GENOMIC PERSPECTIVES ON EVOLUTION IN BRACKEN FERN

by

Joshua P. Der

A dissertation submitted in partial fulfillment

of the requirements for the degree

of

DOCTOR OF PHILOSOPHY

in

Biology

Approved:

! ! !
Paul G. Wolf! ! Michael E. Pfrender
Major Professor! ! Committee Member

! ! !
Carol D. VonDohlen! ! Karen Mock
Committee Member! ! Committee Member

! ! !
Paul Cliften! ! Byron Burnham
Committee Member! ! Dean of Graduate Studies

UTAH STATE UNIVERSITY

Logan, Utah

2010
 ii
ABSTRACT

Genomic Perspectives on Evolution in Bracken Fern

by

Joshua P. Der, Doctor of Philosophy

Utah State University, 2010

Major Professor: Paul G. Wolf

Department: Biology

The fern genus Pteridium comprises a number of closely related species

distributed throughout the world. Collectively they are called bracken ferns and have

historically been treated as a single species, Pteridium aquilinum. Bracken is notorious

as a toxic weed that colonizes open fields and poisons livestock. Bracken is also easily

cultured and has become one of the most intensively studied ferns. Bracken has been

used as a model system for the study of the fern life cycle, fern gametophyte

development, the pheromonal mechanism of sex determination, toxicology, invasion

ecology, and climate change. This dissertation places bracken within a global

evolutionary perspective and establishes bracken as an emerging system for

evolutionary genomics in ferns. Bracken samples from around the world were examined

for chloroplast DNA variation to infer historical phylogenetic and biogeographic

evolutionary events. New high-throughput DNA sequencing technologies and

bioinformatic approaches were used to determine the complete chloroplast genome

sequence in bracken, to identify novel RNA editing sites in chloroplast transcripts, and to

identify gene sequences that are expressed in the gametophyte stage of the fern life
 iii
cycle. These data represent an important genomic resource in ferns and were examined

within a functional and evolutionary perspective. Several novel approaches and analyses

were developed in the course of this research. Results presented in this dissertation

provide novel insights into fern biology and land plant evolution.

(105 pages)
 iv

I dedicate this dissertation to my family.

To Kristal, whose love and support has made this work possible.

To Cora and Tyler who have shown me the love and joy of fatherhood.

To my parents who fostered in me a sense of curiosity and a love of nature.

To Jessica, whose friendship helped shape the man that I have become;

your memory lives on in my heart.

 v
ACKNOWLEDGMENTS

I wish to thank my advisor, Paul Wolf, and my committee members, Michael

Pfrender, Carol vonDohlen, Paul Cliften, Eugene Schupp, and Karen Mock. I also wish

to thank my coauthors on published and future manuscripts resulting from this work for

significant contributions to the research presented in this dissertation. In addition to Paul

Wolf, these people include John Thomson and Jeran Stratford (Chapter 2); Aaron Duffy,

Matt Kusner, Chen Gu, and Paul Overvoorde (Chapter 3); and Michael Barker, Norman

Wickett, and Claude dePamphilis (Chapter 4).

Numerous people contributed plant material used in Chapter 2 and are credited

in Appendix B. Jessie Roper, Mark Ellis, Aaron Duffy, Jacob Davidson, Jeran Stratford,

Amanda Grusz, Eric Wafula, Paul Cliften and Michael Pfrender assisted with lab or

bioinformatics work. Alan Smith, Laura Forrest, Mark Ellis, Aaron Duffy, and Kristal

Watrous provided useful comments on portions of text or complete manuscripts included

in this dissertation.

