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PHYLOGENETICS OF PLEUROTHALLIDINAE:
resolving generic limits in a large group of small orchids



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PROJECT DESCRIPTION

Results from Prior NSF Support

1. DEB-0234064 to NH Williams and WM Whitten, Systematics of Maxillariinae (Orchidaceae): Generic delimitation, pollinator rewards, and pollination. 2003-2005. $300,000. Field work in the first two years was conducted with 3 graduate students in Panama, Costa Rica, and Ecuador, plus attendance at international orchid congresses in Costa Rica and Ecuador. Over 500 accessions representing over 300 species have been photographed, vouchered, and sequenced for ITS, matK, and the atpB-rbcL intergenic spacer with 90% taxon coverage for Central American taxa and Brazil. ITS data are yielding a well-resolved phylogeny that is being supplemented with plastid data. Keys are being constructed using Lucid software. Field work in 2005 is planned for Colombia, Ecuador, and possibly Peru. Publications: Williams & Whitten 2003; Ojeda et al. 2003.

2. DEB-9815821 to NH Williams, Molecular and morphological systematics of Oncidiinae (Orchidaceae). 1999-2003. $25,000. Sequencing within Oncidiinae was increased to 634 species representing about 90 generic concepts for ITS and plastid regions (trnL-F, matK, atpB-rbcL intergenic space) were sequenced for a subset of 190 taxa. Well supported cladograms have allowed numerous clarifications and changes to the taxonomy of the group and have revealed that floral traits are highly homoplasious in deceit-pollinated clades and have misled earlier workers with their classification systems. Publications: Williams et al. 2001; Sosa et al. 2001; Chase & Williams 2001; Chase et al. 2005; Williams & Whitten 2001; Williams, Chase, & Whitten 2001; Dressler & Williams 2000; Koehler et al. 2002.

3. DBI-0138615 to W.E. Higgins and B.K. Holst, Improving and Computerizing the Spirit-Preserved Collection of Orchid Flowers at Selby Gardens. 2002-2003 $22,626. Funds were provided to restore the Spirit Collection of the Marie Selby Botanical Gardens by replacing vials, lids, liners, and chemicals, by storing bottles in plastic trays, and by identifying type specimens. Notices of the restoration and availability of the Spirit Collection have been sent to the biological research community via professional newsletters and the Selby Vignette webpage.

4. DBI-0447401 to B.K. Holst and W.E. Higgins. Improvement and Relocation of Selby Gardens Preserved Collections. 2005-2008. $76,356. This grant will help defray moving expenses and will provide a curatorial assistant to pack/unpack specimens, curate collections. This move of the SEL Herbarium to the main campus of the Gardens provides an opportunity for much-needed collections improvement and expansion.

I. Introduction - The Neotropical orchid subtribe Pleurothallidinae contains over 4,000 species, which represents 16% of Orchidaceae. This hyper-diverse group is distributed from Florida to Argentina with interesting disjunctions in Brazil and the Lesser Antilles, but is most diverse in Colombia, Ecuador, Peru, and Bolivia (Jorgensen and Len-Ynez 1999, Dodson 2004, Vsquez and Ibisch 2000).

Pleurothallids exhibit a broad array of growth habits (epiphytic, terrestrial, rheophyte, lithophyte) and occupy many habitats (e.g., nearly all Neotropical forests, paramo, mangroves, deserts). A typical pleurothallid is an epiphyte with a restricted distribution, frequently endemic, that lives in sympatry with other pleurothallids in extremely moist forests in the Andes (1800-2800 m) and is pollinated by flies (Valencia et al. 2000, Ibisch et al. 2003, Christensen 1994, Blanco and Barboza 2005, Borba and Semir 2001, Borba et al. 2001a, 2001b, 2002). Figures 1 and 2 illustrate the vegetative and floral diversity in Pleurothallidinae. Several pleurothallid species are pioneers and tend to appear and dominate major disturbance landscapes (e.g., road constructions, grounds covered by volcanic ashes after eruptions, coffee and cacao plantations).

Based on molecular data, pleurothallids form a monophyletic group that is firmly positioned as a member of the Epidendreae (van den Berg et al. 2005). Despite the great variation in size (although many pleurothallids are quite small, see Fig 1.) and form, pleurothallids are easily identified by an articulation between the ovary and the pedicel. Another distinctive feature of pleurothallids is a stem (ramicaul) bearing one leaf that always lacks pseudobulbs (Luer 1986).

Figures 1 and 2. This opens a WORD file with the figures.

Pleurothallid genera have been traditionally circumscribed on the basis of morphological characters, e.g., number of pollinia (8,6,4, or2), presence or absence of an active lip, presence of a funnel-like lepanthiform sheath that surrounds the stem (known as the ramicaul by many pleurothallid workers), the degree to which the column protrudes beyond the lip, among other features. Even though the classification solely based on morphological characters has been of great utility, experts of the group as well as users of the classification have recognized several problems at generic and infrageneric levels, especially in the genus Pleurothallis s.l., the largest genus of the subtribe (Luer 2002, Dressler 1993). Pridgeon et al. (2001) presented the first molecular evidence for a reclassification of the Pleurothallidinae, which was followed by many premature taxonomic changes (Pridgeon and Chase 2001) that created a great deal of controversy (Luer 2002, 2004).

The nomenclatural instability of the group has greatly affected two activities. 1) Horticulturally, pleurothallid orchids have a high commercial value and are regulated by the Convention on International Trade in Endangered Species (CITES). The implementation of CITES is very difficult, as many stakeholders (e.g., customs personnel, commercial growers, USDA inspectors) cannot agree on which classification system to use. 2) Moreover, conservation efforts (e.g., the creation of Red Lists of Ecuador and Peru) not only have become a taxonomic nightmare, but they are also delaying research and obstructing fund raising for researchers and NGOs interested in population and habitat protection. It is the aim of this collaboration to produce a more stable and predictive classification for this group of orchids that form the core of Neotropical orchid diversity.

