Phylogenetic Analysis of () using the Plastid Sequence ycf1

A senior thesis submitted to the Department of Biology, The Colorado College

By: Nina Kiyomi Sheade

Bachelor’s of Arts Degree in Biology May 2012

Date: May 21st, 2012

Approved by:

Primary Thesis Advisor: Dr. Mark Wilson

Secondary Thesis Advisor: Dr. Tass Kelso Table of Contents

Abstract……………………………………………………………………………………3

Introduction………………………………………………………………………………..4

Materials and Methods…………………………………………………………………...12

Results……………………………………………………………………………………20

Discussion………………………………………………………………………………..26

Acknowledgements………………………………………………………………………29

Literature Cited…………………………………………………………………………..30

2 Abstract

The appropriate taxonomic organization of Pleurothallis (subtribe Pleurothallidinae, family Orchidaceae) and its subgenera has been debated for more than a century. Recent phylogenetic studies have suggested that subgenera previously elevated to the level of genus based on morphological studies are most appropriately considered subgenera of Pleurothallis. This study analyzed the hypothetical open reading frame ycf1, a plastid gene, in order first to determine its utility in phylogenetic study at the generic and infrageneric level, and second to test support for the existing models or suggest a new model for taxonomic organization of Pleurothallis. A 1,200 bp 5’ region and a 1,500 bp 3’ region of the ycf1 gene were sequenced for representative species of each of the morphologically-based groups within Pleurothallis sensu lato and phylogenetic trees were generated for each region using maximum-parsimony analysis. The tree generated from the 5’ region exhibited minimal topological structure, suggesting either that the species sequenced are too closely related to be segregated into unique genera or the 5’ region of the gene did not contain enough parsimony informative sites to be useful at this level of study. The 3’ ycf1 gene tree had considerably more topology but the small number of species sequenced limits the conclusions that can be drawn from the phylogenetic tree.

3 Introduction

Phylogenetics has emerged in recent decades as the preferred method for the analysis of evolutionary relationships of taxa in the animal and kingdoms. Its methodologies rely on genes that lie on a variability spectrum; for example, highly conserved genes such as the plant plastid gene rbcL have been sequenced with nearly universal primers in thousands of species as a way of studying higher levels of taxa (Judd et al., 2002). To study evolutionary relationships at the generic or infra-generic level, however, much more variable sequences are required. In , highly variable plastid genes, introns, and spacers are typically used because they have been found to be the most variable DNA regions in the plant genome (Judd et al., 2002). Historically, the plastid maturase gene matK (Cuénoud et al., 2002), the ribosomal internal transcribed spacer (ITS) (Pridgeon et al., 2001), and the trnL-F intron and spacer (Sang et al., 1997) were found to be sufficiently variable to study plant systematics at the generic level in most taxa.

The large plant family Orchidaceae (Orchid family) is typically divided into subfamilies, tribes, and subtribes that represent divisions based on morphological similarities (Pridgeon et al., 2006). The largest subtribe is the Pleurothallidinae, consisting of 29 genera and over 4,000 species (Luer, 1986). The largest genus within the subtribe is Pleurothallis, which at one time consisted of over 2,000 species categorized into 32 subgenera that were initially based on morphology (Luer, 1986).

Former subgenera of Pleurothallis such as Acianthera, Anathallis, Crocodeilanthe and

Dracontia have since been raised to the level of genus (Luer, 1986). The remaining subgenera left in the genus are referred to as Pleurothallis sensu lato though Luer (2005)

4 continued to split Pleurothallis s.l. by elevating subgenera to the level of genus based on study of the floral morphology (Table 1).

Pridgeon, Solano, and Chase (2001) used DNA sequencing for a novel approach to reorganizing the Pleurothallidinae. Their study encompassed 185 pleurothallids (the colloquial name for the subtribe) with sequencing data from the internal transcribed spacer (ITS), the plastid gene matK, and the plastid trnL-F intron and spacer. While much of their data agreed with Luer’s categorization of the subtribe, major differences arose in the approach to the genus Pleurothallis as the study suggested that many of the subgenera

Luer had elevated in recent years, such as Acronia, Elongatia, and Lindleyalis, should remain lumped as subgenera (Table 1).

