phylogeny of miconia (melastomataceae) - The University of Chicago ...

Int. J. Plant Sci. 169(7):963–979. 2008. Ó 2008 by The University of Chicago. All rights reserved. 1058-5893/2008/16907-0014$15.00 DOI: 10.1086/589697

PHYLOGENY OF MICONIA (MELASTOMATACEAE): PATTERNS OF STAMEN DIVERSIFICATION IN A MEGADIVERSE NEOTROPICAL GENUS Renato Goldenberg,1 ,* Darin S. Penneys,y Frank Almeda,z Walter S. Judd,y and Fabiań A. Michelangeli§ *Universidade Federal do Parana´, Departamento de Botaˆnica, Campus do Centro Politećnico, Curitiba PR 81540-560, Brazil; yDepartment of Botany, University of Florida, Gainesville, Florida 32611-8526, U.S.A.; zDepartment of Botany, California Academy of Sciences, San Francisco, California 94103-3098, U.S.A.; and §New York Botanical Garden, Bronx, New York 10458, U.S.A.

Phylogenetic relationships within Miconia and other genera in the Neotropical tribe Miconieae were investigated using a maximum parsimony analysis of nuclear internal transcribed spacer and ndhF nucleotide sequences. Included were all sections in Miconia (212 species, ;20% of the genus) and 12 of the 15 remaining genera assigned to the tribe (an additional 239 species). Given the tribe’s reputation for problematic generic distinctions, it was not surprising that most traditionally recognized taxonomic groups—both genera and sections—were shown to be polyphyletic or paraphyletic. Nevertheless, Miconia is composed of several distinct monophyletic groups, with a large majority of the species belonging to only four clades. Some of these groups represent parts of sections proposed in the last revision of the genus, but most of the diversification seems to have occurred in geographical areas that are more restricted than would have been predicted by the distribution of these sections. Moreover, parallel evolutionary trends are seen in anther form, i.e., shifts from elongate to shorter anthers and from minute-pored to large-pored or slitlike dehiscent anthers. These changes may relate to pollinator shifts, especially from buzz pollination to nonvibrational pollination. Thus, the major evolutionary diversifications within the tribe have been obscured by convergence in stamen morphology, leading to many arbitrary generic and sectional circumscriptions. Keywords: internal transcribed spacer (ITS), megadiverse genus, Melastomataceae, Miconia, Miconieae, ndhF, phylogeny, stamen morphology. Online enhancement: appendix.

Introduction

species are common in secondary environments, providing natural or human-induced forest regeneration (Ellison et al. 1993; Baider et al. 1999). While most species are shrubs or small trees, usually preferring very moist, hilly, and forested terrain (Ruokolainen et al. 1997), some are vines, hemiepiphytes, or epiphytic shrubs, and a few, such as M. poeppigii, are large trees, up to 30 m (Wurdack et al. 1993; Almeda, forthcoming). Some species occur in areas with seasonal rainfall, and many grow in the perpetually wet lowland rain forests. A smaller number of species occur in deciduous forests and savannas, but Miconia is virtually absent in the dry South American Chaco and the Caatinga of northeastern Brazil. Most species prefer acidic soils (Ruokolainen et al. 1997), but in the Antilles (Judd 2007) and northern Mesoamerica, a number of species grow on limestone or limestone-derived soils. The majority of the species are pollinated by bees that extract the pollen by vibrating the poricidal anthers from nectarless flowers (Renner 1989), but there are records of pollination by bats, birds (Dent-Acosta and Breckon 1989; Penneys and Judd 2003; Judd 2007), flies (Goldenberg and Shepherd 1998), and wasps (I. Varassin, personal communication) on nectar-producing flowers (Stein and Tobe 1989), whose anthers open by slits or large pores that do not need vibration to release the pollen. Agamospermy occurs in some species (Dent-Acosta and Breckon 1989; Renner 1989; Goldenberg and Varassin 2001)

Miconia Ruiz & Pavoń is the largest genus of Melastomataceae, with about 1050 species (Goldenberg 2000), and is probably the largest exclusively New World genus of flowering plants, perhaps rivaled by only Pleurothallis R.Br. (Orchidaceae) before its breakup (Frodin 2004). These species range from western Mexico and the Caribbean to Uruguay and northern Argentina, growing from sea level to the Andean paramos. A few species, notably Miconia calvescens, have been introduced to the Pacific Islands (Meyer 1996; Meyer and Florence 1996; Medeiros and Loope 1997), where they have become troublesome invasives. Apart from general uses, such as low-quality wood and fuel, species of Miconia are not economically important. Nevertheless, they play an important ecological role in tropical and subtropical forests as a food resource for the fauna because their fleshy fruits are largely consumed by birds and other animals (Snow 1965; Magnusson and Sanaiotti 1987; Levey 1990; Stiles and Rosseli 1993; Figueiredo and Longatti 1997). As fast-growing shrubs or small trees that develop dense populations in disturbed areas, these 1

Author for correspondence; e-mail: [email protected].

Manuscript received September 2007; revised manuscript received December 2007.

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and apparently is related to polyploidy and hybridization (Solt and Wurdack 1980; Almeda and Chuang 1992; Goldenberg and Shepherd 1998), although it is not clear how widely these mechanisms are distributed within the group. Self-incompatibility has also been reported (Renner 1989; Goldenberg and Shepherd 1998), while dioecism occurs in 37 species (Almeda and Dorr 2006). Within the Melastomataceae, Miconia belongs to the tribe Miconieae sensu stricto, as circumscribed by Michelangeli et al. (2004), i.e., excluding Henriettea DC., Henriettella Naudin, Loreya DC., and Bellucia Raf., as these represent a distinct new tribe (Penneys et al. 2004). Huilaea Wurdack and Chalybea Naudin should also be removed from the Miconieae and transferred to the Blakeeae (Penneys 2007). The Miconieae are an exclusively Neotropical clade whose species share tissues with druse crystals (but lacking megastyloids), cymose inflorescences that are terminal or axillary but usually not cauliflorous, anthers lacking pedoconnectives, unappendaged, or with only small dorsobasal connective appendages, fleshy fruits, and noncochleate seeds. Within the Miconieae, the genus Miconia is clearly paraphyletic (Judd and Skean 1991; Michelangeli et al. 2004; Martin et al. 2008). It has long been known that genera within the tribe are poorly characterized, often difficult to discern, or even arbitrarily defined (Baillon 1887; Cogniaux 1891; Gleason 1925a, 1932b, 1958; Macbride 1941; Wurdack 1972, 1980; Judd 1986, 1989; Judd and Skean 1991). Perhaps the best definition for Miconia came from Judd and Skean (1991, p. 54), who, paraphrasing Macbride (1941), stated that ‘‘if a particular species does not fit into any of the related, segregate genera it should be placed in Miconia.’’ The little information about chromosomes suggests a base number of x¼17 for the entire tribe (Solt and Wurdack 1980; Almeda and Chuang 1992; Almeda 1997), with polyploidy also occurring in scattered species. Following Cogniaux (1891) and Judd and Skean (1991), we could define the genus Miconia as including members of Miconieae that are woody and have these traits: nonformicarial leaves; terminal inflorescences; hypanthium not apically constricted; calyx of small lobes, not forming a stout, conical, circumscissile cap; free petals with obtuse, rounded, or emarginate apices; and anthers without a bifurcation at the base. As in many other large angiosperm genera (Kress et al. 2005), one is forced to recognize Miconia only by eliminating other genera; i.e., it is distinguished only by the plesiomorphic characters within the tribe (Judd and Skean 1991; Judd 2007). If we follow the generic alignments proposed by Cogniaux (1891), the genera most closely resembling Miconia are Clidemia D.Don, Tetrazygia Cogn., Leandra Raddi, and Charianthus D.Don. All five have paniculate, multiflowered, terminal, or pseudolateral inflorescences. However, Clidemia usually has pseudolateral inflorescences and unappendaged stamens; many but not all species have long external calyx teeth; Tetrazygia has a long hypanthium, with a constricted apex; Leandra (including Platycentrum Naudin and Pleiochiton Naudin; following Wurdack 1984; Judd and Skean 1991) has acute petals; and Charianthus has red pseudocampanulate corollas and rimose anthers and lacks druse crystals (Penneys and Judd 2005). Also related to Miconia are Tococa Aubl. and Maieta Aublet (both usually with leaves bearing formicaria), Anaectocalyx Triana (with anthers bifurcate at the base), Conostegia