Current status of Pleurothallidinae taxonomy - The lifetime work of Carl Luer M.D., widely regarded as the expert on this subtribe (he has described over 2,000 species and 30 genera), has been published in 26 volumes of the Icones Pleurothallidinarum, which are part of the series of Monographs in Systematic Botany of the Missouri Botanical Garden. During the last 30 years, Luer has prepared alpha-taxonomic treatments of the entire subtribe, with the exception of the genera Octomeria, Stelis, and many Brazilian species. Luer (1985) stated that the taxonomy of Pleurothallidinae was fraught with problems and unnatural groups have been recognized as unambiguous synapomorphies have eluded researchers. At that time Luer believed that Pleurothallidinae would contain over 4000 species in 30 genera.

These numbers are amazingly close to the Chase et al. (2003) classification (3999 species in 32 genera). The current classification by Luer (2004 and in prep.) recognizes 4100+ species in 60 genera. The classification system of Chase et al. only recognizes 32 genera including two that were moved out of Laeliinae. When the classification schemes of the subtribe are compared, we find that the differences are more substantial than just splitting and lumping (Pridgeon and Chase 2001; Luer 2002). Taking into account other treatments (see Table 1), an additional 20 monotypic genera have been described.

A new classification of Orchidaceae based on both molecular and morphological evidence is highly desirable (Chase et al. 2003). Currently the two major conflicting classifications for Pleurothallidinae, Pridgeon and Chase (2001, 32 genera), Luer (ongoing studies, 60 genera) result in 89 generic names in current use, but with different circumscriptions for the genera. A classification based on total evidence and including morphology will produce clades with specific morphological characteristics. Thus it will be more user-friendly and bring order to the artificial megagenus Pleurothallis (with over 1,500 species) and a subtribe that has confounded taxonomists since the time of Lindley (1799-1865).

Previous Research - Preliminary morphological (Neyland et al. 1995) and molecular (Pridgeon et al. 2001) studies of the subtribe have been conducted and these studies demonstrated the need for additional phylogenetic research. These independent studies differed in the source of characters and the resulting phylogeny. Neyland et al. (1995) performed a cladistic analysis of subtribe Pleurothallidinae based on 45 anatomical/morphological characters. Their ingroup comprised 24 genera (34 species); the large genus Pleurothallis consisted of two subgenera and ten species complexes. Their hypothesis that subtribe Pleurothallidinae has undergone a unilinear reduction in the number of pollinia was not supported by their study. Their cladistic analysis suggests that Pleurothallis is not a natural genus and may be divided into several discrete genera.

Pridgeon et al. (2001) evaluated the monophyly of subtribe Pleurothallidinae and the component genera based on molecular data. They sequenced the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) and 5.8S gene for 185 taxa. In addition, to improve the overall assessments along the spine of the topology, plastid sequences from matK, the trnL intron, and the trnL-F intergenic spacer were added for a representative subset of those taxa in the ITS study. The sequence data from all three data sets were combined in a separate analysis of 58 representative species (28 genera). There was support in most analyses for the monophyly of Pleurothallidinae and in some support for inclusion of Dilomilis and Neocogniauxia (formerly in subtribe Laeliinae). Although most genera in the nine clades identified in their analyses were monophyletic, all data sets revealed the polyphyly of Pleurothallis and its constituent subgenera as presently understood (Fig 3 A,B).

Both studies concluded that Pleurothallis sensu Luer is not monophyletic. These studies represent preliminary phylogenetic research of the subtribe and the low sampling (28 genera and 58 taxa) may not reflect the evolutionary history of the group. Relationships in Trichosalpinx remain unresolved, and extensive DNA sampling will be required before taxonomic conclusions can be made with confidence (Pridgeon and Chase 2001).

The DNA-only phylogeny of Pridgeon et al.(2001) sampled only 185 (4.6%) out of 4000 species for one gene region (ITS) and 58 taxa (1.4%) for the 3-gene analysis. The low sampling of the ITS study resulted in a largely unsupported/unresolved phylogeny. Recent molecular research (Pridgeon et al. 2001) indicates that several genera, as presently defined, are paraphyletic. The cladogram in Figure 3 shows a polyphyletic Pleurothallis. Many of their results support many genera proposed by Luer's morphology-based taxonomy. On the other hand, some of their results deviate so greatly from it that they invite skepticism (Jost and Endara 2004, and Fig. 3B).

Generic limits have been changed with little regard to use and current taxonomic treatments offer little hope of stability unless the revisions are based upon a well-sampled molecular/morphological phylogenetic hypothesis. Preliminary molecular data (Pridgeon et al. 2001) confirm the suspected paraphyly of some genera in Pleurothallidinae. Neotropical orchid floras must confront such taxonomically difficult genera in their treatments, but such treatments are hampered by conflicting, arbitrary, and unsupported non-monophyletic generic concepts proposed by various taxonomists. Previous work has demonstrated that combined molecular/morphological data sets can yield highly supported cladograms that can serve as the basis for stable, objective classifications (Higgins 2000, Albert 1994).

Objectives - The goals of this project are to bring the various taxonomic approaches into a unified analysis to produce 1) a stable nomenclature at the generic level; 2) to produce a user-friendly classification scheme (generic and subgeneric/sectional) that is presented via an interactive polyclave key for genera of Pleurothallidinae, that will allow scientists, commercial breeders, control authorities for CITES, USDA plant inspectors, and hobbyists to identify species to genus and section in this group of orchids. The expected outcome of this project is a classification system based on total evidence in a rapidly evolving group of organisms that will further our understanding of the evolutionary history of morphological traits and to stimulate research on their ecology and biogeography.

