Understanding the Historical Diversification of Valerianacea

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Senior Honors Theses Undergraduate Showcase

12-2016

Understanding the Historical Diversification of alerianaceaV

Taylor LeBourgeois University of New Orleans

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UNDERSTANDING THE HISTORICAL

DIVERSIFICATION OF VALERIANACEAE

An Honors Thesis

Presented to

the Department of Biological Sciences

of the University of New Orleans

In Partial Fulfillment

of the Requirements for the Degree of

Bachelor of Science, with University High Honors

and Honors in Biological Sciences

Taylor LeBourgeois

December 2016

Acknowledgments

I would like to thank Dr. Charles Bell for taking on the task of being my thesis advisor and the extensive help and knowledge he has given me. I would also like to thank Dr.

Carla Penz with her help with this thesis. I want to acknowledge the Honors department, especially Dr. Sarwar, for their guidance. I want to recognize my family for their love and support throughout this journey. Finally, I want to thank and dedicate this accomplishment to my late Grandmother, without her none of this would be possible.

Table of Contents

Acknowledgments……………………………………………………………………………ii

List of Tables………………………………………………………………………..……….iv

List of Figures……………………………………………………………………….……….v

Abstract……………………………………………………………………………….……...1

Introduction…………………………………………………………………………….……2

Methods and Materials………………………………………………………………….…..6

DNA EXTRACTION AND PCR REACTIONS…………………………….…….6

DNA SEQUENCING AND PHYLOGENY BUILDING…………………………7

Results…………………………………………………………………………….…………9

Conclusion………………………………………………………………………………….10

References…………………………………………………………………….……………11

iii

List of Tables

Table 1: Sequence Success per Amplified Gene in Accordance with Each Species……….14

List of Figures

Figure 1: Strict Consensus Tree of 8 MPT (23728 steps)....…………………………………15

Figure 2: Bootstrap Consensus Tree…………………………………………………………16

Abstract

Ecologists and evolutionary biologists have long recognized that species diversity is unequally distributed among angiosperm lineages. For example, various plant clades found in the Andes have been proposed as examples of rapid radiations, and the Andes are recognized as one of the Earth’s biodiversity “hotspots”. Species within Valerianaceae, found in the

South American Andes, appear to be an example of such a rapid radiation. Although much attention has been paid to the phylogeny of the South American species of Valerianaceae, there is still a great deal of uncertainty regarding species relationships. Several subgroups within the Andean Valerians have, either not been included, or poorly sampled in previous phylogenetic analyses. One example of such a group is section Porteria. Knowing the phylogenetic placement of this group would go a long way in our understanding of Andean biogeography, as well as morphological and floral evolution in Valerianaceae. In this study, I increase the sample size of section Porteria beyond what has been previously done. A number of genomic regions were amplified and sequenced for 6 species belonging to the section. Sequences were then aligned to previously published sequence data for homologous regions for other species in Valerianaceae. A “supermatrix” of 146 species was then analyzed using maximum parsimony. Resulting trees recovered a monophyletic section Porteria, however statistical support was weak for this conclusion. Although “supertrees” resulting from the analysis of “supermatrices” may be powerful tools for testing hypotheses concerning the evolution of species, there is still much work to be done on Valerianaceae.

Keywords: angiosperm lineages, Valerianaceae, Porteria, “supermatrix”, “supertrees”

Introduction

Ecologists and evolutionary biologists have long recognized that species diversity is unequally distributed among angiosperm lineages (e.g., Barraclough and Savolainen, 2001;

Magallón and Sanderson, 2001; Magallón and Castillo, 2009). Some lineages seem to have experienced a pronounced acceleration in net diversification, and these are often attributed to rapid radiation. To explain shifts in diversification, investigators have long tried to identify

“key innovations.” Numerous key innovative hypotheses have been put forth to link the incredible diversity of angiosperms with life history, physiological, and/or morphological traits (e.g., Magallón and Sanderson, 2001).

Traits such as flower color (Smith et al., 2009), pollination syndromes (Strauss and

Whittall, 2006), and seed dispersal mechanisms have been proposed as traits that might be driving diversification within specific angiosperm clades. However, some investigators have found shifts in diversification rates are not correlated with any observed morphological trait.

Studies of Valerianaceae (Bell et al., 2015) and Orobanchaceae (Uribe-Convers and Tank,

2015) have suggested that bursts in diversification rates appear to be tied to the movement, or dispersal, into new geographic areas.

Numerous plant clades found in the Andes have been proposed as examples of rapid radiations (Crowl et. Al, 2016; Lagomarsino et al., 2016), and the Andes are recognized as one of the Earth’s biodiversity “hotspots.”. Many plant lineages that occur here appear to have diversified rapidly since their origin. For example, Hughes and Eastwood (2006) have shown that species of Lupinus (Fabaceae), occurring in the Andes, are much more diverse than their North American ancestors. A recent study of the Neotropical bellflower

(Campanulaceae) has shown substantial evidence in which higher altitudes do correlate with rapid radiation and diversification (Lagomarsino et al., 2016).

