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Spring 5-11-2012

A Phylogeny of Samydaceae Based on Nuclear GBSSI and EMB2765 DNA Sequences

Chelsa Williams University of Southern Mississippi

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The University of Southern Mississippi

A Phylogeny of Samydaceae Based on Nuclear GBSSI and EMB2765 DNA Sequences

by

Chelsa N. Williams

A Thesis

Submitted to the Honors College of The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Bachelor of Science in the Department of Biological Sciences

May 2012

Approved by

______

Mac H. Alford, Associate Professor

Department of Biological Sciences

______

Glen Shearer, Jr., Chair

Department of Biological Sciences

______

David R. Davies

Honors College, Dean

iii

ACKNOWLEDGMENTS

I would like to take this opportunity to express my gratitude to some key people without whom this thesis would have never been possible. Thanks for the opportunities, your time, and your patience, Mrs. Paula Mathis and Dean Dave Davies of the

University’s Honors College. Thanks to Mrs. Liliana Hernández for being there to answer any questions I may have had along the way. A big thank you and mention of recognition is due for Mrs. Tharanga Samarakoon for being a mentor and huge help to me throughout this project, meeting me when it was not convenient and answering all of my questions.

The biggest thank you goes to Dr. Mac H. Alford for a multitude of reasons: for giving me the opportunity for this thesis, for the meetings, for answering all of my questions, for the patience shown, for the countless returns of e-mail during e-mail correspondence, and for being a mentor.

iv

ABSTRACT

Samydaceae are a tropical family of woody flowering , poorly known because they were once included in the ‘dustbin’ family and because there are few economically important . The family has recently been studied using DNA sequence data, but most of the data were from plastid DNA and some relationships had low statistical support. For this study, two nuclear regions, EMB2765 and GBSSI, were sequenced in order to affirm or refute the relationships inferred from plastid DNA and in order to find stronger statistical support for relationships. The data collected mostly affirm previously collected data from plastid DNA.

v

TABLE OF CONTENTS

Title Page...... i

Approval Page ...... iii

Acknowledgments ...... iv

Abstract...... v

Table of Contents...... vi

Chapter 1: Introduction...... 1

Chapter 2: Materials and Methods...... 5

Chapter 3: Results...... 11

Chapter 4: Discussion...... 17

References ...... 19

vi 1

Chapter 1: INTRODUCTION

Plants, especially the great majority of tracheophytes considered to be flowering plants, are extremely important to humans in the areas of nutrition, medicine, food, shelter, tools, dyes, and overall welfare (Judd et al., 2007). They have impacts on us ecologically and economically. systematics is the science of plant diversity in which the fundamental aim is to study the biological diversity of plants on Earth today and their evolutionary history. A secondary aim is to convey this knowledge about diversity and relationships in the form of phylogenies and classifications (Judd et al.,

2007). According to Judd et al. (2007), this science works by “putting forward hypotheses about the existence of branches of the of life and testing them with evidence.” While some think that systematics is separate from other areas of biology, this area plays an important role in other disciplines such as linguistics, philosophy, ecology, and molecular biology (Judd et al., 2007). Judd et al. (2007) also maintain that

“systematics is essential to our understanding of and communication about the natural world.”

While there are many different ways in which to construct a phylogeny, we attempt to construct one based on evolutionary relationships and root it by using a relative of the group known as an outgroup. The members of the ingroup, the group being studied, are more closely related to each other than they are to the outgroup(s) being used

(Judd et al., 2007). In this study, the outgroups used are aggregatum

(), Hasseltia floribunda, and Prockia crucis (). The ingroup members originally came from a very encompassing, pantropical family known as 2

Flacourtiaceae (Chase et al., 2002). Warburg (1894), Gilg (1925), Hutchinson (1967),

Lemke (1988), and Takhtajan (1997) are all well-known botanists who worked on

Flacourtiaceae, and all of them recognized that relationships within the family and relationships of the family to other families was difficult to ascertain. The family has always been difficult to define and characterize because of various reasons, including predominant character states shared by many other families and flowers too small to be easily examined by hand, even with a hand lens (Chase et al., 2002). Over the years,

Flacourtiaceae have earned a reputation as a “catchall” or “dustbin” group. Alwyn Gentry

(2003) expressed, “If you don’t have any idea what family it is, try Flacourtiaceae or

Euphorbiaceae,” and Williams (1965) proclaimed, “When in doubt, put it in the

Flacourtiaceae.”