D.Don (with fused calyx lobes that fall away as a circumscissile cap), Pachyanthus A.Rich. (with reduced inflorescences with flattened axes, robust hypanthia, and calyx teeth, and often asymmetrical and clawed petals), and Calycogonium D.Don (with four-merous flowers and shoots that produce only one or two nodes before each ends in a few-flowered inflorescence). Several genera, i.e., Killipia Gleason, Kirkbridea Wurdack, Mecranium Hook.f., Ossaea DC., and Sagraea DC., are easily distinguished from Miconia because of their axillary (i.e., lateral) inflorescences (see Judd 1989). Heterotrichum DC. has not been cited above because it has serious delimitation and nomenclatural problems (Gleason 1925b): several species were transferred to Miconia sect. Octomeris by Wurdack (1972), who thought that the genera should be merged, while Judd and Skean (1991) and Liogier (2000) considered it within Clidemia. Miconia was described by Ruiz and Pavon in 1794, with three species. It was under Naudin (1851) that the genus acquired its present circumscription through the inclusion of several genera. Some of these were then used as the basis of named infrageneric taxa, such as Miconia sects. Jucunda, Cremanium, Chaenopleura, and Glossocentrum. Triana (1871) and Cogniaux (1888, 1891) ‘‘improved’’ on Naudin’s sectional classification (table 1), attempting to recognize what they perceived as related species groups. Cogniaux (1891) published what would be the last complete revision of the genus, including 518 species. From then on, Miconia has been studied only in regional floras, the most important being those from Mesoamerica (Almeda, forthcoming); Cuba (Liogier 1963); Jamaica (Proctor 1972); Hispaniola (Liogier 2000); the Lesser Antilles (Howard 1989); Trinidad and Tobago (Williams 1928); Venezuela (Wurdack 1973); the Guianas (Wurdack et al. 1993); Ecuador (Wurdack 1980); Peru (Macbride 1941); and the states of Parana´ (Goldenberg 2004), Santa Catarina (Wurdack 1962), and Saõ Paulo (Martins et al. 1996) in Brazil. These studies documented the ever-expanding melastome inventory for these geographic areas, but they made no serious attempts to provide a better circumscription of Miconia or its sections. The only notable exceptions to this floristic emphasis are the recent revisions of sect. Hypoxanthus (former Chaenanthera; Goldenberg 2000) and of the Antillean clade of Miconia sect. Chaenopleura (including a phylogenetic analysis; Judd 2007). Detailed systematic work has also been provided for some species groups within Jucunda, Tamonea, and Adenodesma (Gleason 1932a). We note that the related genera Tococa (Michelangeli 2005) and Charianthus (Penneys and Judd 2005) have also been recently monographed, and these studies have included phylogenetic analyses involving species of Miconia. Mecranium, which has also been revised (Skean 1993), was the first clade within the tribe to be the subject of a phylogenetic analysis. The sections proposed by Cogniaux (1891) were based primarily on stamen morphology, but he also used hypanthium and calyx morphology (as well as other reproductive characters; see table 2). These sections differ markedly in size, ranging from six to 248 species, and distinctions among some of them are frequently unclear. Nevertheless, much of the taxonomic confusion results from species that either were clearly misplaced by Cogniaux or are poorly known (and thus difficult to place). There are also species (or species groups) that have unexpected character combinations that preclude clear

GOLDENBERG ET AL.—PHYLOGENY OF MICONIA (MELASTOMATACEAE)

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Table 1 History of the Infrageneric Classification of Miconia de Candolle (1828) Miconia sections: 1. Leiosphaera 2. Eriosphaera 3. Miconia Other genera: Cremanium Diplochita Chaenopleura

a

Naudin (1851)

Triana (1871)

Cogniaux (1891)

Miconia subgenera: 1. Adenodesma 2. Diplochita 3. Jucunda 4. Laceraria 5. Miconia 5.1 Haplostachyae 5.2. Diplostachyae 5.3. Impetiolares 5.4. Glomeratiflorae 5.5. Stenostachyeae 5.6. Seriatiflorae 5.7. Paniculares 6. Amblyarrhena 7. Arrhenotoma 8. Cremanium 8.1. Pseudocremanium 8.2. Chiloporus 9. Hartigia 10. Chaenanthera 10.1. Euchaenanthera 10.2. Chaenopleura 10.3. Dichaena

Miconia sections: 1. Jucunda 2. Laceraria 3. Octomeris 4. Diplochita 5. Miconia 6. Glossocentrum 7. Hypoxanthus 8. Amblyarrhena 9. Cremanium 10. Chaenopleura

Miconia sections: 1. Jucunda 2. Tamonea 3. Adenodesma 4. Octomeris 5. Laceraria 6. Miconia 6.1. Aplostachyae 6.2. Diplostachyae 6.3. Impetiolares 6.4. Glomeratiflorae 6.5. Seriatiflorae 6.6. Paniculares 7. Glossocentrum 8. Chaenantheraa 9. Amblyarrhena (incl. Hartigia) 10. Cremanium 11. Chaenopleura

Other genus: Pterocladum

Synonymized under sect. Hypoxanthus by Goldenberg (2000).

assignment to any existing section. Finally, most sections have no designated types, except for sects. Chaenopleura and Tamonea, whose names came from genera that were described based on single species, and sect. Hypoxanthus, whose type has been designated only recently (Goldenberg 2000). For example, sect. Jucunda comes from the genus described by Chamisso (1834), with six species, all of them synonimized under three other species by Cogniaux (1891), of which only two were placed by him within sect. Jucunda; he considered the third in sect. Miconia. Section Miconia was established by de Candolle (1828) with 51 species, and these were placed in several different sections in the classification proposed by Cogniaux (1891). Given these taxonomic factors, it is not possible to use the type species as stakes to position the sections in the trees. Instead, we have adopted, for the purposes of this article, the sectional circumscriptions of Cogniaux (1891; as slightly modified by Goldenberg [2000]) because his monograph is the only current comprehensive taxonomic treatment of Miconieae. The most compelling rationale for attempting this study is that we now have modern techniques at our disposal to critically analyze difficult paraphyletic groups and make progress in identifying their component clades. While poorly defined, Miconia has an astonishing number of species, and these are often conspicuous members of the Neotropical flora. In the absence of modern taxonomic treatments, we lack a phylogenetic perspective not only of variation in traditionally employed morphological features (e.g., inflorescence architecture, hypanthium shape, or staminal features such as the form of the pores/slits or connective appendages) but also of more difficult-to-access features such as pollen, secondary chemis-

try, and chromosome numbers. As systematists, we are faced with some practical difficulties. How do we begin to determine (and diagnose) the most significant clades within an obviously nonmonophyletic Miconia? And how are we going to deal with this large slice of melastome—and also angiosperm—diversity? Because we cannot work on a ‘‘ground zero’’ basis, i.e., simply throw away the taxonomic information that is available, we present here the available phylogenetic data on Miconia and compare them with the existing infrageneric classification. In addition, various clades within Miconia will be compared to other genera in the tribe.