This research was funded in part by National Science Foundation grant

DEB-0228432 to Paul Wolf. The Genomics Education Partnership with grant 52005780

from the Howard Hughes Medical Institute to Professor Sarah Elgin (Washington

University), with additional support from the National Human Genome Research

Institute, and the The Genome Center at Washington University School of Medicine

contributed a portion of the genome sequence data used in Chapter 3. Jeffrey Boore and

Brent Mishler assisted with the collection of another portion of the genome sequence

data. Kenneth White and Ninglin Yin donated resources from Center for Integrated

Biosystems (CIB) at Utah State University (USU) to collect additional genome sequence

data.
 vi
The CIB provided initial funding for my proposal to sequence expressed genes in

bracken gametophytes with a student research grant. Additional funds from the Office of

the Vice President for Research at USU, in a research catalyst grant to Paul Wolf,

enabled me to utilize the Roche 454 sequencing technology to analyze the complete

gametophyte transcriptome in bracken. Keithanne Mockaitis supervised the cDNA library

preparation and transcriptome sequencing at the Center for Genomics and

Bioinformatics at Indiana University.

Computer time from the Center for High Performance Computing at Utah State

University is acknowledged. The computational resources were provided through the

National Science Foundation under Grant No. CTS-0321170 with matching funds

provided by Utah State University.

I also want to thank the friends I have made during my stay in Utah: Ellen Klinger,

Mark Ellis, Craig Faulhaber, Debi Fisk, Jake Davidson, Ryan OʼDonnell, Maure Smith,

Dave Brown, Sid Wright, and the Strange, Scoville, Spaulding, Latta, Wilson,

Burningham, and Duffy families. You all have encouraged me and helped me pass the

time, each in your own special ways.

Joshua P. Der
 vii
CONTENTS

Page

ABSTRACT!........................................................................................................................ ii

DEDICATION!.................................................................................................................... iv

ACKNOWLEDGMENTS!.................................................................................................... v

LIST OF TABLES!.............................................................................................................viii

LIST OF FIGURES!........................................................................................................... ix

CHAPTER

1. INTRODUCTION!....................................................................................................1

2. GLOBAL CHLOROPLAST PHYLOGENY AND BIOGEOGRAPHY OF

BRACKEN (PTERIDIUM: DENNSTAEDTIACEAE)...............................................
! 7

3. COMPLETE CHLOROPLAST GENOME SEQUENCE AND HIGH LEVELS

OF RNA EDITING IN THE FERN PTERIDIUM AQUILINUM DETERMINED
BY MASSIVELY PARALLEL PYROSEQUENCING.............................................
! 32

4. DE NOVO CHARACTERIZATION OF THE GAMETOPHYTE

TRANSCRIPTOME IN BRACKEN FERN, PTERIDIUM AQUILINUM..................
! 49

5. CONCLUSIONS!...................................................................................................79

APPENDICES

A. PERMISSION TO REPRINT CHAPTER 2!........................................................... 82

B. VOUCHER INFORMATION FOR CHAPTER 2!................................................... 88

C. ADDITIONAL FILES FOR CHAPTER 4!............................................................... 91

CURRICULUM VITAE!......................................................................................................92
 viii
LIST OF TABLES

Table! Page

4-1! Transcriptome sequence statistics!....................................................................... 53

4-2! Assembly summary statistics!............................................................................... 55

4-3! Transcriptome coverage estimates!...................................................................... 57

4-4! Taxonomic distribution of unigene blastx hits in the nr database!......................... 59

 ix
LIST OF FIGURES

Figure! Page

2-1! Bayesian phylogeny ............................................................................................

! 17

2-2! Biogeography of bracken.....................................................................................

! 19

3-1! Gene map of the Pteridium aquilinum chloroplast genome!................................. 39

3-2! Chloroplast genome sequence coverage!............................................................. 41

3-3! Density of RNA editing sites!.................................................................................43

4-1! Overview of P. aquilinum transcriptome sequencing and assembly ....................

! 54

4-2! Unigene accumulation curve!................................................................................58

4-3! Distribution of GO-slim functional categories!....................................................... 61

4-4! Unigene overlap with Arabidopsis, Physcomitrella, and Selaginella....................