With the coming of age of molecular biology, relationships of Flacourtiaceae have begun to be clarified because DNA provided more characters. Molecular studies have used DNA from three different cellular sources: mitochondrial DNA, plastid DNA, nuclear DNA, or some combination thereof. Mitochondrial DNA, in general, is not variable enough to be useful in plants, and copies of nuclear genes are far less abundant and more complicated than plastid genes. Thus, most studies of plants have, until recently, utilized plastid DNA as the major data set for studies of relationships.

Based on plastid DNA data, Chase et al. (2002) demonstrated a split into two major lineages within Flacourtiaceae, and Alford (2003, 2005, in prep.) later reported the best division of one of those lineages was into the two major families of Samydaceae and

Salicaceae sensu medio. 3

According to Alford (2003, 2005, in prep.), Samydaceae include about 256 species of the former Flacourtiaceae. Many plants within this “new” family have been reported to have medicinal, insecticidal, fungicidal, and cytotoxic components (Anaya et al., 2004; Beutler et al., 2000; Itokawa et al., 1990; Jullian et al., 2005), to accumulate metals at some of the highest levels ever recorded (Jaffré et al., 1979), and to be common elements of tropical forests worldwide (Phillips & Miller, 2002; Guilherme et al., 1998;

Silvra Jr. et al., 1998). Unfortunately, the family has received very little attention seemingly due to its lack of economically important members. Chase et al. (2002) only sampled one species from this newly resurrected family, not giving much insight into this family. However, some species of have been shown to have inhibitory activity against snake venom phosphordiesterase I (Chai et al., 2010) and to have anti-ulcer, anti- inflammatory, anti-ophidian, and antitumor potentialities (Ferreira et al., 2011). Some species within Samydaceae are also sporadically used for lumber (Chudnoff, 1984;

Richter & Dallwitz, 2000). The potential for economical uses is out there.

Currently, the ongoing research goal is to infer the phylogeny of and revise the classification of the Samydaceae. Previously, plastid genes were used in an attempt to disentangle existing relationships, but these genes posed a multitude of problems. Due to hybridization, plants can contain plastids from other species, thus potentially resulting in misleading phylogenies. Plastid DNA also evolves moderately slowly, resulting in data that may not be variable enough for inferring relationships between closely related genera and species. This study aims to use nuclear regions to provide more insight and resolution to the existing relationships within Samydaceae, with the hope that the nuclear DNA has more variation than the plastid DNA. Each cell contains only one nucleus with two 4 genomic sets, one from “mom” and one from “dad.” The genes chosen for this study presumably have only one copy per set, though the potential for two or more alleles does exist. Alford et al. (unpubl. results) have found Casearia to be polyphyletic based on previous plastid DNA, and those data have shown that there are several other genera nested within the large . Because nuclear DNA regions in other plants have shown great variability and exhibit a different mode of inheritance than plastids, the hypotheses for this study are:

1. Nuclear DNA regions in Samydaceae are expected to shower greater variation

than plastid regions, providing more data for inference of phylogenetic history,

and

2. Analyses of the nuclear DNA data will reveal a pattern of relationships identical

to or more resolved than (i.e., not conflicting with) those from analyses of plastid

DNA.