Material and Methods Taxon Sampling The appendix in the online edition of the International Journal of Plant Sciences contains all of the names, voucher information, and GenBank accession numbers for the 481 sequences of the internal transcribed spacer region of the nuclear ribosomal DNA (nrITS) and the 247 sequences of ndhF included in this study. The nrITS data set includes 21 species of Merianieae, one species of Eriocnema, and 459 accessions from the Miconieae. These 459 sequences correspond to 449 species because multiple infraspecific taxa were included for three species and five widely distributed and highly variable species of Miconia (Miconia calycina, M. dodecandra, M. minutiflora, M. mirabilis, and M. theaezans) are represented by multiple sequences. Additionally, another 22 species were sequenced for

Very short, obovate or cuneate, apex obtuse, pore ventrally prolonged into one or two slits, .4–3.2 mm

Very short, subcuneate, straight, apex rounded to retuse, two to four large pores, .3–2.7 mm

Very short, wide, obovate to oblong, straight to slightly arcuate, apex obtuse to retuse, one minute pore, .8–4.3 mm Very short, wide, obovate to oblong, straight to slightly arcuate, apex obtuse to retuse, one minute pore, 1.3–1.9 mm

Short, linear, straight, one or two rimose, .4–2.3 mm

Short, linear, straight, apex truncate, one or two large pores, .7–4 mm

Elongate to linear-subulate, not glandulose, one minute pore, .9–2 mm Short, linear, straight, apex attenuate, one or two minute pores, 1–5 mm

Elongate, linear-subulate, not glandulose, one minute pore, 1.1–10 mm

Elongate, linear-subulate, flexuose, glandulose, one minute pore, 2.6–8 mm

Elongate, linear-subulate, falcate, not glandulose, one minute pore, 2.7–9 mm Elongate, linear-subulate, falcate, not glandulose, one minute pore, 2–11 mm

Anthers (shape, glands, dehiscence, length)a

Suburceolate, 1.3–6 mm

Wide-campanulate, .7–4.5 mm

Campanulate, 1.3–2.2 mm

Campanulate, 1.3–6 mm

Campanulate to hemispheric, .9–2.4 mm

Campanulate to hemispheric, .8–4 mm

Campanulate to hemispheric, 1.2–4 mm

Campanulate, 1–2.3 mm

Campanulate, 2.5–8 mm

Oblong, 3.8–6.5 mm

Oblong, 2–7.5 mm

Oblong, 2–7 mma

Hypanthium (shape, length)

Open in bud, lobes short

Open in bud, lobes obsolete or short



Open in bud, lobes obsolete or triangular

Open in bud, lobes short

Closed in bud, opening through irregular lobes Open in bud, lobes obsolete or triangular

Open in bud, lobes irregular or triangular

Open in bud, lobes absent or short

Open in bud, lobes absent or denticulate

Open in bud, lobes triangular to lanceolate

Calyx (dehiscence, lobes)

Panicles scorpioid

Panicles regular

Connective lacking or only with a dorsal appendage

Connective lacking or only with a dorsal appendage

Connective with ventral appendages

Leaves sessile

Petals obtuse to retuse

Petals frequently subacute

Others

a

Note. The number of species appears in parentheses after each section name (following Goldenberg 2000). Measurements based on work by Wurdack (1962, 1973, 1980), Martins et al. (1996), Goldenberg (2000), Judd (2007), and Almeda (forthcoming). b Cogniaux (1891) recognized six subsections (see table 1) based on inflorescence and leaf morphology. The distinctions of these subsections are insignificant, and subsequent students of Miconia did not assign new species to any of the subsections. c Hartigia has been treated as a subsection of Amblyarrhena by Cogniaux (1891) but is discussed apart from it in this article.

Chaenopleura (Rich. ex DC.) Hook.f. (87)

Cremanium (D.Don) Hook.f. (209)

Hartigia Griseb. (8)c

Amblyarrhena (Naudin) Triana (206)

Hypoxanthus (Rich. Ex DC.) Hook.f. (17)

Glossocentrum (Crueger) Hook.f. (84)

Miconia DC. (248)b

Laceraria (Naudin) Triana (21)

Octomeris (Naudin) Benth. & Hook.f. (58)

Adenodesma (Naudin) Cogn. (6)

Tamonea Cogn. (71)

Jucunda (Cham.) Triana (24)

Section

Sectional Features as Defined by Cogniaux (1891)

Table 2

GOLDENBERG ET AL.—PHYLOGENY OF MICONIA (MELASTOMATACEAE) multiple accessions but are represented here by a single sequence each because there was no intraspecific variation in those cases. Within the Miconieae, the sampling spans 16 genera (only Catocoryne, Killipia, and Kirkbridea were not sampled). The four genera (Cyphostyla, Allomaieta, Alloneuron, and Wurdastom) assigned to tribe Cyphostyleae (Gleason 1929) and placed in Miconieae by Renner (1993) also were not sampled here. Miconia is represented by 218 sequences from 212 taxa and 208 species (;20% of the species of Miconia). All sections of Miconia were sampled (sectional placement following Goldenberg 2000): Adenodesma (3 spp. sampled), Amblyarrhena (24 spp.), Hypoxanthus (9 spp.), Chaenopleura (29 spp.), Cremanium (28 spp.), Miconia (52 spp.), Glossocentrum (22 spp.), Hartigia (4 spp.), Laceraria (4 spp.), Octomeris (11 spp.), Tamonea/Jucunda (19 spp.), and incertae sedis (3 spp.). The ndhF data set is a subset of the nuclear data set containing 13 species of the Merianieae and 234 species of the Miconieae (145 from Miconia; again, all sections were sampled). The tribe Merianieae has been chosen here as the outgroup because it has repeatedly been shown to be sister to the Miconieae sensu stricto (Clausing and Renner 2001a, 2001b; Renner et al. 2001; Fritsch et al. 2004; Michelangeli et al. 2004; Renner 2004) and including taxa from more distantly related tribes rendered the alignment of the nrITS data problematic. Preliminary analyses of more than 300 sequences of ndhF spanning the entire family confirmed the sister relationship of Merianieae and Miconieae. Additionally, Eriocnema, a capsular-fruited taxon from Brazil that has been traditionally placed in the Microliceae, was included in this analysis because it has been shown to be sister to the remaining members of the Miconieae (Fritsch et al. 2004; Martin et al. 2008). Most of the genomic DNA used in this study was isolated from silica-dried leaf tissue, but herbarium material was used for ;8% of the taxa. DNA extractions were performed using the Qiagen DNeasy plant mini kit, following the manufacturer’s protocol with the addition of 30 mL of Proteinase K (20 mg/mL) and 30 mL of b-mercaptoethanol to each sample along with the AP1 buffer. The homogenate was incubated at 42°C for 12 h, with slow rocking. Alternatively, the glass milk DNA extraction method, as outlined by Struwe et al. (1998), was employed. For some samples that were hard to amplify, the glass milk extraction method was used to clean DNA isolated with the DNeasy kit. The nrITS was amplified using specific primers, as detailed by Michelangeli et al. (2004). PCR was performed in a 25-mL volume with the following reaction components: 0.8 mL template DNA (;30 ng/mL), 2.5 mL 10X Ex Taq buffer (Takara Bio), 0.2 mM dNTP mixture, 6.5 mg BSA, 1 mmol of each primer, 2 units of Takara DNA polymerase (Takara Bio), and 5% (v/v) of DMSO. The PCR parameters were 94°C for 5 min and 35 cycles of 94°C for 10 s, 50°C for 45 s, and 72°C for 50 s, followed by 72°C for 10 min. The ndhF gene was amplified with primers developed by Olmstead and Sweere (1994). Following Clausing and Renner’s (2001b) study, we amplified the 39 end of the gene between positions 972 (i.e., codon 305 of solanaceous sequences) and 1955, using forward primer ndhF-972F, reverse primer ndhF1955R, and one or two pairs of internal primers (ndhF-1318F, ndhF-1318R, ndhF-1603F, and ndhF-1603R). Sequencing condi-