The Pridgeon et al. (2001) paper drew criticism based on its limited sampling of species studied and the small percentage of the orchid genome assessed (Luer, 2002; Jost and Endara, 2003). Luer (2002) criticized its emphasis on monophyletic clades, a concept that has since been heavily supported by the biological systematics community.

Jost and Endara (2003) questioned the objectivity of the classification based on some subjective interpretation of the phylogenetic trees. This controversy has led to further phylogenetic study of the subtribe Pleurothallidinae.

Using the Pridgeon et al. (2001) reclassifications of the subtribe as a starting point, several additional phylogenetic studies have explored Pleurothallidinae genera.

The genus Stelis has been defined and redefined in the past decade (Karremans, 2010;

Solano, 2005) based on morphology, geographic distribution, and extensive phylogenetic study as have the genera Dracula (Meyer & Cameron, 2009), Masdevallia (Abele, 2007;

Matuszkeiwicz & Tukallo, 2006), and Scaphosepalum (Endara et al., 2011).

5 Sequencing of matK and ITS in Pleurothallis s.l. species has continued in further depth as well. Nearly 150 species of Pleurothallis had the ITS region sequenced first

(Kenyon, 2008). Several dozen species had the matK gene sequenced and constructed into a gene tree (Shum, 2011). This led to phylogenetic trees that are more detailed than the one set forth in Pridgeon et al. (2001) (Fig. 1; Fig. 2). Based on the phylogenetic gene trees constructed, it is hypothesized that there are more clades than either of the other models suggest, however the level in the taxonomic hierarchy of these clades is unclear

(Table 1).

Ycf1, a hypothetical chloroplast open reading frame, was first used by Neubig et al. (2009) in a phylogenetic study of the entire family Orchidaceae. Ycf1 strikes a balance between coding sequences that are too conserved to be useful in plant systematics and introns or intergenic sequences that are littered with insertions and deletions and therefore difficult to align. Of a total length of 5,500 bp, only a ~1200 bp

5’ region and a ~1500 bp 3’ region were sequenced and analyzed (Fig. 3). In

Scaphosepalum, 3’ ycf1 has been found to have more parsimony informative sites than matK per 100 base pairs and therefore ycf1 is phylogenetically valuable at lower taxonomic levels (Endara et al. 2011).

The primary objective of this study was to generate sequence data from the ycf1 gene in order to produce a ycf1 gene tree for comparison to the ITS and matK trees. The goal was then to analyze these gene trees to aid in the understanding of the evolutionary history of Pleurothallis and to assess support for the current hypotheses regarding the various current taxonomic classifications of Pleurothallis or alternatively, to suggest additional hypotheses.

6 Table 1. Spectrum of taxonomic models for the group Pleurothallis sensu lato

“Splitter” model (favored by Luer, 1986)

Genus Acronia/Zosterophyllanthos Genus Elongatia Genus Lindleyalis Genus Loddigesia Genus Pleurothallis sensu stricto Genus Ancipitia Genus Colombiana Genus Mirandopsis Genus Rhynchopera Genus Talpinaria “Lumper” model (favored by Pridgeon et al., 2001)

Genus Pleurothallis sensu lato Subgenus Acronia Subgenus Elongatia Subgenus Lindleyalis Subgenus Loddigesia Subgenus Pleurothallis (inc. Ancipitia, Colombiana, Mirandopsis) Subgenus Rhynchopera Subgenus Talpinaria Model as informed by ITS and matK (Shum, 2011 and Wilson unpublished)

Clade Acronia/Zosterophyllanthos/Macrophyllae-Fasciculatae Clade Ancipitia and Colombiana/Scopula Clade Acroniae: I (inc. P. allenii, P. luctuosa, P. neglecta, P. pallida, and P. rowleei) Clade Acroniae: II (inc. P. forceps-cancri, P. quadricaudata etc.) Clade Acroniae: III (inc. P. phalangifera, P. alvaroi, P. stricta, and P. gomezii) Clade Antenniferae Clade Elongatia Clade Lindleyalis/Restrepioidia Clade Loddigesia Clade Mirandopsis/Mirandia Clade Pleurothallis sensu stricto (inc. Longiracemosae, Macrophyllae-Racemosae, and the Mesoamerican clade) Clade Rhynchopera Clade Talpinaria

7 Figure 1. ITS tree based on data from Kenyon (2008) and unpublished data from Mark Wilson’s lab, Biology Department, Colorado College. Maximum-parsimony analysis. The numbers represent bootstrap support values. Continued onto the next page.