5

Chapter 2: MATERIALS AND METHODS

Species of Samydaceae and three outlier species from Lacistemataceae and

Salicaceae were previously collected, had DNA extracted using a Qiagen DNeasy® Plant

Mini Kit (QIAGEN Sciences, Maryland), and were frozen at -20°C. A list of species used, including vouchers, can be found in Table 1. Primers chosen for this experiment included GBSSI forward and reverse primers and EMB2765 forward [9F2] and reverse

[9R] primers. GBSSI is a 714 bp part of the nuclear gene granule bound starch synthase

(called waxy in grasses) which was developed in lab using the genomes of Populus,

Manihot, and Ricinus (Goodstein et al. 2012). The GBSSI forward primer is 5’-

ACTGTRAGCCCTTACTATGC-3’, and the GBSSI reverse primer is 5’-

GTTCCATATCGCATAGCATGC-3’. EMB2765 is a low-copy nuclear gene where exon 9 was chosen because it was easy to amplify and proved to be most successful for amplification across a range of and distant outgroups (Wurdack and Davis,

2009). The forward primer, EMB2765ex9F2, was 5’-

TATCCAAATGAGCAGATTATGTGGGA-3’ and the reverse primer, EMB2765ex9R, was 5’-TTGGTCCAYTGTGCWGCAGAAGGRT-3’, following Wurdack and Davis

(2009).

Thawed samples of DNA were mixed in a 0.2 mL Fisherbrand PCR tube with attached cap according to the following recipe: 25 µL of Takara ExTaq Premix (Otsu,

Shiga, Japan), 10 µL of PAR enhancer, 8 µL of sterile, deionized distilled water, 2.5 µL of forward primer, 2.5 µL of reverse primer and 2.0 µL of sample DNA. Once combined, the 50 µL samples underwent amplification with a PCR regime consisting of 94°C for 3 6 minutes, then 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 min, then 72°C for 2 minutes, and finally stored at 4°C on a Thermo Electron

Corporation PCR Sprint Thermal Cycler (Milford, Massachusetts). The PAR enhancer used here is a polymerase chain reaction additive reagent developed in a study by

Samarakoon et al. (unpubl. results). This enhancer is intended to overcome the effects of

PCR inhibitors, and it is composed of trehalose, bovine serum albumin, and polysorbate-

20 (Tween-20) (Samarakoon et al., unpubl. results).

After amplification, DNA samples went through electrophoresis. The gel used was about 30 mL of 1.0% agarose. 4 µL of the DNA sample was mixed with 1 µL of loading dye and loaded into the wells. 1.5 µL of Promega BenchTop PCR markers

(Madison, Wisconsin) was mixed with 1 µL of loading dye and loaded into the first well to create a lane of standard fragments for determining the size of amplified fragments.

The gel was run at 100 V for approximately 20 minutes and then soaked in a Pyrex dish of distilled water containing 3 µL of ethidium bromide 1% solution for at least 30 minutes. This allowed for staining of any present nucleic acid.

Once the ethidium bromide was allowed to stain the nucleic acids, the gel was fluoresced using a Fisher Scientific ultraviolet light box (Dubuque, Iowa). DNA samples fluorescing orange were noted, double-checked for appropriate size, and put aside for purification and DNA sequencing. These samples were purified using a Qiagen

QIAquick® PCR Purification Kit (QIAGEN Sciences, Maryland) and then packaged for sequencing by Eurofins MWG Operon in Huntsville, Alabama.

Sequences were provided in .abi format from Eurofins and were loaded in

Sequencher 4.7 – Build 2946 (Ann Arbor, Michigan), where they were proofread. 7

Unusable sequence was discarded, and computer-labeled bases were double-checked; those mislabeled or not labeled were corrected, and contigs were formed by automatically assembling the data of the forward and reverse strands. Some samples were not able to form contigs because of unusable forward or reverse sequences, but the usable forward or reverse strands were still used.

With the program ClustalX (Thompson, 1997), sequences were aligned using

“complete alignment.” Using these alignments, bases were again checked against the other DNA samples at particular bases and against the chromatograms in Sequencher.