! 63
 CHAPTER 1

INTRODUCTION

Evolutionary studies are increasingly looking to molecular genetic data to inform

inference because of the wealth of historical information archived in the genome. Early

evolutionary genetic studies relied on markers such as DNA restriction sites or various

types of DNA fragment profiles, so called "genetic fingerprints” (Avise, 2004). Direct

access to the historical record in the genome has been enabled by methods for

sequencing DNA (Hillis et al., 1996; Soltis et al., 1998), allowing us to connect an

organismʼs genotype with variation in form and function in cells, tissues, and whole

organisms. New methods in molecular biology are rapidly advancing our knowledge of

gene and genome function using tools such as gene expression microarrays, genetic

knockout mutants, and CHip- and Methyl-seq enrichment procedures. Despite the

advances these methods are driving in biotechnology, it is critical to place these

discoveries within an evolutionary context to fully comprehend these complex systems.

Evolutionary studies, especially those using genetic data, often face a trade-off

between resources devoted to sampling breadth (i.e. number of units to be surveyed)

and sampling depth (i.e. the quantity of data collected about each unit). For example,

when inferring the evolutionary relationships among members of a group, a researcher

must decide how many individuals/taxa to sample, often at the expense of the number of

character traits that can be collected and analyzed. How this balance shifts will largely

be determined by the research objectives, the scope and scale of the project, and the

available resources to conduct the research.

 2
As the cost of DNA sequencing drops with new advances in high-throughput

technologies (Shendure and Ji, 2008; Ansorge, 2009), the trade-off between sampling

breadth and depth is becoming less important for classic types of evolutionary studies.

However, high-throughput DNA sequencing is enabling new kinds of evolutionary

analysis on a whole-genome scale (Rokas and Abbot, 2009). Ironically, studies using

massive throughput sequencing technologies continue to push the limits of what is

possible within the bounds of current technology and available resources, bringing the

trade-off between sampling breadth and depth to a whole new scale.

Often, different sampling strategies are required to examine evolutionary

processes in different aspects of a study system. These alternative sampling strategies

can be complementary, and yield results that provide insight for interpreting the results

from other components of the research program. This dissertation seeks to highlight the

reciprocal illumination garnered by utilizing dramatically different approaches to broadly

and deeply sample genetic data to infer historical and contemporary evolutionary

processes. I present three studies, which use traditional, and emergent, high-throughput

DNA sequencing approaches to sample genetic information to examine evolution in the

bracken fern, Pteridium.

Pteridium is a cosmopolitan fern genus comprised of several closely related

species, which are well differentiated from other genera in the family Dennstaedtiaceae

(Tryon, 1941). Bracken is notorious as a weed in open fields and is toxic to people and

livestock (Holm et al., 1997). Despite its toxicity, bracken is eaten as a delicacy in

several parts of the world and, due to its often high local abundance and large coarse

stature, is sometimes used as thatching or packing material. Because bracken is

common, easily cultured and manipulated, and can impose large economic
 3
consequences, it has become one of the most intensively studied ferns. Bracken has

been used as a model system for the study of the fern life cycle (Webster and Steeves,

1958; Gottlieb, 1959; Takahashi, 1961; DeMaggo and Raghavan, 1972; Sheffield and

Bell, 1981; Sheffield, 1984, 1985, 2008; Wynn et al., 2000), gametophyte development

and the pheromonal mechanism of sex determination (Steeves et al., 1955; Näf, 1958;

Whittier, 1962; Sobota and Partanen, 1966; Bell and Duckett, 1976; Ashcroft and

Sheffield, 2000; Robertson, 2002; Schneller, 2008), cyanogenesis (Cooper-Driver and

Swain, 1970), carcinogenesis (Potter and Baird, 2000; Alonso-Amelot and Avendano,

2002; Beniston and Campo, 2003), invasion ecology (Schneider, 2004; Rodrigues da

Silva and Matos, 2006; Ghorbani et al., 2007), and climate change (Pakeman and Marrs,

1996).