8 9 Figure 2. matK gene tree based upon data from Shum (2011) and unpublished data from Mark Wilson’s lab, Biology Department, Colorado College. Maximum-parsimony analysis. The numbers represent bootstrap support values. Continued onto the next page.

10 11 Figure 3. The hypothetical open reading frame ycf1 showing the 5’ and 3’ regions along with the primers used to amplify and sequence those regions with arrows pointing in the direction of nucleotide addition by PCR.

Materials and Methods

DNA extraction

DNA samples were obtained from fresh or frozen (stored at -20oC) plant material.

The plant material was ground up under liquid nitrogen until a fine powder was achieved.

The DNeasy Plant Mini Kit (QIAGEN inc.) protocol was used to extract total DNA. A sample of the total plant DNA was then run on a 0.8% agarose 1X TAE buffer gel at 100

V for 15 min. In order to determine the concentration of extracted DNA, the plant samples were run against Lambda DNA at 10 ng, 25 ng, 50 ng, 75 ng, 100 ng, or 150 ng per well.

Designing new polymerase chain reaction primers for 5’ ycf1

PCR amplification

Primers 1F and 1200R (Neubig et al., 2008), designed for a study of the

Orchidaceae, did not reliably amplify the 5’ ycf1 segment in Pleurothallis and were used in this study to help design new primers. Primers 1F and 1200R were used in initial polymerase chain reaction (PCR) amplifications. DNA of one plant from every sub-

12 group of Pleurothallis being studied was amplified using these primers (Table 2). A master mix was prepared using 1μl of 1F primer, 1 μl of 1200R primer, 2 μl of DMSO, and 16 μl of molecular biology grade water for a total of 20 μl per reaction. The concentration of template DNA was adjusted, where necessary, to 2 ng/μl. PuReTaq

Ready-To-Go PCR beads (GE Healthcare) were combined with 20 μl of master mix and

5 μl of template DNA for a final volume of 25 μl and a final DNA quantity of 10 ng.

Three PCR reactions were set up per plant. The tubes were then placed in an iCycler

(BioRad) thermal cycler for PCR amplification using the following program:

ycf1 thermal cycler program

1 cycle 94.0oC 3 min 8 cycles 94.0oC 30 s 60.0oC 1 min (Touchdown 1 degree/cycle to 52oC) 72.0oC 3 min 30 cycles 94.0oC 30 s 50.0oC 1 min 72.0oC 3 min 1 cycle 4.0oC hold

Excising PCR products

The three PCR reactions were combined with 15 μl of Gel Loading Dye

(BioLabs) for a total volume of 75 μl. The product was then run on a 1.8% agarose gel in

1X TBE buffer alongside a 100 bp ladder in order to verify the length of the product

(Table 3) and separate the product from any nonspecific bands. The gel was run for 1 h at 100 V. The gel was photographed using an ultraviolet light imager (UVP). The band of the correct length containing the PCR product was excised from the gel using a clean razor blade and then weighed.

13 Table 2. List of the plants and their Pleurothallis sub-group used in the design of the new 5’ ycf1 primers. Subgroups of Pleurothallis follow Luer (1986).

Accession # Species Name Pleurothallis Sub-group PL194 Pleurothallis anthrax Ancipitia PL297 Pleurothallis restrepioides Elongatia PL397 Pleurothallis macrophylla Elongatia PL295 Pleurothallis quadrifida Loddigesia PL356 Pabstiella tripterantha Pabstiella PL364 Pabstiella tripterantha Pabstiella PL226 Pleurothallis gomezii Pleurothallis-Acroniae PL454 Pleurothallis ptychophora Pleurothallis-Antenniferae PL393 Pleurothallis inflata Pleurothallis-Macrophyllae-Racemosae PL460 Pleurothallis megalotis Pleurothallis-Macrophyllae-Racemosae PL003 Pleurothallis ruscifolia Pleurothallis-Pleurothallis PL473 Pleurothallis nuda Restrepioidia PL477 Pleurothallis medinae Restrepioidia PL192 Pleurothallis scoparum Scopula

Table 3. A list of the expected PCR product sizes associated with each primer pair.