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tions and reagents were similar to those used for the internal transcribed spacer (ITS), but the annealing temperature was set to 48°C, and DMSO was not included. Alternatively, a similar PCR program was run with 10 initial cycles with an annealing temperature of 45°C, followed by 25 cycles with an annealing temperature of 48°C. Cycle sequencing reactions were performed with the same PCR primers, using ABI Prism Big Dye 3.1 terminator cycle sequencing kit (Perkin Elmer, Norwalk, CT), and were then analyzed with an Applied Biosystems 377XL automated DNA sequencer. Resulting sequences were edited using Sequencher 4.5 (GeneCode, Ann Arbor, MI).

Sequence Alignment and Phylogenetic Analyses The complete matrix was initially aligned using CLUSTAL X (Thompson et al. 1997) and then adjusted manually, especially at the edges of large insertions/deletions (indels). Data editing, manual alignment, tree viewing, and character optimization were performed in WinClada (Nixon 1999–2002). Indels were coded using the ‘‘simple gap coding’’ method of Simmons and Ochoterena (2000), as implemented in 2xRead (Little 2005). Three phylogenetic analyses were conducted using the parameters described below: the nuclear (ITS) and plastid (ndhF) data sets were each analyzed separately and in a combined analysis, using all nrITS and ndhF sequences. All maximum parsimony analyses were performed with TNT (Goloboff et al. 2001). One thousand random addition sequence replicates holding 20 trees per replicate were conducted. Each starting tree was first exhaustively swapped with subtree pruning and regrafting, then tree bisection reconnection (TBR), and was then subjected to a 200-iteration ratchet (Nixon 1999). The ratchet used a probability of 5 for both up-weighting and down-weighting; 10% of the informative characters were reweighed for each replicate (other parameters were set to default). After the random addition sequences were complete, trees were swapped to 750,000 trees using TBR. Strict consensus jackknife support values were calculated from 1000 replicates (Farris et al. 1996). Each replicate was performed with 20 random taxon entry sequences and swapped using the parameters described above. Branches were collapsed if supported ambiguously. The consensus tree was retained from each replicate.

Results The aligned nrITS data matrix is 1050 bp long, of which 387 sites are potentially informative. Simple gap coding yielded 321 indels, of which 183 are potentially informative. The aligned ndhF data matrix is 1000 bp long, with 154 potentially informative sites. Nineteen gaps were coded, and only four are potentially informative. All phylogenetic analyses were stopped once 750,000 most parsimonious trees (MPTs) were stored and memory capacity was reached. MPTs of the nrITS analysis have length ðLÞ ¼ 3171, consistency index ðCIÞ ¼ 0:27, and retention index ðRIÞ ¼ 0:75, and in the strict consensus, 256 nodes are collapsed (not shown). The ndhF analysis yielded MPTs of L ¼ 486, CI ¼ 0:47, and

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RI ¼ 0:79, and 181 nodes (for 247 taxa) are collapsed in the strict consensus (not shown). In both analyses, Miconieae (including Eriocnema) is resolved as monophyletic. In the ndhF analysis, there is very little resolution within Miconieae, but the resulting clades are not in conflict with those present in the strict consensus of the nrITS analysis. The combined analysis yielded MPTs of L ¼ 3818, CI ¼ 0:28, and RI ¼ 0:74, and in the strict consensus, 209 nodes are collapsed (figs. 1–3). Even though jackknife support values are not particularly high along the spine of the tree, they are considerably higher in the combined analysis than in the nrITS analysis alone for all clades. Because there is no hard conflict between the nuclear analyses and the plastid analyses or between these and the combined analysis and because the combined analysis both is the best resolved and has the highest support values, from here on, we will discuss only the results of the combined analysis. Species of Miconia are present in almost every clade within Miconieae(figs. 1–3). They are absent only from the Leandra þ Ossaea (scorpioid), Tococa, and Ossaea p.p. clades, which will not be discussed in detail here. On the other hand, they can be regarded as the major components of at least four clades (Miconia I-IV) and one grade (Miconia V). In the other parts of the cladogram, species of Miconia occur in small subclades or are scattered among species from other genera, as in the Caribbean þ Conostegia, Mecranium þ allies, and Leandra s.s. clades and the Clidemia grade. Approximately 85% (188 out of 220) of the terminals sampled here that belong to Miconia are in the five clades and grade (Miconia I–V), which also include all species from sects. Tamonea/Jucunda, Laceraria, Chaenopleura, Glossocentrum, Hypoxanthus, and Adenodesma; almost all species from sects. Cremanium and Amblyarrhena (excluding Hartigia); and most terminals from sect. Miconia. Thus, the only Miconia groups that are absent or unimportant in these clades are sect. Hartigia (formerly in sect. Amblyarrhena) and sect. Octomeris (including the species transferred from Heterotrichum). The Miconia I and II clades are the basalmost branches in the tribe, and both are composed exclusively of species of Miconia. Miconia I has three species from sect. Tamonea and also one from sect. Miconia. Miconia II has three out of the four sampled species from the small sect. Laceraria. The Caribbean þ Conostegia clade consists of a grade made up of mainly species and small clades from the Antilles, from the genera Miconia and Ossaea (with some of the latter often considered within Leandra), and all sampled species of the genera Calycogonium, Tetrazygia, Pachyanthus, and Charianthus. This clade also has a subclade with northern South American and Central American species, with all sampled species of Conostegia included, plus a few species of Leandra, Clidemia, Miconia, and Tococa. The species of Miconia in this large clade belong to sects. Cremanium and Octomeris, both at the base of the grade, and also a small clade with six Cuban and Puerto Rican species from sects. Octomeris, Amblyarrhena, and incertae sedis, along with two species from other genera. In the Conostegia subclade, there are four species of Miconia, from sects. Miconia and Octomeris, and one incertae sedis. The Mecranium þ Anaectocalyx þ allies clade contains a grade made up of primarily species and small clades from Miconia, Tococa, and Clidemia, plus all sampled species of Anaectocalyx and Mecranium. The species of Miconia belong to