Relations to work in progress - The PIs have ongoing research programs related to this proposal. PI (Higgins) has worked on total evidence analyses in Encyclia (in press) and the phylogeny of Laeliinae (van den Berg et al. 2005). PIs Williams and Whitten have worked on the systematics of Oncidiinae, Maxillariinae, Zygopetalinae, and Laeliinae. This proposal relates to longer-term goals of the PI's ongoing research projects. This project is a key component of the Selby Gardens Orchid Identification Center's long-term goals of studying the systematics of Orchidaceae to support conservation efforts worldwide and the molecular systematic program at UF to produce a stable classification of Orchidaceae. PIs Williams and Whitten have a pending collaborative proposal for the Assembling the Tree of Life program at NSF for a large study of the family Orchidaceae to try to resolve tribal and subtribal level relationships in the family. This current proposal complements that proposal and if both are funded will result in both a better tribal level understanding of the family and a generic level understanding of subtribe Pleurothallidinae.

Figure 3. This opens a WORD file with the figures. The cladograms are best viewed by increasing the size to 150% or so.

A. Preliminary Pleurothallidinae phylogeny based on molecular data (Pridgeon et al. 2001). B. The discordant elements of Pleurothallis in its traditional sense are shown in red. Segregate genera shown in other colors.

II. Plan for sampling and data collection - PI Higgins at SEL will supervise the morphological portion of the project and conduct the morphological analysis. He will combine the DNA and morphological matrices for the total evidence analysis and will run the total evidence analyses. He will train the botanist hired to build the morphological matrix in MacClade and construct the Lucid3 interactive key. He will also supervise the four internships from Neotropical institutions. PIs Williams and Whitten at UF will supervise the DNA portion of the project and conduct the DNA analysis. The graduate student and collaborator Pridgeon will extract DNA, sequence 350 species per year, and build the DNA matrix.

High quality DNA cannot be reliably extracted from herbarium specimens (except for recently collected material Pridgeon & Whitten, pers. obs.) so fresh material is required of most species. Because of intense horticultural interest in the group, a large portion of the species are available in cultivation, both by members of the Pleurothallid Alliance (see letters of collaboration) and in several botanical gardens in the USA. Additional specimens will be acquired from several commercial firms in both the USA and foreign countries (see letters). We will do field collecting in Panama, Costa Rica, Ecuador, and Bolivia to obtain critical species (see letters and Budget Justification). Active research/collecting permits are in hand for these countries and permits in other countries are pending; we anticipate no problems because of our existing collaborations in those countries.

Choice of Taxa and Data - Taxa: We have planned a careful sampling strategy based on previous phylogenetic work within the subtribe and on the concepts of Luer (collaborator, see letters). The largest phylogenetic studies of orchids published thus far have sampled fewer than 150 genera for two or three loci (Cameron in press; Freudenstein et al. 2004). Previous and ongoing work on Oncidiinae, Maxillariinae, Zygopetalinae, Stanhopeinae by Williams and Whitten involve comparable sample sizes and have been achieved in a similar time frame. The goals of our project are reachable and the data generated by it will contribute enormously to the current knowledge of orchid phylogeny.

TABLE 1. Generic names in current use; number of species/number species to sample. Authority names omitted to save space. Acianthera 220/22; Acostaea 9/5; Anathallis 118/12; Ancipitia 27/10; Andinia 12/6; Andreettaea 1/1; Antilla 12/6; Apoda-prorepentia 7/4; Apatostelis 33/10; Areldia 1/1; Atopoglossum 3/3; Barbosella 39/12; Barbrodria 1/1; Brachionidium 72/15; Chamelophyton 1/1; Colombiana 6/3; Condylago 1/1; Crocodeilanthe 66/30; Cryptophoranthus 46/20; Cucumeria 1/1; Didactylus 4/2; Dilomilis 8/2; Diodonopsis 5/2; Draconanthes 2/1; Dracontia 17/6; Dracula 145/20; Dresslerella 11/6; Dryadella 48/12; Echinosepala 8/4; Elongatia 10/5; Empusella 1/1; Epibator 3/2; Frondaria 1/1; Gyalanthos 2/2; Jostia 1/1; Kraenzlinella 17/5; Lepanthes 1133/200; Lepanthopsis 54/20; Lindleyalis 7/3; Lueranthos 1/1; Luerella 1/1; Madisonia 1/1; Masdevallia 898/120; Masdevalliantha 2/2; Mirandopsis 1/1; Mixis 1/1; Myoxanthus 60/12; Mystacorchis 1/1; Neocogniauxia 2/1; Octomeria 201/20; Ophidion 4/2; Pabstiella 6/3; Phloeophila 20/4; Physosiphon 30/10; Physothallis 3/2; Platystele 110/20; Pleurothallis 2083/250; Pleurothallopsis 1/1; Porroglossum 35/15; Proctoria 1/1; Pseudolepanthes 10/5; Restrepia 107/25; Restrepiella 1/1; Restrepiopsis 21/6; Rhynchopera 10/5; Rodrigoa 9/5; Rubellia 1/1; Salpistele 6/4; Sarracenella 2/2; Scaphosepalum 67/35; Specklinia 140/50; Stelis 1051/116; Talpinaria 1/1; Teagueia 11/8; Tomzanonia 1/1; Tribulago 1/1; Trichosalpinx 157/35; Trisetella 25/12; Tubella 72/15; Unciferia 10/5; Xenosia 2/2; Zootrophion 20/15; Zosterophyllanthos 53/20; Total to sample=1398

We plan to sample approximately 1400 species and will use the results of previous generic-level studies, admittedly imperfect, (see Luer citations in references) to the extent that they exist to guide our selection of species in large genera. We plan to sample all of the monotypic genera. For small genera, we plan to sample a representative group of species (including the type species whenever possible) to test for monophyly. For medium-sized genera we plan to sample approximately 30% of the species, again to test for monophyly. For large genera, such as Lepanthes, Stelis, Pleurothallis, Masdevallia, we plan to sample all of the currently recognized subgeneric taxa for a minimum of two species per subgeneric taxon. Table 1 shows the currently recognized genera with number of species and number of species we propose to sample. Specimens will also be selected to represent the diversity of the geographic range of Pleurothallidinae. Beyond this, sampling will concentrate on species that have been transferred among multiple genera and be guided by the recommendations of collaborator Luer who has described many of the species.