Gene Forward Primer Reverse Primer PCR product size 5' ycf1 1F 1200R ~1200 bp 5' ycf1 5'-ycfNina-f 5'-ycfNina-r ~800 bp 3' ycf1 IntF 5500R ~1000 bp 3' ycf1 3720F IntR ~850 bp 3' ycf1 3'-ycf-for 1 3'-ycf-rev1 ~1350 bp

Purification of PCR products

The excised piece of gel containing the PCR product was purified using the

QIAquick Gel Extraction Kit (QIAGEN). Columns were incubated for 1 minute at room temperature using molecular biology grade water to elute the product. Concentrations were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific).

14 Sequencing

Two samples of each of the PCR products were sent at a concentration of 10 ng/μl to Sagagene along with primers 1F and 1200R so that two forward sequences and two reverse sequences would be generated. Electropherograms received from Sagagene were imported into FinchTV (Geospiza) and analyzed for overlapping signals or low amplitude signals and then edited for the longest but most highly supported sequence possible. The sequences were exported to the program Se-Al for alignment with the other sequences for that plant. A consensus sequence was generated with care taken to ensure that there were at least two nucleotides supporting the consensus nucleotide at each position. If there were ambiguities, a nucleotide was designated based upon majority rule. If the sequences did not overlap or an irresolvable ambiguity emerged, additional sequencing data was obtained from Sagagene.

Creating new primers

One consensus sequence from each sub-group of Pleurothallis (Luer, 1986) was imported into a single Se-Al file and a consensus sequence was created from all of the sequences. This consensus sequence was then imported into an online primer generator,

Primer3 (http://frodo.wi.mit.edu/primer3/). Several sets of primers were returned and each was analyzed for specificity to the ycf1 gene and to orchid plants in general using

BLAST search (NCBI). Two new primers were selected and named 5’ycf-Nina-f

(GTGGTCGGGCTCTATTATGG) and 5’ycf-Nina-r (CCCAATTCCCAATTCTCTTG)

(Figure 4). The oligonucleotides were synthesized by IDT.

15 Figure 4. Diagram of the 5’ region of the ycf1 gene. The arrows indicate where the old primers annealed and where the new primers anneal. The arrows are pointing in the direction of nucleotide addition by PCR.

Designing new PCR primers for 3’ ycf1

Primers 3720F and 5500R were designed by Neubig et al. (2008) to be used in

PCR amplification of 3’ ycf1, and primers IntF and IntR (Neubig et al., 2008) were designed as internal primers to aid in sequencing the entire 1500 bp segment of the ycf1 gene. However, the primer pair 3720F and 5500R did not reliably amplify 3’ ycf1 in

Pleurothallis. Primer pairs 3720F and IntR, and IntF and 5500R were used instead to amplify the ends of 3’ ycf1 in order to sequence those ends and design new primers.

PCR amplification and purification

PCR Master Mix (Promega) was used as the source of Taq polymerase, nucleotide bases, and buffer. A master mix was created for the 3720F and IntR primer pair using 1 μl 3720F, 1 μl IntR, 12.5 μl PCR Master Mix, and 5.5 μl molecular biology grade water for a total of 20 μl. A second master mix was created for the IntF and 5500R primer pair using 1 μl IntF, 1 μl 5500R, 12.5 μl PCR Master Mix, and 5.5 μl molecular biology grade water. The 20 μl of mastermix and 5 μl of DNA template at 2 ng/μl were

16 combined in a 200 μl tube (USA Scientific) for a total reaction volume of 25 μl and a total DNA quantity of 10 ng. A plant from every Pleurothallis sub-group was amplified

(Table 4). Three reactions were set up for each primer pair per plant. The same protocol was followed as above for the PCR amplification, excision, and purification.

Table 4. List of the plants and their Pleurothallis sub-group used in the design of the new 3’ ycf1 primers

Accession # Species Name Pleurothallis sub-group PL204 Pleurothallis eumecocaulon Ancipitia PL469 Pleurothallis quadrifida Loddigesia PL222 Pleurothallis stricta Pleurothallis-Acroniae PL207 Pleurothallis pruinosa Pleurothallis-Longiracemosae PL393 Pleurothallis inflata Pleurothallis-Macrophyllae-Racemosae PL003 Pleurothallis ruscifolia Pleurothallis-Pleurothallis

Sequencing

The purified PCR products were sent for sequencing and the electropherograms were processed in the same manner as above. A consensus was created for the section between the 3720F primer and the IntR primer for all of the plants that were sequenced.