sects. Amblyarrhena, Hartigia, and Octomeris. The species in this clade either are restricted to the Greater Antilles (Mecranium) or northwestern South America (Miconia pulvinata, M. spinulosa, and M. ulmarioides) or are widespread but with a strong Antillean and Central American presence (M. racemosa, M. ciliata, and M. lacera). Miconia III is made up by mainly Andean, Central American, and Antillean species of Miconia, from sects. Chaenopleura, Cremanium, and Amblyarrhena, plus single species from sects. Octomeris and Miconia and a few from Leandra and Clidemia. All species sampled from Chaenopleura are included in this clade, with all Greater Antillean species except for two (M. selleana and M. stenobotrys) plus one from Central America (M. grandidentata) in one subclade. The Andean and Central American species of sects. Chaenopleura, Cremanium, and Amblyarrhena are interdigitated and dispersed across the grade at the base of the clade, with at least four species of sect. Chaenopleura in a monophyletic subgroup (including the most recent common ancestor [MRCA]—of M. bullata and M. latifolia). Almost all species from sects. Cremanium and Amblyarrhena sampled in this study belong to this clade, with some exceptions in clades Miconia IV and Caribbean þ Conostegia for Cremanium and in several places for Amblyarrhena. The cladogram shows no resolution among species from these two sections or among the Andean Chaenopleura. Miconia IV has 69 terminals, with all but four (all from Clidemia) belonging to Miconia. These consist of species mainly from sects. Tamonea/Jucunda, Miconia, and Glossocentrum but also part of sect. Hypoxanthus, the extra-Andean South American species from sect. Cremanium, and one species each from sects. Laceraria and Octomeris. The distribution of the species based on traditional sectional assignments does not follow a clear pattern. For instance, the five extra-Andean members of sect. Cremanium are in three different subclades, while the three species of sect. Hypoxanthus are in two subclades. Nevertheless, species from sect. Tamonea/Jucunda always seem to cluster with species from sect. Miconia, and similarly, species of sect. Glossocentrum often are placed as close relatives of others in sect. Miconia. This group has a strong geographical presence in extra-Andean South America, with some widespread species reaching Central America and the Caribbean and one small lineage (M. striata and allies) from the Lesser Antilles and adjacent South America. The subclades including the MRCAs of M. rubiginosa/M. ligustroides and M. discolor/ M. cinerascens have mostly eastern Brazilian and Amazonian species, along with some widespread ones (i.e., M. chrysophylla, M. albicans, and M. stenostachya). The subclade including the MRCA of M. smaragdina and M. magdalenae contains the type of the genus, M. triplinervis, and is composed mostly of species from sect. Miconia from the Andes, Mesoamerica, or the Antilles; however, some species within this subclade are currently assigned to other sections or occur in extra-Andean South American regions. The Miconia V grade is composed of three clades. The first is composed of Clidemia heptamera and two species of Miconia, these from sects. Amblyarrhena and Miconia. The second is made up of species of mainly Leandra (mostly sect. Tschudya; see Martin et al. 2008) and M. ceramicarpa (sect. Miconia), mostly from the Guayana Shield. The large third clade has species of Miconia, mainly representatives of sects. Tamonea/

Fig. 1 Strict consensus tree resulting from the combined analyses of nrITS and ndhF sequence data. Numbers above the branches correspond to estimated jackknife support. Sectional assignments within Miconia are indicated by color (see legend at the bottom of the figure). All names in black belong to other genera. One asterisk indicates the type of the genus Miconia; two asterisks indicate the type of a section.

Fig. 2 Strict consensus tree (continued). 970

Fig. 3 Strict consensus tree (continued). 971

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Jucunda and Miconia, and also species of sects. Glossocentrum and Hypoxanthus, all three species sampled from sect. Adenodesma, and one eastern Brazilian species of sect. Amblyarrhena. The geographical distribution of the species follows the same pattern found in Miconia IV: all but one (M. ernstii, from Dominica) are from extra-Andean South America, plus several widespread species. The Clidemia grade has many isolated species and small clades composed of species of Clidemia, Leandra, Miconia, Leandra, Tococa, and Maieta, plus a large clade (including the MRCA of Sagraea capillaris and Clidemia septuplinervia) with several species of Clidemia, plus some from Ossaea, Sagraea, and the monospecific Necramium. All the eight species of Miconia in this grade belong to sect. Miconia, but these species are not placed together in the cladogram. Finally, the Leandra s.s. clade has only a single sampled species of Miconia (sect. Octomeris). The remaining species of this clade, which are from eastern Brazil, belong mainly to Leandra, but a few species of Clidemia, Ossaea, and Pleiochiton are also placed here.

Discussion The best way to evaluate morphological character evolution would be to produce a complete morphological character matrix parallel to the molecular matrix, and this is currently under way. Because of the large number of taxa in this study and the fact that most of the anatomical and seed morphology portion of this study is just beginning, at this point, we will be discussing only those morphological characters that have been traditionally used in delimiting genera within the Miconieae and sections within Miconia or characters currently known to support more inclusive clades. The sections of Miconia provide an efficient way to summarize morphological characters and can be used as a proxy for character distribution through the group (see table 2). This is especially true for the stamens, which have been extensively used to define these groups (Triana 1871; Cogniaux 1891; Goldenberg et al. 2003; Judd 2007). For instance, long, subulate, and minute-pored anthers occur in sects. Jucunda, Tamonea, Octomeris, and Adenodesma (fig. 4A, 4B, 4D), the latter also bearing glands on the connective. Shorter and linear anthers occur in sects. Miconia (still minute pored and with connective appendages ventrally projecting; fig. 4P, 4Q), Glossocentrum (broad pored and connective unappendaged or with a dorsal appendage; fig. 4G, 4H), and Hypoxanthus (broad, ventral pores resembling slits that expose two locules; fig. 4L–4O). Finally, short and cuneate or obovate anthers occur in sects. Amblyarrhena and Hartigia (with tiny apical pores; fig. 4C), Cremanium (with two to four broad pores; fig. 4E, 4F), and Chaenopleura (opening through slits that expose four locules; fig. 4I–4K).

The Sections of Miconia with Elongate Anthers Sections Jucunda, Tamonea, Adenodesma, and Octomeris have been regarded as basally branching groups within the genus or even the tribe as a whole (J. J. Wurdack, personal

notes; Judd and Skean 1991). This hypothesis was probably based on their relatively large flowers (table 2), somewhat resembling those in many genera outside Miconieae, and the absence of specialized features (mostly involving the stamens). In part, these authors were correct because Miconia I is composed mostly of species of sect. Tamonea, but it is difficult to explain why the other species sampled from the first three sections are positioned in Miconia IV and V. Nevertheless, these species are frequently positioned in basal grades or basally within each clade or subclade in our trees. The same pattern can be found, with some exceptions, in sect. Octomeris, in the Caribbean þ Conostegia and Mecranium þ Anaectocalyx þ allies clades. Wherever there is a species of sect. Tamonea/Jucunda at the base of a clade, there is not one from sect. Octomeris, and vice versa. Also, wherever there is a species of sect. Tamonea/ Jucunda at the base of a grade or clade, it is almost always related to species from sect. Miconia and rarely related to species in the predominantly West Indian/Andean clades with species from sects. Cremanium, Amblyarrhena, and Chaenopleura. The opposite is true for the basally placed species of sect. Octomeris, which are almost never related to species from sect. Miconia but are frequently related to species from Cremanium, Amblyarrhena, and Chaenopleura. If one considers the position of these species of sects. Tamonea/Jucunda and Octomeris, then it may be logical to assume that they could have retained an array of ancestral character states (for the tribe), and thus, the traditional circumscription of these sections may have been based on these symplesiomorphic characters. Judd and Skean (1991) stated that almost every specialized line in Miconieae has basal species that in some way resemble those in sect. Tamonea/Jucunda or Octomeris. The same authors also noted that the difficulties found in the delimitation of groups within Miconieae are due to the lack of morphological gaps, which could be related to rapid diversification from a basal complex, coupled with the lack of extinctions. The difficulty in defining the boundary between the Tamonea/ Jucunda complex and sect. Miconia was emphasized by Goldenberg (2000). These problematic boundaries could be the result of mistaken/careless sectional placements of species by previous authors. We suspect, however, that a more likely explanation is that the lack of a clear distinction between these three sections (as evidenced by the placement of their species on our cladograms) relates to multiple and parallel evolutionary shifts in the size and shape of flowers and stamens, probably correlated with pollination modes. This may have led to the independent and convergent origin of several lineages with smaller flowers and stamens (leading to the divergent placements of such species that we see in our trees). The small sect. Adenodesma may be monophyletic; it is supported by at least one distinctive morphological synapomorphy, glands on the stamen connective (see Gleason 1932a). It has been regarded as a part of the Tamonea/Jucunda complex (Gleason 1925a, 1932a), but in our cladogram, it is more closely related to species of sect. Miconia, within a grade that includes some species of the Tamonea/Jucunda complex. From a phylogenetic perspective, Miconia sect. Octomeris makes little sense, and the species traditionally assigned to this section are placed throughout several clades and are almost never paired with other members from the same section. Judd and Skean (1991) suggested the transfer of some of its species to Clidemia and others to early-divergent lineages within