Two questions deserve answers: 1) Why sample so many species? 2) Can we meaningfully analyze such large data sets? Taxon sampling is critical to producing correctly resolved phylogenetic trees (Zwickle and Hillis 2002; Rokas and Carroll 2005). Maximization of the number of taxa sampled is the strategy favored by most phylogeneticists, although its necessity remains the subject of debate. The effects on phylogenetic accuracy, resolution, and clade support of adding taxa and/or characters have been explored by Graybeal (1998), who found that when the total number of characters is held constant, accuracy is much higher if the characters are distributed across a larger number of taxa. Similar conclusions, namely that denser species sampling greatly improves the ability of an analysis to reconstruct phylogeny, have been reached by others (Lecointre et al. 1993; Lecointre et al. 1994; Hillis 1996; Purvis and Quickie 1997). Rokas and Carroll (2005) report that that increasing taxon number correlates with a slight decrease in phylogenetic accuracy, although their empirical study utilized very small taxon samplings (76 yeasts, 42 mammals, and 16 angiosperms). Mitchell et al. (2002) found that adding taxa actually decreased resolution and support by inadvertently introducing more long nodes, and that probably even more taxon sampling is required to achieve resolution ( but their study compared 49 vs. 77-taxon data sets for noctuid moths, a family with over 45,000 species). Soltis and Soltis (2004) showed that even 61 plastid gene sequences fail to place Amborella correctly unless taxon sampling is sufficiently dense.

The primary reason for dense taxon sampling of pleurothallids is that preliminary studies (Pridgeon et al. 2001) show that some currently recognized genera are non-monophyletic; the conflicting current classifications are an insufficient guide of which exemplar taxa to sample. We feel that dense taxon sampling of Pleurothallidinae is required not only to provide phylogenetic resolution, but also to evaluate the distribution of potentially homoplastic morphological characters mapped onto a relatively complete molecular tree, to reveal biogeographic patterns of cladogenesis, and to produce a stable classification.

Can we meaningfully analyze such large data sets? Salamin et al. (2005) examined the performance of different methods of phylogenetic analysis for large angiosperm data sets and concluded that the distribution of branch lengths rather than the rate of evolution was found to be the most important factor for accurately inferring these large trees. A tree containing 13,000 taxa was created to represent a hypothetical tree of all angiosperm genera and the efficiency of phylogenetic reconstructions was tested with simulated matrices containing an increasing number of nucleotides up to a maximum of 30,000. Even with such a large tree, their simulations suggested that simple heuristic searches were able to infer up to 80% of the nodes correctly.

Morphological data - Morphological characters, in particular those of the flower and more specifically those of the anther, have been the foundation for orchid classification for centuries. Characters such as anther orientation, pollinium number and orientation, and types of pollinium stalks have been used to define taxa from subfamily to genus (Dressler 1993). Other floral characters used to distinguish genera include number of stigma lobes, sepal connation, resupination, and similarity of perianth parts (Luer 1986). Luer also placed great weight on the evolution of lip mobility and segregated species having any one of the various mechanisms in which this trait has independently evolved (Luer 1986, 1987, 2000). Recent molecular studies of the orchids have demonstrated the homoplasious nature of these characters (e.g., Freudenstein and Chase 2003), which is not surprising since they are intimately involved in pollination mechanisms. This level of homoplasy is not a problem per se, since it is now clear that highly homoplasious data sets can still be highly structured (Kllersj et al. 1999). Given the small size of the morphological data sets constructed thus far, it has not been possible to obtain significant resolution within the subtribe based solely on morphological data.

We will conduct a morphological analysis of the same species that are used for molecular data collection using species as terminals (Kron and Judd 1997). Previous cladistic analyses of the family scored relatively few characters, which is typical for morphological data sets in general. For example, Burns-Balogh and Funk (1986) scored 70 characters for 37 terminals within the family, whereas Freudenstein and Rasmussen (1999) used 71 characters for 98 generic level exemplars from across the family. Given that we are sampling a much larger number of taxa in a more narrowly defined group than any previous morphological study of orchids, we propose an increase in the number of characters for the analysis. This is in part because the high sampling density will make characters that previously would have been autapomorphies (and were therefore excluded in the sparsely sampled data set) now relevant as potential synapomorphies. Although the total number of morphological characters will probably be relatively small compared to the molecular data, they can still play an important role in affecting and describing the topology (e.g., Baker et al. 1998). Careful morphological analysis will allow us to diagnose the clades recovered and characterize them in a user-friendly key, which cannot be done using molecular data alone.

Our morphological data set will include both floral and vegetative characters and most of the characters traditionally emphasized in orchid classification (e.g., Kurzweil 1987, 1988; Freudenstein and Rasmussen 1996, 1997; Freudenstein et al. 2002). See Table 2 for a starting list of 95 characters.

We will focus on features that can be scored from herbarium specimens, living cultivated plants, and spirit-preserved material already in collections, and supplemented by targeted field collecting. The main collections for morphological character scoring are the living collections at the University of Florida, Marie Selby Botanical Gardens, Lankester Botanical Gardens, Costa Rica, and the horticultural collections of collaborators; and the herbarium collections at Missouri Botanical Gardens (MO), University of Florida (FLAS), and Marie Selby Botanical Gardens (SEL). We wish to emphasize our own examination of specimens to assure consistent interpretations of characters.

Pridgeon (1982) conducted an anatomical survey of 200 species in 22 genera of subtribe Pleurothallidinae and indicated which vegetative characters were of diagnostic value and at what taxonomic level. The established subtribal phylogenetic trends of reduction in number of pollinia and specialization of the perianth may be correlated with particular morphological trends, which are either reductionary or involve specializations directly related to the water relations of the epiphytic habit. The morphological analysis of Freudenstein and Rasmussen (1999) showed little resolution at lower taxonomic levels. Morphological data alone are not sufficient because of the relatively low number of characters compared to the large number of taxa to be studied (Chase et al. 2003).