Chapter 2 sets the stage for all subsequent evolutionary work in the genus

Pteridium by providing a sound phylogenetic and biogeographic framework from which I

can appropriately sample taxa for detailed studies. I adopted a broad and shallow

sampling strategy to examine ancient historical events in Pteridium, which led to

speciation and the spread of bracken around the world. This study examined chloroplast

DNA variation within Pteridium in a global phylogenetic context to ascertain evolutionary

patterns of divergence, dispersal and colonization. These data were also used to

examine the maternal ancestry of two tetraploid taxa hypothesized to be of hybrid origin.

Chapter 3 utilized a focused and deep sampling strategy to obtain the complete

chloroplast genome sequence of Pteridium aquilinum ssp. aquilinum which, combined

with deep RNA sequencing, enabled a detailed examination of RNA editing in the

chloroplast transcriptome. The complete nucleotide sequence of the chloroplast genome

 4
enabled us to take a comparative genomic approach to assess structural and sequence

level changes in the chloroplast genomes among leptosporangiate ferns.

Chapter 4 examined the same deep RNA sequencing data set presented in

Chapter 3, but takes advantage of a physical sample preparation procedure (cDNA

normalization) and a shift in perspective to broadly sample expressed genes in the very

large nuclear genome of Pteridium (nearly 10 Gb, three times the size of the human

genome). These sequences were derived from the gametophyte stage of the plant life

cycle, providing a fertile data set for investigation into the evolution of life history and

reproduction in ferns. These data also provide the first systems-level view of genome

composition in ferns, which I examined within a comparative evolutionary genomics

framework. I also bioinformatically mined these sequences to infer the functional

composition of genes expressed in this life stage.

Together these three studies illustrate the complementarity of different sampling

strategies to develop a research program in evolutionary genetics and genomics in a

non-model organism.

LITERATURE CITED

ALONSO-AMELOT, M. E., AND M. AVENDANO. 2002. Human carcinogenesis and bracken

fern: a review of the evidence. Current Medicinal Chemistry 9: 675-686.

ANSORGE, W. J. 2009. Next-generation DNA sequencing techniques. Nature

Biotechnology 25: 195-203.

ASHCROFT, C. J., AND E. SHEFFIELD. 2000. The effect of spore density on germination and
development in Pteridium, monitored using a novel culture technique. American Fern
Journal 90: 91-99.

AVISE, J. C. 2004. Molecular markers, natural history, and evolution, second edition.
Sinauer Associates, Sunderland, Massachusetts, USA.
 5
BELL, P. R., AND J. G. DUCKETT. 1976. Gametogenesis and fertilization in Pteridium.
Botanical Journal of the Linnean Society 73: 47-78.

BENISTON, R. G., AND M. S. CAMPO. 2003. Quercetin elevates p27(Kip1) and arrests both
primary and HPV16 E6/E7 transformed human keratinocytes in G1. Oncogene 22:
5504-5514.

COOPER-DRIVER, G. A., AND T. SWAIN. 1970. Cyanogenic polymorphism in bracken in

relation to herbivore predation. Nature 260: 604.

DEMAGGO, A. E., AND V. RAGHAVAN. 1972. Germination of bracken fern spore.

Experimental Cell Research 73: 182-186.

GHORBANI, J., M. G. LE DUC, H. A. MCALLISTER, R. J. PAKEMAN, AND R. H. MARRS. 2007.

Effects of experimental restoration on the diaspore bank of an upland moor degraded
by Pteridium aquilinum invasion. Land Degredation and Development 18: 659-669.

GOTTLEIB, J. E. 1959. Development of the bracken fern, Pteridium aquilinum (L.) Kuhn.—
II. stelar ontogeny of the sporeling. Phytomorphology 9: 91-105.