Valeriana is a group of flowering plants that are indigenous to Europe and Asia.

Among this group of plants are Valerianaceae and Caprifoliaceae. It is composed roughly of

300 species, and is said to have a foul odor associated with the roots of these plants.

Valerianaceae has been theorized to have originated in Asia, it is predominately located in the Southern Andes. Of the 300 recognized species, approximately 75% are endemic to the

Andes where they are represented at high, mid, and low elevations (depending on latitude).

Although much attention has been paid to the phylogeny of the South American species of Valerianaceae (e.g., Bell and Donoghue, 2005), there is still a great deal of uncertainty regarding species relationships. Much of this uncertainty can be attributed to the limited sampling of particular subgroups within the South American valerians. One such subgroup is Valeriana section Porteria, which was originally described as a separate genus

(Porteria) due to several characteristics (shortened calyx, overlapping bracts, and spurs at the base of the corolla) (Pittier, 1926). However, there were other species believed to be mistakenly grouped with the previously declared species because of divergent characteristics.

As decades passed, many botanists have reviewed this genus (Porteria), adding similar species as they were discovered. As of 1914, seven Venezuelan species were described by the botanist Ellsworth P Killip. The following four of those seven were focuses of this research: Valeriana bractescens, Valeriana spicata, Valeriana phylicoides and Valeriana triplinervis. Two recently described species that are also concentrations are Valeriana rosaliana and Valeriana quirorana. They are found throughout the Andes, specifically

Venezuela. Valeriana spicata also occurs in Columbia and Valeriana bractescens is

4 additionally distributed in Peru and Ecuador. There have been studies published identifying key characteristics that distinguish the six species from one another. Valeriana bractescens is identified as having entire lance-shaped leaves, a corolla longer than 1 centimeter, and heart- shaped bracts being 2 or more centimeters wide (Pittier, 1926). Valeriana rosaliana is classified by its spatulated, notched leaves which are 2 to 3.5 centimeters in length. The bract is 10 to 12 millimeters long and the white corolla is shaped like a funnel (Meyer, 1979).

Valeriana spicata is identified by the absence of the petiole which leads to the direct attachment of the leaves to the stem. The leaves also have round-notched edges, and are tightly packed together. The corolla is a deep yellow color, and the branches lack trichomes

(Pittier, 1926). Valeriana quirorana is classified with leaves 2 cm or longer, that have notched edges (Morillo and Rodriguez, 1993). Valeriana phylicoides is classified by its 6 to

8-millimeter-long corolla, and notched leaves. Valeriana triplinervis is classified by its faintly serrated leaves; the length of the corolla equals 1 centimeter or less; the length of the bracts is 7 millimeters or less; and its leaves are elongated, linear, and sharp at the tip. To date, only a handful of species from section Porteria have been sampled for molecular phylogenetic studies (Hildalgo et al., 2004; Bell and Donoghue, 2005). There is not only uncertainty if the section is monophyletic, but there is further uncertainty where it belongs in the Valerianaceae phylogeny.

The objective of this study was to examine 6 previously un-sampled species from subsection Porteria and determine if this group is monophyletic. In addition, I examined the placement of section Porteria within Valerianaceae. For this task, I compared newly generated gene sequences from section Porteria with previously published sequences from across Valerianaceae exclusively located in the Andes. Lastly, I synthesized all molecular

5 data to construct a “supermatrix” in order to infer a “supertree” for the species Valerianaceae located in the Andes.

Methods and Materials

DNA EXTRACTION AND PCR REACTIONS

Six silica dried plant samples were obtained from Venezuela (Table 1). The leaves were used because these tissues provide the best quality extraction samples. There were several steps undergone to get the purest DNA using the Qiagen DNeasy Plant Mini

Extraction Kit (Qiagen; Valencia, California). Each sample was weighed and placed inside of a tube containing a grinding bead. The grinding bead was used to assist in the disruption of the tissues while convulsing. The granulates were washed with a solvent while centrifuged and after a series of repetitions a supernatant was formed. Once the DNA was properly extracted, a master mix was created containing both forward and reverse primers. The primers were allocated for the amplification of individual regions in a polymerase chain reaction (PCR.) All six samples underwent roughly 20 polymerase chain reactions with specific corresponding primers in a Bio-Rad S100 Thermal Cycler. To properly observe if the PCRs were successful, the samples had to be viewed under an ultraviolet light. A small portion of the DNA was combined with a DNA gel loading dye. This combination was placed into agarose gel wells and on electrophoresis apparatus. A power source is connected on either ends of the unit, and an electric field is administered throughout. Once finished, the gels were observed through the Bio-Rad Universal Hood Molecular Gel Imaging System and the Quantity One program. If the gel showed distinct bands, it signified the reaction was successful and there was DNA present. The PCR product had to be purified of all the extra agents by using ExoSap-IT (USB-Affymetrix; Cleveland, Ohio.)