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Fig. 4 Stamens of Miconia. A, Miconia tomentosa (Rich.) D.Don (Lugo 4144, CAS); B, M. tuberculata (Naudin) Triana (Aymard 1848, CAS); C, M. livida Triana (Wallnoffer 113–21782, US); D, M. jucunda (DC.) Triana (Tozzi 94–232, UEC); E, M. hymenanthera Triana (Asplund 16222, US); F, M. theaezans (Bonpl.) Cogn. (Leitaõ-Filho 11873, UEC); G, M. cinnamomifolia (DC.) Naudin (Goldenberg s.n., UEC); H, M. tetragona Cogn. (Va´squez 6864, US); I, M. hypiodes Urb & Ekman (Ekman 7552, US); J, M. bullata (Turcz.) Triana (Luteyn 6446, US); K, M. melanotricha (Triana) Gleason (Standley 51574, US); L, M. chrysophylla (Rich.) DC. (Plowman, 12294, US); M, M. sellowiana Naudin (Glaziou 8690, BR); N, M. sellowiana Naudin (Arau´jo 723, UEC); O, M. pusilliflora (DC.) Naudin (Goldenberg 37, UEC); P, M. tetrasperma Gleason (Va´squez 4747, US); Q, M. tiliaefolia (Hutchinson 8655, US). Assignment of the species follows Cogniaux’s (1891) sections: A, Adenodesma; B, Octomeris; C, Amblyarrhena; D, Jucunda; E, F, Cremanium; G, H, Glossocentrum; I–K: Chaenopleura; L–O, Hypoxanthus; P, Q, Miconia.

Tetrazygia. Within sect. Octomeris, several species resemble some members of sect. Amblyarrhena (i.e., those having campanulate hypanthia and stout, minute-pored anthers) but have larger flowers and anthers (table 2). Polyphyly explains the proximate positions of some species of sects. Octomeris and Amblyarrhena (and Hartigia) in several places in the cladogram. The small subclade including the MRCA of Miconia pachyphylla and Pachyanthus moaensis in the Caribbean clade is the only part of the cladogram where two species of Octomeris are placed together and in derived positions along with species from other sections. All the spe-

cies from this distinctive clade are from Cuba and Puerto Rico and have moderate-sized flowers, obovate to spathulate petals, strongly fused calyx lobes, few-seeded fruits, and—most notably—either basal or, less commonly, axile placentation with the placental tissue reduced (but nonreduced in Pachyanthus monocephalus; E. Becquer, personal observation). Most are shrubs from strongly seasonal areas and occur on limestone soils, as is also characteristic of many other species within the Caribbean þ Conostegia clade. Finally, the two eastern Brazilian species of sect. Octomeris are resolved in very distant clades: M. octopetala belongs to the Miconia

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IV clade, and its flowers look like larger versions of those of species of sect. Glossocentrum; it also has the hairy leaves and glomerulate inflorescences that apparently are shared among several species in the clade. Miconia plumosa has glandular trichomes and narrow petals with a rounded tip, and it is not surprising that it is resolved within the Leandra s.s. clade, which consists of mainly eastern Brazilian species of Leandra (Martin et al. 2008). The small sect. Laceraria was established for a group of species with a fused, irregularly rupturing calyx, a condition that is known for several other species of Miconia (Kriebel et al. 2005; Almeda, forthcoming) that have been variously assigned to sects. Amblyarrhena, Laceraria, and Miconia. Within the tribe, Conostegia also has a calyx that is closed in bud, but it ruptures cleanly along a well-defined circumscissile line and then falls away as a cap. The fact that three of the four species in the section form a monophyletic group shows that this character may be a useful synapomorphy, although its presence also in M. striata (of Miconia IV), M. friedmaniorum (of the Conostegia clade), and some species of Clidemia and Tococa not sampled here indicates that it has arisen more than once within the genus and tribe. A similar condition is exhibited by M. rubiginosa (of Miconia IV), which has a calyx tube and lobes that are circumscissile and collectively fall away as a ring on fruiting hypanthia. An irregularly rupturing calyptrate calyx is a putative synapomorphy of Mecranium (in the Mecranium þ Anaectocalyx þ allies clade). Although it is evident from the above discussion that the species of Miconieae with elongate anthers constitute an extremely polyphyletic assemblage, having representatives in widely divergent positions in our cladograms (see figs. 1–3), many of these species do form a clade (i.e., those occurring primarily in the Caribbean region). The Caribbean clade exhibits extensive morphological diversity; major subclades include the core Tetrazygia (including the MRCA of Tetrazygia bicolor and Tetrazygia cristalensis), Charianthus (Penneys and Judd 2005), the core Pachyanthus (including the MRCA of Pachyanthus cubensis and Pachyanthus tetramerus), and the basal placentationreduced placenta clade (including the MRCA of M. pachyphylla and P. moaensis; see Becquer-Granados et al. 2008). Finally, Conostegia, a well-defined genus whose species often have pleiostemonous flowers and lack external calyx lobes, with the inner lobes fused into a stout, conical calyptra (Schnell 1996), is also apparently related to these Caribbean taxa.

Miconia Sects. Amblyarrhena, Cremanium, and Chaenopleura and the Andean/Central American/Antillean clade All species from sect. Chaenopleura and the majority from sects. Cremanium and Amblyarrhena belong to a single clade (Miconia III). These three sections account for ;500 species, close to half of the described species of Miconia. Except in the Antillean Chaenopleura subclade, the relationships among the species in this clade are unclear. Nevertheless, this clade exhibits strong geographical cohesion. As mentioned, a distinct subclade is restricted to the Antilles, and the remaining species occur in the Andes or Central America, along with the widespread M. theaezans. The species of sect. Chaenopleura in the Greater Antilles represent a major radiation of the family in