These sequences were then imported into WinClada (Nixon, 2002) and analyzed using parsimony to infer the phylogenetic tree and using jackknife analysis for statistical support of the phylogenetic relationships. Uninformative characters, which are those that were the same for all samples or only different in one sample, were selected and deactivated before beginning analyses. Heuristic analyses were run on data from

EMB2765, GBSSI, and combined EMB2765 and GBSSI, in each analysis saving 5000 total and initiating 500 replications, with 5 trees saved per replication. The most parsimonious tree(s) was(were) found and saved. If there was more than one parsimonious tree, a strict consensus tree was calculated and saved. A jackknife analysis was then run using 500 replications, each replication consisting of 5 individual replications, saving 2 trees per replication. The jackknife values were then mapped onto the most parsimonious tree or strict consensus tree (Figures 2, 3, and 4). Jackknife analysis involves resampling portions of the available data at random without replacement and seeing how frequently or infrequently the same results are obtained; the 8 more frequently a result is obtained, the more data there are to support that relationship.

This is the number reported above branches in the phylogenetic tree (Farris et al., 1996).

9

Table 1. Species sampled from Samydaceae and associated outgroups for phylogenetic analysis.

Sample Species Voucher EMB2765 GBSSI Tube

OUTGROUP

Hasseltia floribunda 28 Alford 2990 (BH) X

Lacistema aggregatum 24 Alford 3019 (BH) X

Prockia crucis 85 Alford 3132 (BH) X X

INGROUP

Casearia aquifolia 232 Michelangeli 1502 (NY) X

Casearia barteri 191 Stone et al. 5060 (MO) X

Casearia calva 251 Harwood 3 (USMS) X

Casearia 158 Salazar 2638 (BH) X X commersoniana

Casearia flavovirens 253 Harwood 5 (USMS) X

Casearia gladiiformis 142 Roberston 7500 (K) X X

Casearia grandiflora 8 Michelangeli 692 (BH) X

Casearia grewiaefolia 252 Harwood 4 (USMS) X X

Casearia hirtella 185 Victor LR-26709 (US) X

Casearia javitensis 81 Alford 3098 (BH) X

Casearia luetzelburgii 207 Marquete et al. 4061 (RB) X

Casearia nitida 43 Alford 3029 (BH) X 10

Casearia oblongifolia 202 Marquete 4044 (RB) X

Casearia obovalis 82 Alford 3102 (BH) X X

Casearia praecox 131 Stevens 22930 (BH) X

Casearia rupestris 204 Marquete 3170 (RB) X

Casearia sp. 249 Harwood 1 (USMS) X

Casearia sp. 250 Harwood 2 (USMS) X

Casearia sp. 195 Walters 937 (MO) X

Casearia sylvetris 26 Alford 2999 (BH) X X

Casearia tremula 163 Machusa 7051 (NY) X

Casearia velutina 255 Harwood 8 (USMS) X

Euceraea nitida 159 Hobbes 213 (BH) X X

Lunania parviflora 69 Alford 3114 (BH) X X

Ryania speciosa 67 Alford 3118 (BH) X X

Samyda dodecandra 38 Alford 3027 (BH) X X

Samyda mexicana 152 Fishbein 5127 (MISSA) X X

Samyda spinulosa 138 Taylor 11678 (MO) X

Zuelania guidonia 52 Alford 3033 (BH) X X

11

Chapter 3: RESULTS

A total number of 32 species had positive results. Fourteen of these species produced data for only GBSSI. Six of these species produced data for only EMB2765.

Twelve of these sequences produced results for both GBSSI and EMB2765. Forward and reverse copies were obtained for each species for every positive result. Not all results were good sequences, and some characters were determined by forming contigs or through alignment in ClustalX.

After mopping uninformative characters, 112 characters were potentially phylogenetically useful (variable) in EMB2765 data, 227 were useful in GBSSI data, and

339 were useful in the merged matrix of both sets of data. See Table 2 for a comparison of variation with other sampled DNA regions. From the table it is clear that these nuclear regions exhibit almost as much or more variability than the previously sampled regions.