Morphological Laboratory Methods: This project will expand on the previous work of Neyland et al. (1995) and Pridgeon (1982). The morphological matrix will be constructed using MacClade and analyzed using maximum parsimony in PAUP* and TNT. Confidence in tree topology will be estimated with bootstrap, jackknife and decay indices.

The morphological data collected will be used to produce an Internet based key using commercial software. Lucid3 is software for construction of polyclave, interactive web-based identification keys. Lucid3 http://www.Lucid3central.org/ represents a major innovation in the delivery of taxonomic information.

Table 2. Morphological characters (95) to use in Pleurothallidinae. Attribute; state 0, state 1, state 2, state 3, state 4.

plant characters:

plant habit: repent, cespitose;
plant height (excluding inflorescence): less than 10 cm, more than 10 cm;
stem habit: superposed, distinct;
rhizome: ascending, prostrate, pendent;

stem characters:

stem: abbreviate, conspicuous;
stem form: not cane-like, cane-like;
stem internodes: 1, 2+;
annulus: absent, present;
cauline sheaths1: laterally compressed, tubular;
cauline sheaths2: not speckled, speckled;
stem sheaths1: enclosed imbricating sheaths, sheath near middle, several at base;
stem sheaths2: not lepanthiform, lepanthiform;
stem sheaths3: not overlapping, overlapping;
stem sheaths4: glabrous, scurfy/pubescent;
stem length1: shorter than leaves, longer than leaves, equal;
stem length2: shorter than rhizome, longer than rhizome, equal;
sheath surface: glabrous, pubescent;

leaf characters:

leaf: not sheathing, sheathing;
leaf base: not cordate, cordate;
leaf surface: glabrous, ciliate/pubescent;
leaf1: sessile, attenuate into a petiole;
leaf2: membranous, coriaceous;
leaf length: less than 3 mm, 5+ mm;
cuticle surface: smooth, papillose;
epidermal papillae: absent, present; embedded inflorescence peduncle: absent, present;
glandular trichomes: absent, 1 surface, both surfaces;
stomatal apparatus: flush with epidermis, raised above epidermis;

inflorescence characters:

spathaceous bract1: not foliaceous, foliaceous, inconspicuous;
spathaceous bract2: not enclosing flowers, enclosing flowers;
floral bracts: not spiculate, spiculate;
flowering: successive, simultaneous, solitary flower;
flower stalk: pedicellate, sessile;
inflorescence emergence point: leaf apex, middle of leaf, terminal on stem, lateral on stem, rhizome;
inflorescence: racemose, paniculate, fasciculate, solitary;
resupination: resupinate, nonresupinate, variable;
perianth shape: parts similar, parts dissimilar;
pedicel surface: not verrucose, verrucose;
peduncle: laterally compressed, terete;
peduncle length: shorter than leaf, longer than leaf;

sepal characters:

sepal veins: 1-veined, 2-3 veined;
sepals1: not verrucose, verrucose;
sepals2: not spiculate, spiculate;
sepals3: not caudate, caudate;
sepals4: not echinate, echinate;
sepal substance: fleshy, membranous;
sepal-sepal fusion1: apices fused, apices free;
sepal-sepal fusion2: basally connate, variously connate below apex, coherent, free;
lateral sepal fusion to dorsal: not fused, fused forming trilobed fan-like calyx;
lateral sepals1: not carinate, transversely carinate at base;
lateral sepals2: without transverse callus, with transverse callus;
lateral synsepal shape: different from dorsal sepal, similar to dorsal sepal;
synsepal: absent, present;
sepal apex: without callus pad, with callus pad;
sepal interior: pubescent, glabrous;

petal characters:

petal apex: not thickened, thickened;
petal margins: entire, fimbriate to lacerate;
petal shape: transversely bilobed, linear;
petal length: subequal to sepals, distinctly smaller than sepals;
petal substance: membranous, fleshy-thickened;
petals1: not transverse, transverse;
petals2: not auriculate at base, auriculate at base;
petals3: no callus along labellar margin, callus on labellar margin;
petals4: not angled, angled;

labellum characters:

labellum attachment: not clawed, clawed;
labellum base1: appressed to column foot, adnate to column foot, free;
labellum base2: arm-like calli, other calli;
glenion: absent, present;
labellum carinate: no, yes;
labellum divided into epichile/hypochile: no , yes;
labellum hinge1: simple to column foot, articulated to bulbous apex of column foot, not hinged;
labellum hinge2: under tension, loose; labellum shape: entire, lobed;
labellum: radiating lamellae, hair-like appendages;
labellum-column relation: not embracing column, embracing column;

ovary, column, anther, stigma characters:

ovary: not ornamented, ornamented;
ovary-pedicel: not articulate, articulate;
column1: arching, straight;
column2: not membranous, membranous;
column3: not winged, winged;
column foot1: absent, present;
column foot2: pedestal-like, not pedestal-like;
column foot3: curved, straight;
column foot4: laterally compressed, not laterally compressed;
column foot5: not bulbous, bulbous;
column apex: not hooded, hooded;
clinandrium: toothed, not toothed;
anther position: apical, incumbent;
pollinia number: 8, 6, 4, 2;
pollinia shape: spherical, ovoid;
pollinia size: unequal, equal;
stigma1: apical, ventral;
stigma2: entire, bilobed;
stigma opening: not hooded, hooded;

Molecular data: We chose loci (Table 3) to provide a range of variability to maximize resolution and support for clades at all hierarchic levels, as well as to represent the perspectives of both nuclear and plastid genomes. Sequences of ITS, trnL-F, matK+trnK, rpL16 intron, atpB-rbcL intergenic spacer will be analyzed with maximum parsimony and support will be estimated with bootstrap, jackknife, and decay indices.

DNA isolation and PCR: The total DNA CTAB extraction method of Doyle and Doyle (1987) will be employed using fresh or recently dried (or silica gel preserved) leaf or floral material. Target sequences will be amplified from genomic DNA using Taq polymerase and cleaned according to established protocols.