A second consensus was created for the section between the IntF primer and the 5500R primer for all of the plant species. The two consensus sequences were then concatenated to produce one continuous sequence.

Designing new primers

The consensus sequence was imported into Primer3. Two new primers were selected using the same method as above: 3’ycf-for1 (ATGCCTAAAGAATGGCGAAA) and 3’ycf-rev1 (TCATTCAAAAATTGCCCACA). Additional internal primers were also designed to aid in sequencing: IntF2 (CATTAACGTAAATCCAAAGAA) and IntR2

(GGTCCTTTTGAATCAGCATTTT) (Figure 5).

17 Figure 5. Diagram of the 3’ region of the ycf1 gene. The arrows indicate where the old primers annealed and where the new primers anneal. The arrows are pointing in the direction of nucleotide addition by PCR.

Sequencing 5’ ycf1

Primers 5’-YcfNina-f and 5’YcfNina-r were used to amplify DNA from a selection of Pleurothallis plants across the 16 sub-groups being studied (Table 5). The same method as detailed above using PuReTaq Ready-To-Go PCR beads was used to amplify the 5’ ycf1, however, because the reaction was so robust, only 1 PCR reaction tube was needed per plant. The amplified sequence was then purified in the same manner as above, and then sent for sequencing. The electropherograms were treated the same as above and a consensus sequence was generated for 5’ ycf1 in each plant.

Sequencing 3’ ycf1

Primers 3’-ycf-for 1 and 3’-ycf-rev 1 were used to attempt to amplify DNA from the same plants as 5’ ycf1, however due to difficulty in amplification, only a selection of plants could be sequenced, as indicated with an asterisk (Table 5). The same method as detailed above using PCR Master Mix was used to amplify the 3’ ycf1. The amplified sequence was then purified in the same manner as above and sent for sequencing. The

18 Table 5. List of all the plants, their species name, and their Pleurothallis sub-group used in the study of 5’ ycf1. Plants with an asterisk next to their accession numbers were also used in the study of 3’ ycf1.

Accession # Species Name Pleurothallis sub-group PL194 Pleurothallis anthrax Ancipitia PL204 Pleurothallis eumecocaulon Ancipitia PL256* Pleurothallis anceps Ancipitia PL297* Pleurothallis restrepioides Elongatia PL362* Pleurothallis restrepioides Elongatia PL397 Pleurothallis macrophylla Elongatia PL294* Pleurothallis quadrifida Loddigesia PL295* Pleurothallis quadrifida Loddigesia PL469* Pleurothallis quadrifida Loddigesia PL012* Pleurothallis radula Macrophyllae-Fasciculatae PL021 Pleurothallis bivalvis Macrophyllae-Fasciculatae PL034 Pleurothallis radula Macrophyllae-Fasciculatae PL215* Pleurothallis rubroinversa Macrophyllae-Fasciculatae PL010 Pleurothallis excavata Mesoamerican PL025 Pleurothallis dorotheae Mesoamerican PL202 Pleurothallis cobriformis Mesoamerican PL356 Pabstiella tripterantha Pabstiella PL364 Pabstiella tripterantha Pabstiella PL067 Pleurothallis forceps-cancri Pleurothallis-Acroniae PL222* Pleurothallis stricta Pleurothallis-Acroniae PL226 Pleurothallis gomezii Pleurothallis-Acroniae PL454 Pleurothallis ptychophora Pleurothallis-Antenniferae PL006 Pleurothallis divaricans Pleurothallis-Longiracemosae PL207* Pleurothallis pruinosa Pleurothallis-Longiracemosae PL323 Pleurothallis xanthochlora Pleurothallis-Longiracemosae PL393* Pleurothallis inflata Pleurothallis-Macrophyllae-Racemosae PL460 Pleurothallis megalotis Pleurothallis-Macrophyllae-Racemosae PL003* Pleurothallis ruscifolia Pleurothallis-Pleurothallis PL331 Pleurothallis tentaculata aff. Restrepioidia PL473 Pleurothallis nuda Restrepioidia PL477 Pleurothallis medinae Restrepioidia PL337 Pleurothallis loranthophylla Rhynchopera PL377 Pleurothallis pedunculata Rhynchopera PL018 Pleurothallis penicillata Scopula PL191 Pleurothallis aspergillum Scopula PL192 Pleurothallis scoparum Scopula PL305 Pleurothallis hitchcockii Talpinaria PL441* Pleurothallis sandemanii Talpinaria PL339 Pleurothallis truncata Truncatae

19 electropherograms were treated the same as above and a consensus sequence was generated for each plant.