For EMB2765 data, parsimony analysis resulted in four most parsimonious trees, with length 217, consistency index 0.62, and retention index 0.68. For the GBSSI data, there was only one most parsimonious tree with a length of 496, a consistency index of

0.62, and a retention index of 0.71. For the merged matrix of both sets of data, there were

1,082 most parsimonious trees, the length was 715, the consistency index was 0.62, and the retention index was 0.70.

12

Table 2. DNA regions tested for phylogenetic utility.

Locus Base Pairs Informative Chars. * % Infor. Chars.* plastid trnL 650 93 14.3 plastid trnL-F 350 33 10.6 plastid 3’ end ndhF 800 167 4.8 plastid matK 1360 157 8.7 plastid trnH-psbA 300 46 6.5 plastid rpL16 1165 136 8.6 nuclear ITS 990 114 8.7 nuclear EMB2765 810 112 13.8 nuclear GBSSI 715 227 31.7

*Based on current sampling. Gaps not included. (All based on Alford & Samarakoon, unpubl. data, except for GBSSI and EMB2765.)

13

100

100 96

98

100 90

81 100 94

70 82

100

60 100

100 60 75

79

73

100

63

Figure 1. Strict consensus tree of Samydaceae based on morphology, 6 plastid, 1 nuclear, and 1 mitochondrial region (6 MPTs, L=1537, RI=0.79). All taxa except coriacea are represented by at least morphology and two DNA regions. Jackknife values ≥60% are above branches. Based on Alford & Samarakoon, unpubl. data. 14

Figure 2. Phylogeny of Samydaceae based on parsimony analysis of nuclear EMB2765

DNA data. Numbers above the branches are jackknife statistical values.

15

Figure 3. Phylogeny of Samydaceae based on parsimony analysis of nuclear GBSSI

DNA data. Numbers above the branches are jackknife statistical values.

16

Figure 4. Phylogeny of Samydaceae based on parsimony analysis of nuclear GBSSI and

EMB2765 DNA data. Numbers above the branches are jackknife statistical values. 17

Chapter 4: DISCUSSION

While there are some similarities between the trees produced with these nuclear data (Figures 2-4) and the previous plastid data (Figure 1), there are also differences. Not every tree contains the same exact species, so it can be a bit difficult to compare simply by looking at the branches. Compared to the plastid DNA data tree, the trees based on nuclear data are much better supported statistically. As Figure 2 shows, every branch in the EMB2765 tree has a jackknife value >58%. Many branches in the GBSSI tree in

Figure 3 also have high values, but four branches are supported by less than 50%. In the combined EMB2765 and GBSSI nuclear DNA data (Figure 4), the phylogeny is not as well resolved, due to missing data, but all branches except one in the strict consensus tree have values >70%.

By looking at the number of variable sites (Table 2) and jackknife values (Figures

2-4), the nuclear DNA data surpass that of the plastid data. More branches in the plastid

DNA data have low jackknife values. The tree based on plastid DNA includes data from more sampled species. However, the trees agree with each other on some broad points.

The one major difference is the occurrence of Casearia velutina at the base of the

EMB2765 and combined trees. Given that Casearia velutina has all of the features of a

‘standard’ Casearia, this result is likely incorrect or based on a mis-identified sample.

Further study of the voucher specimen or a re-amplification of the DNA should resolve this issue.

Consistent with the data from Figures 2-4, there are still some genera nested within Casearia, including Samyda, , and . Some Casearia (C. 18 commersoniana and C. javitensis) are more closely related to , as predicted from morphological data because they have staminodes that occur within the stamens and have a branched style, just like Ryania. Overall, the data yielded by the EMB2765 and GBSSI nuclear genes affirms the previous findings using plastid DNA, but with higher statistical support. Thus, these genes appear to be appropriate regions for the continued study of relationships within Samydaceae. 19

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