this region (Michelangeli et al. 2008) and are resolved as a monophyletic group with an actinomorphic androecium of white stamens with erectly held, obovate anthers that open by two longitudinal slits exposing the contents of the four locules, pale blue berries, and seeds with a smooth testa (Judd 2007). The presence of a radially symmetrical androecium and usually white anthers is shared with the related species of sect. Cremanium. The phylogenetic proximity between Andean/Central American Chaenopleura and Cremanium already has been suggested by Gleason (1925a) and Judd and Skean (1991), but some species, such as M. nigricans (Peru) and M. melanotricha (Costa Rica and Panama), resemble certain Antillean ones in vegetative features. A close relationship between Andean species of sects. Chaenopleura and Cremanium was also supported by the preliminary cladistic analysis of Judd (2007), and in that analysis (based on ITS sequences), the Antillean species of sect. Chaenopleura form a clade nested within a paraphyletic sect. Cremanium. Miconia tetrastoma (an Antillean species of sect. Cremanium) is resolved here within the Antillean Chaenopleura clade but has been previously placed with other species from Cremanium (see Judd and Penneys 2004; Judd 2007). The phylogenetic position of M. desportesii and M. sphagnicola, which may be close relatives of M. tetrastoma, is in need of additional study (Judd and Penneys 2004). As for the rest of the clade, it seems that all species share the characteristic short, stout anthers, but the mode of dehiscence has evolved in at least two different ways: either the two to four broad-pored Cremanium type (sometimes ventrally inclined and resembling longitudinal slits in those species assigned to sect. Chaenopleura) or the single small-pored Amblyarrhena type, although intermediates occur. Broader character sampling among these groups could determine whether the species of Amblyarrhena and Cremanium belong to distinct clades and how many times broad-pored or smallpored anthers have evolved. Also, studies of pollination ecology may help us understand the selective pressures driving changes in anther morphology because these species probably lack nectar and offer only pollen as a reward. The way that pollen is collected from the anthers by visitors must be different because the size of the pores is different. Wind may also help to facilitate pollen removal from broad-pored anthers (F. Almeda, personal observation). Some species of sect. Cremanium that are distributed outside the Andes and Central America are not resolved in Miconia III. The eastern Brazilian species of sect. Cremanium were placed by Cogniaux (1891) in this section because their anthers have two broad pores. However, their anthers are not short and cuneate, as are those of their Andean counterparts, but slender and oblong, and they resemble those of sect. Glossocentrum. In fact, the number of pores, whether one or two, is sometimes difficult to assess because it depends solely on the persistence, thickness, and degree of protrusion of the septum between the two thecae (see Goldenberg et al. 2003). Also M. tetrandra, from the Antilles, has anthers that are more slender than those of core Cremanium, despite its large pores. In the same way, the only Brazilian species from sect. Amblyarrhena (M. ramboi) is placed in the Miconia V grade, along with species from the same region that belong to other sections. This species and other Brazilian Amblyarrhena (M. paradoxa and M. penduliflora; not sampled for this study) have anthers that do not look like their Andean counterparts;

GOLDENBERG ET AL.—PHYLOGENY OF MICONIA (MELASTOMATACEAE) instead they are similar to those of species of sect. Miconia but shorter and thicker.

Section Hartigia and Relatives in the Mecranium, Anaectocalyx, and Allies Clade The distinction of Hartigia as a section apart from Amblyarrhena seems to be correct because all its species belong to a single subclade within the Mecranium þ Anaectocalyx þ allies clade. The distribution of these species agrees with that of their counterparts in the clade: all come from Central America and northern South America, as well as the Antilles, and there is at least one widespread species (M. ciliata). The two morphological characters that define Hartigia occur elsewhere in the tribe but not together. The first one is the scorpioid inflorescence, which has apparently evolved in only two other clades within Miconieae (twice in Miconia IV and in the scorpioid Leandra þ Ossaea clade). This unusual inflorescence architecture may help to position reproductive structures in a way that optimizes pollination and fruit dispersal. In some species with scorpioid inflorescences, the flowers are somewhat congested at anthesis, but the inflorescence axes lengthen in fruit. Thus, flowers can be offered close to each other for pollinators, but the fruits are offered individually for the dispersers. The second character is the short, stout, and minute-pored anthers. Similar anthers apparently evolved at least twice in the Mecranium þ allies clade and perhaps more than once in Miconia III. A related, well-supported clade is represented by Mecranium, a group that is easily distinguished from the species of sect. Hartigia (as well as other species of Miconia) by its axillary inflorescences. Other putative synapomorphies include the calyx lobes fused into a domelike, apiculate cap that ruptures irregularly at anthesis, the reduced external calyx lobes, an androecial fringe, and perhaps the four-merous flowers (Skean 1993). Tococa broadwayi and Tococa perclara form a clade; these species, which do not produce formicaria, are not placed in the Tococa clade, in agreement with the morphological cladistic analysis of Michelangeli (2000). Anaectocalyx is from South America and has conspicuously bracted, six-merous flowers with a calyx composed of prominent, individually caducous lobes and anthers, with the thecae and part of the connective bilobed and prolonged ventrally below the insertion of the filament.

Miconia Sects. Miconia, Glossocentrum, and Hypoxanthus and the South American Lowlands Clades Species from sect. Miconia are restricted only to the large clade that comprises all species from Miconia IV to Leandra s.s., except for a few species placed in Miconia I and Miconia III and in the Conostegia subclade. Nevertheless, they are absent in some large portions of this clade, such as in the Leandra s.s. clade and in a large part of the Clidemia grade. This suggests that most of the species of sect. Miconia have no close relationship to the species in core Cremanium, Amblyarrhena, or Chaenopleura. The species of this section also appear not to be intimately related to the core Leandra or the large Caribbean clade. On the other hand, there are not just one or two clades where the species of sect. Miconia are prevalent, as is the case for sects. Adenodesma, Laceraria, Cremanium, Amblyarrhena, Chaenopleura,

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and Glossocentrum. The species of sect. Miconia are always intermixed with species of sects. Tamonea/Jucunda, Glossocentrum, and Hypoxanthus and the few species of eastern Brazilian Cremanium, Octomeris, and Amblyarrhena. They are also frequently mixed with species of Clidemia, Leandra, and even Conostegia. Cogniaux (1891) divided sect. Miconia into six subsections based largely on inflorescence morphology. An attempt to plot these subsections onto the cladogram (not depicted) showed that they do not deserve further consideration. The Ossaea p.p. clade (¼Ossaea sect. Octopleura) is placed as the sister group of Miconia IV (which includes many species of sect. Miconia), and both groups contain many species with ribbed hypanthia and dorsobasal anther appendages (these are sometimes gland edged). These features could be synapomorphic, and this clade is discussed in more detail by Judd and Skean (1991). The species of sect. Glossocentrum are placed in three different regions of the cladogram: M. rubiginosa and M. discolor subclades in Miconia IV and the large subclade including the MRCA of M. furfuracea and M. rigidiuscula in Miconia V. The species with glabrous to glabrescent leaves and regular panicles (i.e., nonglomerulate and nonscorpioid inflorescences) are restricted to the M. rubiginosa and M. furfuracea subclades, and they have remarkably similar inflorescences and vegetative characters. On the contrary, all the species in the M. discolor subclade have hairy abaxial leaf surfaces and glomerulate or scorpioid inflorescences. The species sampled from sect. Hypoxanthus come out in the same three subclades cited for Glossocentrum (figs. 2, 3). In these three lineages, the species from both sections are always placed together. Goldenberg (2000) found that sect. Hypoxanthus has two distinctive groups: one bearing stamens with long anthers on short to absent basal connective prolongations and the other with very short anthers on long connectives. All of the species with long anthers on short connectives are in the Miconia V grade (M. furfuracea subclade), while the species with short anthers on long connectives are in both subclades in the Miconia IV clade. Thus, these two groups are nonmonophyletic. Finally, species from both sections that have anthers opening through slits (sects. Hypoxanthus and Chaenopleura) are not directly related. According to Cogniaux (1891), they differ only in the shape of the anthers (linear in the former and obovate to cuneate in the latter), but if we exclude the Andean species from Chaenopleura, the species in this section have anthers that expose four locules, while species from Hypoxanthus expose only two (Goldenberg et al. 2003; Judd 2007). Problems regarding the diffuse sectional limits between sects. Miconia and Glossocentrum, between Glossocentrum and Hypoxanthus, and between Glossocentrum and Cremanium have already been noted (Goldenberg 2000; Goldenberg et al. 2003) and certainly point to convergent evolution of staminal morphology.

