea, Vriesea, and Werauhia. Tillandsieae: Ovary partly inferior, capsule septicidal; petal appendages usually lacking; stigma mainly of the conduplicate-spiral or simple-erect type, rarely of the convolute-blade type, occasionally of the unique coralliform type; pollen mainly with diffuse apertures, occasionally of the insulae type, or rarely of the Vriesea imperialis type or inaperturate. Guzmania, Mezobromelia, Racinaea, Tillandsia, and Viridantha. Due to insufficient sampling of core tillandsioid genera in this study, no taxonomic suggestions are made below the tribal level. However, some previous taxonomic changes are supported by the molecular results. For example, most xeric Andean members of Vriesea sect. Vriesea were transferred to Tillandsia by Grant (1993b), and T. insignis has been transferred to Werauhia (Barfuss et al., 2004). Transfer of T. singularis to Vriesea should await a more thorough study of the tribe Vrieseeae. A generic reclassification of the subfamily causing taxonomic and nomenclatural changes will only be made with a significantly enlarged sampling. LITERATURE CITED ANGIOSPERM PHYLOGENY GROUP (APG) An ordinal classification for the families of flowering plants. Annals of the Missouri Botanical Garden 85: ANGIOSPERM PHYLOGENY GROUP (APG) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society 141: BAKER, W. J., C. B. ASMUSSEN, S. BARROW, J. DRANSFIELD, AND T. A. 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