HILLIS, D. M., C. MORITZ, AND B. K. MABLE. 1996. Molecular systematics, second edition.
Sinauer Associates, Sunderland, Massachusetts, USA.

HOLM, L., J. DOLL, E. HOLM, J. PANCHO, AND J. HERBERGER. 1997. World weeds: Natural
histories and distribution. John Wiley and Sons, New York, New York, USA.

NÄF, U. 1958. On the physiology of antheridium formation in the bracken fern, Pteridium
aquilinum (L.) Kuhn. Physiologia Plantarum 11: 728-746.

PAKEMAN, R. J., AND R. H. MARRS. 1996. Modelling the effects of climate change on the
growth of bracken (Pteridium aquilinum) in Britain. Journal of Applied Ecology 33:
561-575.

POTTER, D. M., AND M. S. BAIRD. 2000. Carcinogenic effects of ptaquiloside in bracken

fern and related compounds. British Journal of Cancer 83: 914-920.

ROBERTSON, F. W. 2002. Reproductive behavior of cloned gametophytes of Pteridium

aquilinum (L.) Kuhn. American Fern Journal 92: 270-287.

RODRIGUES DA SILVA, U. D. S., AND D. M. D. S. MATOS. 2006. The invasion of Pteridium

aquilinum and the impoverishment of the seed bank in fire prone areas of Brazilian
Atlantic forest. Biodiversity and Conservation 15: 3035-3043.

ROKAS, A., AND P. ABBOT. 2009. Harnessing genomics for evolutionary insights. Trends in
Ecology and Evolution 24: 192-200.

SCHNEIDER, L. C. 2004. Bracken fern invasion in southern Yucatan: A case for land-
change science. Geographical Review 94: 229-241.
 6
SCHNELLER, J. J. 2008. Antheridiogens. In T. A. Ranker and C. H. Haufler [eds.], Biology
and evolution of ferns and lycophytes, 134-158. Cambridge University Press,
Cambridge, UK.

SHEFFIELD, E. 1984. Apospory in the fern Pteridium aquilinum (L) Kuhn 1: Low
temperature scanning electron microscopy. Cytobios 39: 171-176.

SHEFFIELD, E. 1985. Cellular aspects of the initiation of aposporous outgrowths in ferns.

Proceedings of the Royal Society B 86: 45-50.

SHEFFIELD, E. 2008. Alternation of generations. In T. A. Ranker, and C. H. Haufler [eds.],

Biology and evolution of ferns and lycophytes, 49-74. Cambridge University Press,
Cambridge, UK.

SHEFFIELD, E., AND P. R. BELL. 1981. Cessation of vascular activity correlated with
aposporous development in Pteridium aquilinum (L.) Kuhn. New Phytologist 88:
533-538.

SHENDURE, J., AND H. JI. 2008. Next-generation DNA sequencing. Nature Biotechnology
26: 1135-1145.

SOBOTA, A. E., AND C. R. PARTANEN. 1966. The growth and division of cells in relation to
morphogenesis in fern gametophytes I. Photomorphogenetic studies in Pteridium
aquilinum. Canadian Journal of Botany 44: 497-506.

SOLTIS, D. E., P. S. SOLTIS, AND J. J. DOYLE. 1998. Molecular systematics of plants II:
DNA sequencing. Kluwer Academic Publishers, Boston, Massachusetts, USA.

STEEVES, T. A., I. M. SUSSEX, AND C. R. PARTANEN. 1955. In vitro studies on abnormal

growth of prothalli of the bracken fern. American Journal of Botany 42: 232-245.

TAKAHASHI, C. 1961. Chromosome study on induced apospory in the bracken fern. La

Kromosomo 48: 16021605.

TRYON, R. M. 1941. A revision of the genus Pteridium. Rhodora 43: 1-67.

WEBSTER, B. D., AND T. A. STEEVES. 1958. Morphogenesis in Pteridium aquilinum (L.)

Kuhn - general morphology and growth habit. Phytomorphology 8: 30-41.