DNA SEQUENCING AND PHYLOGENY BUILDING

The DNA sequencing could not begin until all samples underwent PCRs and purification with ExoSap-IT. A master mix was made for every forward primer and reverse primer. When filling a sequencing tray, there were 16 master mixes formulated in total. The mix contained microliters of Big Blue Dye. The 96-well plates were loaded with a forward master mix in the first 6 slots and the corresponding reverse master mix in the following 6 slots. The PCR products were placed into all 12 slots containing the corresponding master mixes. This process was repeated for every primer combination. These were sent to W.M.

Keck Conservation and Molecular Genetics Laboratory at the University of New Orleans to be sequenced through a dye terminator cycle. Once completed all samples were edited.

The forward and reverse primers were dedicated to splitting the double helical DNA into two separate strands. The strands were supposed to perfectly match each nucleotide with the corresponding nucleotide. Adenine (A) matches with Thymine (T), and Guanine (G) matches with Cytosine (C). When the sequences returned, there were some inconsistencies.

Some of the nucleotides did not match, and the code had to be deciphered. The sequence fragments were edited using the computer package Sequencher Version 5.3 (Gene Codes

Corporation; Ann Arbor, Michigan) to build contiguous sequences.

Sequences were then aligned and added to a matrix consisting of 25 molecular markers for 140 additional Valerianaceae samples that were obtained from GenBank. This

“supermatrix”, containing all 146 sampled taxa, was used to infer a “supertree” for

Valerianaceae using PAUP* 4.0 (Swofford, 2002) under the maximum parsimony (MP) criterion. Maximum parsimony searches were conducted using heuristic search methods with tree bisection reconnection (TBR) branch swapping, collapse of zero-length branches, and all

8 characters weighted equally. The analyses were repeated 100 times with the RANDOM

ADDITION option. Sets of equally most parsimonious trees were summarized by a strict consensus tree. Bootstrap tests (Felsenstein, 1985) were performed using 300 replicates with heuristic search settings identical to those of the original search. All parsimony and likelihood analyses were performed using the computer software PAUP* 4.0.

Results

Twenty-two different primer pairs were initially attempted for PCR amplification for all newly sampled species. Out of these twenty-two, 7 did not work for various reasons. The errors could have been introduced early on during the research or during the sequencing. Out of the 15 samples that were submitted for sequencing, 3 of those did not correctly sequence and the data could not be retrieved. There were a total of 12 sequences that could be properly edited and reviewed. The chance of getting a perfect sequence was slight, which required a precise overlook of each nucleotide. Once the single stranded DNA was matched with its corresponding strand from the reverse primer, it was aligned to homologous sequences that were obtained from GenBank. The final “supermatrix” contained a total of 20,577 aligned base-pairs for 146 samples.

A parsimonious heuristic search resulted in 8 most parsimonious trees of the length

23728. These 8 most parsimonious trees were used to generate a strict consensus tree

(“supertree”) presented in Fig. 1.

Results from these analyses are consistent with previous studies on Valerianaceae

(e.g., Bell and Donoghue, 2005) with regards to the overall phylogeny of the group. For the most part, statistical support for species relationships is low (Fig. 2). However, all species sampled belonging to section Porteria did form a clade (Fig. 1, blue box). These results are not supported by the bootstrap analysis (Fig. 2; bootstrap values <50%). Though, there is some support for species relationships within section Porteria (Fig. 2).

Conclusion

Based on these analyses, it is tempting to suggest that section Porteria does form a clade with Valerianaceae. However, bootstrap support for this conclusion is low. This low support might be the result of several factors: 1) amount of missing data in the “supermatrix”,

2) rogue taxa in the alignments, or 3) limited sequence divergence for the molecular markers used in this study. As with many phylogenetic studies, there is still work to be done to fill in the gaps of our knowledge for Valerianaceae. It may take additional taxa and sequencing being to the problem or potentially new (“next generation”) sequencing technologies.

In addition, although “supertrees” resulting from the analysis of “supermatrices” may be powerful tools to testing hypotheses concerning the evolution and diversification of species (Davis, 2010), there is still much work to be done on Valerianaceae. Many interesting hypotheses concerning morphological and floral evolution, as well as biogeographic questions (Bell and Donoghue, 2005), cannot be adequately addressed until we have a better resolved and supported phylogeny.

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Table 1: Sequence Success per Amplified Gene in Accordance with Each Species The “X” represents a successful sequence that could be used to create the phylogeny.