Aligning the consensus sequences and creating a phylogenetic tree

All of the consensus sequences for both 5’ ycf1 and 3’ ycf1 were loaded into separate alignment files in the program MEGA 5.0 (Tamura et al., 2011). The sequences were manually aligned and spaced appropriately to account for indels present in some sequences. The 5’ ycf1 sequences were truncated at the start sequence ATC AGC AAC and the end sequence AAG GAT GAA. The 3’ ycf1 sequences were truncated at the start sequence TCT CGA TTA and the end sequence ATT AAA TTC. Several ycf1 sequences from other pleurothallids (obtained from personal correspondence with Mark Whitten) were added to the alignment. Several ycf1 sequences from species of the genus

Lockhartia were imported from a BLAST search into the alignment and used as an out- group. A phylogenetic tree was then constructed using MEGA 5.0 (Tamura et al., 2011) using maximum-parsimony and 1000 bootstrap iterations. The bootstrap consensus tree was selected for the 3’ ycf1 and the 5’ ycf1.

Results

A 5’ ycf1 gene tree was generated (Fig. 6) and then a second, condensed tree was generated (Fig. 7). Nodes with bootstrap values of less than 50% collapsed in the condensed tree and created a large Pleurothallidinae polytomy (node A). The analysis identified species from the Lockhartia genus as being the out-group with 99% bootstrap support (node B). Within the Pleurothallidinae polytomy, several subgroups with moderate support are identified. Pleurothallidinae species that are not part of

20 Pleurothallis s.l. clustered with 80% bootstrap support, with the exception of

Pleurothallis crocodiliceps (node C). One of the few nodes that correspond to currently recognized subgroups of Pleurothallis, the three species of subgroup Lindleyalis clustered together with moderate 61% bootstrap support (node H). Two sampled individuals of Pleurothallis quadrifida (subgroup Loddigesia) clustered together with moderate 63% bootstrap support, however a third sampled individual of that species is found in the unresolved polytomy. The two individuals in node E correspond to the subgroup Acroniae III as described in the model as informed by matK and ITS (Table 1).

Other nodes in the Pleurothallis polytomy with equally moderate support correspond less well to recognized subgroups. These include nodes D, F, G, I, and J. Notably, the two sampled individuals of Pabstiella tripterantha (subgroup Pabstiella) did not cluster together, with one linked to Pleurothallis hitchcockii (subgroup Talpinaria) with 61% bootstrap support (node I) and the other in the unresolved polytomy.

The 3’ ycf1 bootstrap consensus tree (Fig. 8) and the condensed view of the tree

(Fig. 9) have more highly supported clades than the 5’ ycf1 trees. While some of the topology of the tree collapses in the condensed view, much of it remains. The node that contains all of the Pleurothallis species clustered together with 99% bootstrap support

(node C). Clear distinctions between recognized subgroups are maintained in the condensed tree. The subgroup Pleurothallis clustered with the Ancipitia and the Acroniae with 92% bootstrap support (node D). The Acronia (node E), Loddigesia (node F), and

Elongatia (node G) subgroups clustered together with 97%, 99%, and 99% bootstrap support, respectively. The Pleurothallidinae clustered together with 92% bootstrap support (node A) and the out-group was identified with 100% bootstrap support (node B).

21 Figure 6. Bootstrap consensus tree of 5’ ycf1 using maximum-parsimony analysis and 1000 bootstrap iterations.

22 Figure 7. Condensed view of the bootstrap consensus tree of 5’ ycf1. Only nodes with greater than 50% bootstrap support are displayed.