WHITTIER, D. P. 1962. The origin and development of apogamous structures in the

gametophyte of Pteridium in sterile culture. Phytomorphology 12: 10-20.

WYNN, J. M., J. L. SMALL, R. J. PAKEMAN, AND E. SHEFFIELD. 2000. An assessment of

genetic and environmental effects on sporangial development in bracken [Pteridium
aquilinum (L.) Kuhn] using a novel quantitative method. Annals of Botany 85:
113-115.
 7
CHAPTER 2

GLOBAL CHLOROPLAST PHYLOGENY AND BIOGEOGRAPHY OF BRACKEN

(PTERIDIUM: DENNSTAEDTIACEAE)1

ABSTRACT

Bracken ferns (genus Pteridium) represent an ancient species complex with a

natural worldwide distribution. Pteridium has historically been treated as comprising a

single species, but recent treatments have recognized several related species.

Phenotypic plasticity, geographically structured morphological variation, and

geographically biased sampling have all contributed to taxonomic confusion in the

genus. We sampled bracken specimens worldwide and used variable regions of the

chloroplast genome to investigate phylogeography and reticulate evolution within the

genus. Our results distinguish two major clades within Pteridium, a primarily northern

hemisphere Laurasian/African clade, which includes all taxa currently assigned to P.

aquilinum, and a primarily southern hemisphere Austral/South American clade, which

includes P. esculentum and P. arachnoideum. All European accessions of P. aquilinum

subsp. aquilinum appear in a monophyletic group and are nested within a clade

containing the African P. aquilinum taxa (P. aquilinum subsp. capense and P. aquilinum

subsp. centrali-africanum). Our results allow us to hypothesize the maternal progenitors

of two allotetraploid bracken species, P. caudatum and P. semihastatum. We also

discuss the biogeography of bracken in the context of the chloroplast phylogeny. Our

1 Citation:
Der, J. P., J. A. Thomson, J. K. Stratford, and P. G. Wolf. 2009. Global chloroplast
phylogeny and biogeography of bracken (Pteridium; Dennstaedtiaceae). American Journal of
Botany 96(5): 1041-1049. Permission to reprint in Appendix A.
 8
study is one of the first to take a worldwide perspective in addressing variation in a

broadly distributed species complex.

INTRODUCTION

Describing any biological characteristic of organisms requires adequate sampling

so that statements are universally valid. Thus, one should consider the range of variation

within the taxon (be it taxonomic, morphological, physiological, ecological, genetic, or

geographic) to accurately describe diversity within the group (Hillis, 1998). To capture

such variation, one must sample sufficiently to understand both the limits and typical

ranges of variation. Such sampling means collecting and examining representatives

across the distribution of the taxon. In general, sampling across a taxonʼs range is easier

to do for lower taxonomic levels because successively smaller clades encompass

increasingly narrow ranges. However, sampling across geographic ranges becomes

logistically challenging for species or species-complexes with worldwide (or nearly so)

distributions.

Examples of widespread species for which a global perspective has been taken

include highly mobile birds (Burg and Croxall, 2004), invertebrates (Lee, 2000; Boyer et

al., 2007), and fungi (Hibbett, 2001; Banke and McDonald, 2005). Within green plants,

worldwide distributions are common among “invasive” species, i.e., those with recent

(less than 500 years), human-mediated, widespread distributions. Examples include

aquatic weeds (Barrett, 1989) among others (Holm et al., 1997). At least one study has

examined evolutionary history in a widespread invasive species, Cardamine flexuosa

(Lihová et al., 2006), but few examples, if any, have examined the phylogenetic structure

of a species complex with an ancient (preagricultural; 10,000 years) worldwide

 9
distribution (but see Bakker et al., 1995). For a species to occupy such a broad range, it

must have a wide ecological amplitude (i.e., ecological valence) or be able to adapt

quickly to a wide range of local environmental conditions without speciation. Additionally,

to achieve a wide distribution, a species must have a high dispersal and colonization

ability, or be an old taxon with a broad ancestral distribution, or both. The genus