Stamens and Pollination The conventional delimitation of sections in Miconia has been based largely on stamen morphology. When one looks at the androecium characters in some sections (fig. 4), it seems that there are strong parallel trends in several clades, leading from large anthers in Tamonea/Jucunda to shorter anthers in sects. Miconia and Glossocentrum (Gleason 1925a) and also

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from minute-pored anthers in sects. Tamonea and Miconia to large-pored Glossocentrum-type anthers and finally the large and long-pored (resembling slits) anthers in sect. Hypoxanthus and Antillean sect. Chaenopleura. These trends can perhaps be explained by a slight shift in the pollination mode: the large and minute-pored flowers are usually visited, one by one, by large- to medium-sized buzzing bees, and visits from small bees or other insects are unlikely to result in pollination because they either cannot extract the pollen or cannot touch the stigma while extracting the pollen (Renner 1989). Smaller flowers in dense clusters can be visited by the same large- to medium-sized buzzing bees that vibrate whole inflorescence branches or flower clusters and also individually by smaller bees that would not be able to pollinate large flowers because of their size. Finally, the pollen from broad-pored anthers can be released without vibration (Goldenberg and Shepherd 1998; Goldenberg and Varassin 2001). Nonbuzzing bees and syrphid flies have been observed feeding or gathering pollen from M. pusilliflora (sect. Hypoxanthus; R. Goldenberg, personal observation), while wasps and muscid flies have been observed visiting the nectar-producing M. hyemalis (eastern Brazilian sect. Cremanium; I. G. Varassin, personal communication) and M. pepericarpa (sect. Glossocentrum; Goldenberg and Shepherd 1998), respectively. These species, which are visited by nonvibrating insects, are also visited by buzzing bees, leading us to conclude that the shortening of the anthers and the broadening of the pores has not restricted but actually increased pollinator diversity, which in turn may represent a trend toward a generalization among the relations between plants and pollinators (Gomez and Zamora 2006). In some groups, the enlargement of the dehiscence area of the anthers, coupled with nectar production, has led to either bat (M. sintenisii and perhaps some other Antillean sect. Chaenopleura species; Judd 2007) or bird pollination (M. melanotricha [F. Almeda, personal observation] and Charianthus and Tetrazygia fadyenii [Penneys and Judd 2003, 2005]). These characters related to pollination ecology are under strong selection pressure and therefore are more subject to convergence (Anderson et al. 2002), as seen, for example, in the remarkably similar, bird-pollinated flowers of T. fadyenii and the species of Charianthus (Penneys and Judd 2003, 2005). It seems clear that stamen morphology, at least when used alone, is not a reliable character on which to base taxonomic groups because similar anthers seem to have evolved multiple times in the Miconieae. Other floral features linked to pollinator syndromes (such as petal form and color) are also suspect. It is evident from even a casual examination of figures 1–3 that there has been extensive homoplasy in several floral characters. It is possible that other morphological characters will clarify the circumscription of particular clades because morphology has been shown to be a source of numerous phylogenetically informative characters in several recent analyses of taxa within the tribe (Skean 1993; Michelangeli 2000; Penneys and Judd 2005; Judd 2007; Martin et al. 2008). Characters related to seeds are especially promising because they are highly variable within the tribe (Groenendijk et al. 1996) and have been used successfully as phylogenetic characters in Melastomataceae (Whiffin and Tomb 1972; Fritsch et al. 2004) and even as synapomorphies within groups of Miconieae (Michelangeli 2000; Martin et al. 2008; E. Becquer, personal communication).

Geographical Trends and Local Diversification This study shows that the lineages within the Miconieae are strongly correlated with geography. Some taxa that until now have been regarded as widespread are in fact restricted to particular regions, with geographically distant members actually belonging to other clades. This is readily apparent in the case of sect. Cremanium, with a clearly Andean/Central American– based diversity (Miconia III clade) and whose extra-Andean species actually belong to other groups. A similar pattern was found for sect. Chaenopleura, where the Antillean species are not closely related to the Andean ones, the latter being placed within a Cremanium/Amblyarrhena complex. Outside Miconia, the genus Leandra has a clade based in eastern Brazil (Leandra s.s.) and others in other parts of Central America or northern South America (scorpioid Leandra þ Ossaea, Leandra clidemioides subclade at the base of the Clidemia grade; see Martin et al. 2008). A large array of Caribbean species traditionally placed in Pachyanthus, Calycogonium, Tetrazygia, or Charianthus, and even with a few species placed in Clidemia or Miconia, was found to constitute a clade—here called the Caribbean clade—which again highlights the close connection between phylogeny and geography. Much of the diversity of Miconia (and Miconieae) found in some geographical areas reflects only local diversification and not multiple arrivals or introductions of distantly related groups. The best example of this pattern is the diversity of Miconieae within the Caribbean. The Antillean species are derived mostly from only three clades: the large Caribbean clade, Miconia sect. Chaenopleura in Miconia III, and a portion of the Mecranium þ allies clade. However, a fourth clade may also be significant in terms of Antillean diversity within Miconieae, i.e., the axillary-flowered members of Clidemia and Ossaea in the Clidemia grade (i.e., species treated as Sagraea; Judd and Skean 1991). Another good example comes from clades Miconia IV and V, both encompassing most of the morphological and species diversity of Miconia in eastern Brazil, regardless of their current sectional placement. These two clades consist of species that have been placed in at least eight sections (Tamonea, Jucunda, Octomeris, Miconia, Cremanium, Amblyarrhena, Glossocentrum, and Hypoxanthus). Speciation in these evolutionary lines has apparently involved extensive morphological differentiation and convergence. This has led students of the family to assign some species to geographically distant groups to which they really do not belong. Moreover, almost all the diversity of the Miconieae (including species placed in Miconia, Leandra, Clidemia, Pleiochiton, and Ossaea) that is centered in eastern Brazil belongs to only three clades (the ones cited above and the Leandra s.s. clade). All eastern Brazilian species belonging to other clades are either widespread (i.e., M. dodecandra, M. theaezans, and Clidemia hirta) or part of widespread species complexes (i.e., from the scorpioid Leandra þ Ossaea clade). Major taxonomic realignments, such as changes in the circumscriptions of the sections or genera in Miconieae, are premature. They must await a more detailed and highly resolved phylogeny that includes well-defined morphological characters. Nevertheless, the traditionally recognized genera within Miconieae, which have been diagnosed largely on the basis of very few characters (drawn from highly homoplasious floral features) and are largely

GOLDENBERG ET AL.—PHYLOGENY OF MICONIA (MELASTOMATACEAE) widespread (with a few exceptions, mostly in the Caribbean), will be replaced by the geographically cohesive clades discovered in our phylogenetic analyses. Work in progress will allow the more accurate delimitation and morphological characterization of these clades and will then form the basis of a new phylogenetically based classification of Miconieae.

Acknowledgments We thank the following individuals for providing plant material: J. Dan Skean Jr., Claire Martin, Eldis Becquer, Andrea K. dos Santos, Andre´ Amorim, Ludovic Kollmann, Karen Redden, Mac Alford, Larry Kelly, Robbin Moran, Scott Mori, Maria E.

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Morales, John Clark, Susanne Renner, and Ken Wurdack. This work was funded in part by the National Science Foundation (grants DEB-0515665 to F. A. Michelangeli and R. Goldenberg and DEB-0515636 to W. S. Judd). Additional funding came from the Lewis B. and Dorothy Cullman Program for Molecular Systematics Studies at the New York Botanical Garden. R. Goldenberg received grants from the Conselho Nacional de Pesquisas e Desenvolvimento (Brazil; Po´s-Doutorado no Exterior and Bolsa-Produtividade). We thank Dan Skean Jr., Eldis Becquer, Claire Martin, and Isabela Varassin for their insight on phylogeny, characters, and pollination of Miconieae and Paulo Labiak and Vale´ria Muschner for helpful comments on the manuscript.

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