Sequencing: Sequences will be generated from purified PCR template using the PE Biosystems reagents and run on ABI 3130 capillary sequencers at UF. Sequence contigs will be assembled and examined against their electropherograms using Sequencher (Gene Codes, Ann Arbor, MI) to check accuracy and correct any obviously incorrect base-calls. We now routinely obtain sequence reads of 700-800 bases per reaction.

Table 3. Proposed regions to sequence and relevant data on each region.FORMATTING IN PROGRESS ITS 1 & 2 +5.8S trnL-F matK +trnK atpB-rbcL intergenic spacer* rpL16 intron # taxa sampled to date for region 300 200 100 400 25 # included positions in matrix 759 1346 1917 2031 1300 # variable sites 389 495 678 678 201 # phylogenetically informative sites 293 (39%) 228 (17%) 327 (17%) 327 (16%) 32 (2.5%)** *data from orchid subtribe Maxillariinae, but should be comparable for Pleurothallidinae **this should increase as we increase taxon sampling

Molecular Laboratory Methods:

Molecular Data Analysis - Sequence alignment:
Alignment will be straightforward for trnK + matK, a protein- coding region with few indels. The spacer regions atpB-rbcL, trnL-F, and ITS region presents the greatest challenge for alignment because of the high levels of variability, which is the same reason that they are useful at lower taxonomic levels. Our experience with these regions in a large number of subtribes, including Pleurothallidinae, make us confident we can align these regions by eye for the subtribe Pleurothallidinae.

We will use indels as well as base substitutions as phylogenetic characters. Gaps will be coded using simple indel coding as implemented in Gapcoder (Young and Healey 2003). Some have argued that indels are more reliable characters than base changes (Lloyd and Calder 1991). Simmons et al. (2001) supported the use of indels in phylogenetic studies, but they did not conclude that they were superior to base substitutions. Freudenstein and Chase (2001) found them to be a particularly important component of the nad1b-c dataset.

Phylogenetic analysis: We will analyze each data set individually, combined by genome, and combined in a total molecular analysis. Conducting individual and various combined analyses will allow us to determine whether there is conflict at the level of locus or genome. Incongruence between nuclear (ITS) and plastid data sets are rarely reported in orchids (van der Niet et al. 2005, Shipunov et al. 2005) and usually from subfamily Orchidoideae where allopolyploidy is common. Previous phylogenetic analyses of Orchidaceae utilizing sequences from the three genomes have not revealed notable incongruence (e.g., Cameron in press; Freudenstein and Chase 2001; Goldman et al. 2001). However, we will examine the data sets for incongruence using the ILD test (Farris et al. 1994) in order to have a more objective, quantitative approach in addition to our manual comparisons. Although this procedure, which is implemented in TNT and PAUP*, has been criticized as not being an accurate indicator of congruence (Yoder et al. 2001, Darlu and Lecointre 2002, Dowton and Austin 2002), it remains the best available tool for exploration. If we find incongruence, we will attempt to localize it to particular taxa by performing an Adams consensus (Adams 1972) on trees from analyses for each gene locus done separately, since that procedure allows individually conflicting terminals to drop to the node of conflict without losing the remaining structure in the tree. This will allow us to identify and reexamine problematic taxa to determine if lab errors could be responsible for data disagreement.

Parsimony analysis under equal weights as implemented in TNT (Goloboff et al. 2000) and PAUP* will be used as the primary tree search strategy for all analyses because this approach can handle large data sets in a reasonable time frame and because of its accommodation of heterogeneity among base positions relative to model-based approaches (Kolaczkowski and Thornton 2004). Heuristic searches will be performed using strategies to explore the data as completely as possible to search for islands of most parsimonious trees (Maddison 1991), given that the problem of local optima can be particularly troublesome for very large data sets (Goloboff 1999). In particular, TNT has implemented a number of innovative strategies such as the Parsimony Ratchet (Nixon, 1999), sectorial searches, tree-drifting, and tree-fusing (Goloboff 1999) that improve the thoroughness of tree search. Individual data sets will be combined directly (Miyamoto 1985; Cracraft and Mindell 1989; Kluge 1989; Kluge and Wolf 1993; Nixon and Carpenter 1996), as opposed to consensus combination (Penny et al. 1982; Bull et al. 1993; Jones et al. 1993), because it allows full character interaction, yielding, to the extent of the heuristic analysis, a maximally parsimonious solution. Given that each data set will undoubtedly have homoplasy, this can be important in revealing "underlying signal" among the data sets. This is done with the knowledge that independent genomes could be functioning essentially as single characters (Doyle 1992; de Queiroz 1993) and that they may be reflecting different patterns due to incongruent transmission and fixation. Nonetheless, the combined analysis will represent our best phylogenetic estimate.

Because of the size of the datasets we will be analyzing, model-based approaches such as Maximum Likelihood (ML) and Bayesian analysis are computationally more problematic for use as primary tree search methods. However, we will employ these approaches in particular cases in which their properties might be especially useful. These include situations where long branches are to be expected and where the parsimony analyses indicate significant rate heterogeneity among branches. In these cases, smaller clades with potentially problematic taxa will be submitted for model-based analyses. Maximum Likelihood analysis will be performed using PAUP*. Modeltest (Posada and Crandall 1998) will be used to select the evolutionary model that best fits the data on starting trees obtained from the parsimony analyses (typically GTR + I + G), with parameter values estimated from the data. Bayesian implementation of likelihood in MrBayes (version 3.1; Ronquist and Huelsenbeck 2003) using the Metropolis Coupled Monte Carlo Markov Chain approach will also be used to reconstruct trees under the likelihood model.

Assessing tree robustness and support: Jackknife analysis (Farris et al. 1996) will be used to assess tree support for parsimony analysis of individual datasets and all combined analyses. A minimum of 5000 replicates will be performed, each of which will include a small amount of branch swapping in each jackknife replicate, as it can significantly improve results (Freudenstein et al. 2004). As stated above, Bayesian analysis also provides an indication of support in its posterior probability values. Sensitivity analysis will be used to explore the datasets for robustness to alignment variation.