D

E

F A G

H

I

J

C

B

23 Figure 8. Bootstrap consensus tree of 3’ ycf1 using maximum-parsimony analysis and 1000 bootstrap iterations.

24 Figure 9. Condensed view of the bootstrap consensus tree of 3’ ycf1. Only nodes with greater than 50% bootstrap support are displayed

D

E C

F

G

A

B

25 Discussion

Examining infrageneric genetic profiles of Pleurothallis and the potential classification of its subgenera strikes at the heart of the subjectivity of phylogenetics.

Gene trees are not species tree, and are always open to more than one interpretation; this study is no exception. The 5’ ycf1 gene tree collapses into one large polytomy in its condensed form. This can be interpreted in one of two ways. The first interpretation is to say that this is a “soft polytomy” (Madison, 1989), a case in which there are insufficient parsimony informative sites from this section of the gene and therefore the nodes cannot be resolved. This interpretation of the data would suggest that 5’ ycf1 is not sufficiently variable to study the subtleties of Pleurothallis at the generic or infrageneric level. A second interpretation looks at the pleurothallid polytomy as a “hard polytomy” (Madison,

1989), one in which all of the subgenera split through speciation from a single node in a recent radiation event and therefore cannot be readily distinguished even by highly variable genes. This interpretation of the data suggests that the plants are all very closely related and therefore might not be meted out into multiple genera, at least on the basis of the 5’ region ycf1 gene. This interpretation would not support any of the existing models because very few of the recognized subgroups of Pleurothallis are identified as discrete clades in the gene tree.

This second interpretation of the 5’ ycf1 data is consistent with the ITS (Fig. 1) and matK (Fig. 2) gene trees. While the gene trees have some clades of subgenera that are well supported with high bootstrap values, such as Loddigesia and Lindleyalis, many others are rather poorly supported. In condensed views of the ITS and matK gene trees, many of the clades would collapse and form large polytomies. The sequence data

26 currently available suggest that all of the subgenera of Pleurothallis are equally related to the most recent common ancestor stemming from a well-supported node. In this context, if one subgenus is elevated to the level of genus, all of the subgenera must be elevated as well.

The limited data from the 3’ ycf1 gene trees suggest that this region of the gene is sufficiently variable to be used in phylogenetic analysis of Pleurothallis at the generic and infrageneric level. Recognized subgroups of Pleurothallis are clustered within well- supported nodes. There may be an argument for elevating the subgenera that stem from node C containing all of the Pleurothallis plants in the condensed view of the tree (Fig.

9). Node D, containing the plants from the subgenera Pleurothallis, Ancipitia, and

Acroniae, would form a new conscription for Pleurothallis s. s. because the type species for genus Pleurothallis, Pleurothallis ruscifolia, falls amongst them. Talpinaria, Acronia,

Loddigesia, and Elongatia could all be elevated to genera as well. All of the clades are well supported with upwards of 90% bootstrap support and they are equally distantly related to the most recent common ancestor. However, many more plants would have to be sequenced including plants from all of the other subgenera before this could be considered a plausible hypothesis for the classification of Pleurothallis. At this time the phylogenetic data can neither support nor contradict the universal elevation of subgenera as put forth by Luer in his splitter model (Table 1).

Future research would focus on further sequencing of the 3’ ycf1 gene in order to construct a tree based on more extensive data. Additional sampling of individuals and subgenera are also necessary. These data could then be combined with matK and ITS data to give a high-resolution picture of Pleurothallis. With the data from all three highly

27 variable genes perhaps much more highly supported clades will emerge that could support the elevation of the subgenera to the generic level. Alternatively these perspectives may reveal dissonance between gene trees that suggest the evolutionary history of Pleurothallid orchids is complex and/or recent enough to warrant caution in its taxonomic classification

28 Acknowledgements

I would like to thank the Colorado College Biology Department, the Keller Family

Venture Grant, the Figge-Bourquin Grant, and the Faculty-Student Collaborative Grant for providing me with the funds to carry out this research. I am especially grateful for the advice, support, and time that Dr. Mark Wilson has given to helping me throughout my research and thesis writing. I would like to thank Dr. Tass Kelso for her indispensable advice during the thesis writing process. I would also like to thank my lab mates Qunyh

Nguyen, Lily Shan, Caleb Volz, Leah Kellog, and Margo Simon - without whom I would have only half of the data I was able to analyze. I would also like to extend my gratitude to Donna Sison for helping me stay organized and informed throughout this process. And finally I would like to thank all my family and friends for their unconditional love and support.

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