The possibility that two data sets may have different phylogenetic histories has become an argument against combining data in phylogenetic analysis. However, two data sets sampled for a large number of taxa may differ in only part of their histories. This is a situation in which the relative advantages of combined, separate, and consensus analysis become much less clear. Wiens (1998) proposed a simple methodology for dealing with this situation that involves performing separate analyses of the data sets and combining the data but considering unresolved those parts of the combined tree that are strongly contested in the separate analyses until a majority of unlinked data sets support one resolution over another. Computer simulations suggest that the accuracy of combined analysis for recovering the true phylogeny may exceed that of either of two separately analyzed data sets, particularly when the mismatch between phylogenetic histories is small and the estimates of the underlying histories are imperfect (high homoplasy). Combined analysis may provide a poor estimate of the species tree in areas of the phylogenies with different histories but gives an improved estimate in regions that share the same history. Thus, when there is a localized mismatch between the histories of two data sets, the separate, consensus, and combined analyses may all give unsatisfactory results in certain parts of the phylogeny. Similarly, approaches that allow data combination only after a global test of heterogeneity will suffer from the potential failings of either separate or combined analysis, depending on the outcome of the test.

Total evidence has the merit of producing the least assumption-burdened estimate of genealogy, and it maximally explains and describes all the available character evidence (Eernisse and Kluge 1993). During phylogenetic reconstruction, the influence of different evolutionary processes needs to be considered (Donoghue and Sanderson 1998). Unique topologies are sometimes found when combining matrices. A combined analysis increases the number of characters, which is known to increase the support (Bremer et al. 1999; Kron and Judd 1993). Eernisse and Kluge (1993) adopted the position that data sets need to be analyzed both separately and combined simultaneously to potentially increase the descriptive efficiency and explanatory power of the data. Combined analyses of morphological and molecular data in Orchidaceae, such as examining the cladistic relationships of the slipper orchids (Cypripedioideae), have been successful (Albert 1994).

Translating trees into a classification, and deciding among alternative possible classifications. Entwisle and Weston (2005) suggested criteria for deciding among alternative taxonomies for large, complex taxa, such as Pleurothallidinae. These criteria include: 1) Where possible, named taxa should be monophyletic based on current reliable evidence. That is, we should not knowingly name or accept paraphyletic or polyphyletic taxa unless as an interim step while we gather and assess more knowledge for a 'radical' change, or where there is a high probability of further change in the short term (e.g., 10 years). 2) Minimize taxonomic change to minimize nomenclatural changes. The primary reason to minimize nomeclatural changes in pleurothallids is that they constitute a significant fraction of neotropical floras; unnecessary name changes cause difficulties in using existing floras, checklists, and biodiversity databases. 3) Avoid epithets already in use in possible congeners. Rejected criteria include: 1) that genera should always be easy to recognize morphologically; and 2) that size matters (avoid monotypic or large genera; Scotland and Sanderson 2004). Within pleurothallids, a subgeneric classification scheme can be created that will convey phylogenetic information yet will minimize nomenclatural disruption.

Dissemination of results and archiving of data products: Results will be disseminated in a number of ways. The phylogenetic tree will be posted on the UF website. Because of the large number of taxa and nodes, we have chosen to employ special software to make viewing the tree and recovering information from it as easy as possible. Inxight VizServer software (already purchased at UF) allows users to navigate easily through the tree using a hyperbolic tree. By clicking and dragging on nodes, users can stretch portions of the tree and follow a path of their own choosing through the tree. (See demo at http://www.flmnh.ufl.edu/orchidatol/). Clicking on terminals and nodes brings up pages of information on those groups. These pages will include descriptions of the taxa, images, literature references, links to GenBank accession numbers for individual DNA sequences, and links to other relevant orchid websites.

The data will be shared with other scientists. Total DNAs will be archived at UF for use by other qualified scientists (in compliance with the Convention on Biological Diversity). Listings of all Pleurothallidinae genera and species will be posted on the SEL webpage reflecting their taxonomic status. Results will be published in peer-reviewed scientific journals. All DNA sequences and aligned DNA matrices will be deposited in GenBank and accession numbers will be provided with the appropriate taxon on our website, which will be maintained at the University of Florida. All morphological character observations will be derived from voucher specimens, which will be deposited in public herbaria (FLAS, SEL, MO, QCA, USJ, UEC, VASQ as appropriate) and documented on our websites. The phylogenetic tree will be deposited in TreeBase(http://www.treebase.org/treebase/) and on the Tree of Life project (http://tolweb.org/tree/phylogeny.html) website. The morphological data matrix will be input into the Lucid3 (http://www.Lucid3central.com) software package (University of Queensland) to create polyclave keys to the genera (and subgenera and sections of the larger genera) of Pleurothallidinae with illustrated character states posted on the web; this Delta-compatible key to genera can be easily revised as additional taxa or characters are added. A member of our group (UF) has already assembled such a key for the orchid genus Maxillaria for Central America. This research complements the Genera Orchidacearum project at Royal Botanic Gardens, Kew, and All Species Foundation (http://www.all-species.org) effort to identify all living organisms.

III. Education, Training, and Outreach - Undergraduate and Postgraduate Opportunities - Some of the most important components of this proposal are the educational opportunities available for parataxonomists and graduate students. Funding is requested to provide stipends for interns at MSBG as well as for one graduate student position at UF. One graduate student (Lorena Endara) has a specific dissertation topic and a potential new student will formulate a topic later. These students will be trained broadly in the area of systematics with aspects of their research being integrated into this project.

Promoting teaching, training, and learning: Training of parataxonomists at Selby Gardens (internships), graduate students at UF, and undergraduate research projects at both will be integral parts of the project. This approach provides both an invaluable structure and learning aid to detailed knowledge of plant diversity and allows teaching of theoretical concepts of phylogenetic systematics throughout the project. The proposed activity will broaden the participation of underrepresented groups by combining Latin Americans, parataxonomists, women, developing nations, and botanical gardens with university researchers on an integrated molecular/morphological systematics project. The value of a graduate student practicum at MSBG is the integration of theoretical principals with practical application of systematic botany and the additional resources available at a botanical garden.

The project will enhance the infrastructure for research at MSBG with the addition of Lucid3 software and an additional computer. The joint project will enhance the research collaboration and communications among the institutions involved in this research. The opportunity for Latin American students to study in the US will enhance their educational experiences.

Broader impacts: Because orchids are such a popular and increasingly economically important group of plants, and in view of their diversity and myriad specializations, orchids are a natural group with which to capture the interest of the public and to convey the importance of systematics, evolution, biodiversity, and especially conservation. This project is designed to capitalize on this opportunity by providing outreach at various levels, including institutional (museums, universities, and botanical gardens) and most broadly through web-based tools. The project will provide for training of undergraduates, graduate students, and parataxonomist fellows, and will foster collaboration among scientists, horticulturists, and the enthusiastic hobbyist community. It will increase collaboration among a worldwide network of orchid systematists, including professionals from throughout Latin America. Men and women students from Brazil, Colombia, Ecuador, Bolivia, and Costa Rica will broaden the representation of scientists from Central / South America and bring gender, ethnic and geographical diversity to this collaborative project.

Herbaria in Latin America and the Caribbean hold over 18 million specimens, a large number but still less than one per km2 of land. Also, there is less than one botanist per 10,000 km2 in Latin America and the Caribbean. Undetermined Pleurothallidinae specimens at SEL will be identified and annotated as part of the project and parataxonomists will be trained. Botanists from developing nations will be trained through internships at MSBG. Intern curriculum will include techniques for alpha-taxonomy, interactive key construction, and matrix construction and analysis.

Research integration: The project integrates research and education by including various students under the supervision of the PIs. The project involves undergraduate research projects, 4-year support for a graduate student, and 4 internships. The internships will be practical hands-on projects based on morphological characters focusing on a monophyletic, manageable clade, e.g., Teagueia. Results will involve an oral presentation, a written report, and a web deployable Lucid key. Graduate student support allows academic training in botany and molecular systematics and provides the resources necessary to fund graduate research advancing discovery and understanding while at the same time contributing to the success of this project.

Potential benefits to society at large: With the increasing need to inventory the planet's flora and fauna and changes over time, a stable taxonomy and nomenclature is critical to floristic inventorying and databasing. It is the aim of this collaboration to produce a more stable and predictive classification for this group of orchids that form the core of Neotropical orchid diversity and to resolve problems created by nomenclatural instability. Horticulturally, pleurothallid orchids have a high commercial value and are regulated by the Convention on International Trade in Endangered Species (CITES). The implementation of CITES is very difficult, as many stakeholders (e.g., customs personnel, commercial growers, USDA inspectors) cannot agree on which classification system to use. Moreover, conservation efforts (e.g., the creation of Red Lists of Ecuador and Peru) not only have become a taxonomic nightmare, but they are also delaying research and obstructing fund raising for researchers and NGOs interested in population and habitat protection. This project will resolve many of those problems.

Workshops, Symposia, Presentations: The results of the project will be broadly disseminated through scientific articles, popular articles, web pages, and an interactive key to enhance scientific understanding of the evolution of the subtribe and the technological methods of total evidence approach to phylogenetic reconstruction. Our goal is to reach out to the general public and to disseminate the information resulting from this project to professional plant biologists. To this end we will present results regularly at national and international scientific and horticultural meetings at all stages of the project. These meetings will include BSA meetings each year and the 19th World Orchid Conference(WOC) in Miami (2008). The WOC attracts several thousand commercial orchid growers, scientists, hobbyists, and interested lay people from around the world with lectures, plant sales, and exhibits. The PIs will attend this meeting and organize a workshop on the progress of the project. Phylogenetic trees, databases, and web sites will be demonstrated, and the principles of molecular systematics, cladistics, and phylogenetic classification will be presented. The PIs have found that this audience of well-educated enthusiasts is eager to adopt new ideas if they can be explained without the use of jargon and presented in a non-patronizing manner.

Tasks for each team member and time scheduling:

Year1-2006

Year 2-2007

Year 3-2008

Year 4-2009

Williams

Exchange DNAs; begin seq. & database

Matrix construction; analysis; imaging

Matrix construction; analysis; imaging;

Coordinate website and publications for Botany 2010 symposium;GenBank submissions; coordinate publications for Monocots IV symposium.

Whitten

receive loans; verify voucher identifications

Sequencing & databasing

Sequencing & databasing

Sequencing & databasing; cooridinate website

Pridgeon

Sequencing

Sequencing and matrix construction

Sequencing and matrix construction

Publishing; outreach materials, manuscript writing

UF graduate student

Phylogeny based project

Phylogeny based project; Sequencing, morphological analyses; databasing

Phylogeny based project; Sequencing, morphological analyses; databasing

Phylogeny based project; Sequencing, morphological analyses; databasing

UF undergraduate student

Phylogeny based project

Phylogeny based project

Phylogeny based project

Phylogeny based project

Higgins

Morphological char. selection/refinement; receive specimens; verify vouchers; train biologist and intern

Morphological data coll.; verif vouchers; work with interns

Morphological data coll.; verif vouchers; work with interns

Combined DNA+morphological analyses; publish outreach materials

MSBG Biologist

Start morphological table; fieldwork Colombia, Ecuador, Peru

Morphological data coll.; fieldwork Brazil and Bolivia

Morphological data coll. and analyses; fieldwrok Costa Rica

Construct and post Lucid3 key; develop educational materials

Holst

Process vouchers

Sequencing & databasing

Sequencing & databasing

Sequencing & databasing

UF Bioinformatics Tech (1/2 time)

Construct database

Database implementation & refining

Database implementation & refining

Database implementation and refining; database finalization; web hyperbolic tree finalization



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Page last updated: 7 September 2005