Pteridium Gled. ex Scop. (bracken fern, Dennstaedtiaceae) represents one such taxon,

having achieved a natural worldwide distribution and occupying diverse habitats (Page,

1976, 1986; Holm et al., 1997). Fossil evidence indicates that bracken had achieved a

worldwide distribution by the Oligocene, ~23.8 mya (reviewed by Page, 1976), and

several lines of evidence indicate that bracken can disperse and establish following long

distance dispersal by spores (Punetha, 1991; Rumsey et al., 1991).

Pteridium is often treated as a monotypic genus after Tryon (1941), but most

contemporary systematists recognize the genus as a species complex in need of

taxonomic revision (Page, 1976; Brownsey, 1989; Page and Mill, 1995; Thomson,

2000b). Bracken ferns are easily recognized and well differentiated from other genera in

Dennstaedtiaceae, and many infrageneric taxa within Pteridium have high levels of

geographically based morphological structure. However, great confusion in defining

infrageneric taxa has resulted from the fact that bracken has high levels of phenotypic

plasticity, few diagnostic morphological characters, and the presence of intermediate

phenotypes where different morphological forms come into contact, demonstrating that

reproductive barriers are incomplete (Page, 1976). These factors, coupled with local

taxonomic judgments based on geographically biased sampling, have led to a large

number of local forms being described as new species, subspecies, or varieties,

resulting in a multiplicity of names (Tryon, 1941; Page, 1976; Thomson, 2000a). The
 10
genus is distributed worldwide and is notorious as a weed because of its exceptional

ability to grow rhizomatously in dense patches, overgrowing open fields and pasture

(Tryon, 1941; Holm et al., 1997).

Bracken ferns have a long and complex taxonomic history. The first bracken

species were described by Linnaeus in the genus Pteris L. (Linnaeus, 1753). Later

authors followed this generic circumscription, but Agardh (1839) was the first to examine

specimens worldwide and set the brackens apart as section Ornithopteris J.Agardh

(Tryon, 1941; Brownsey, 1989). Later, Hooker (1858), in a comprehensive treatement of

Pteris, subsumed all brackens as varieties of Pteris aquilina L. Various authors

segregated the brackens from Pteris, but it was not until Kuhn (1879) defined Pteridium

aquilinum (L.) Kuhn that the brackens were widely accepted as a distinct genus (Tryon,

1941). The last global revision of the genus was Tryonʼs (1941) monograph, in which he

reduced more than 135 previously named variants into a single species, with two

subspecies containing 12 varieties. Subsequent authors have continued to modify the

taxonomy of bracken, but most works reflect a geographically limited perspective

(Brownsey, 1989; Page and Mill, 1995; Wolf et al., 1995; Speer, 2000; Thomson and

Alonso-Amelot, 2002; Gureyeva and Page, 2005; Thomson et al., 2005, 2008). Recent

evidence suggests that two Pteridium taxa have allopolyploid hybrid origins, and these

two tetraploids are currently recognized as segregate species (Tan and Thomson,

1990b; Thomson, 2000a, b; Thomson and Alonso-Amelot, 2002).

Recent work has reexamined the systematic utility of morphological characters

(Thomson and Martin, 1996), characterized the structure of the chloroplast genome (Tan

and Thomson, 1990a; Tan, 1991), and used genetic fingerprinting to examine

evolutionary history in Pteridium (Thomson, 2000a, b). Much of this work contributes
 11
toward a taxonomic revision of the genus. The typification of Pteridium aquilinum (L.)

Kuhn has been revisited, and nomenclature within ʻlatiusculumʼ morphotypes (N.

America, Europe and northeast Asia) has been clarified (Thomson, 2004; Thomson et

al., 2008).

Global perspectives on phylogeography can be inferred using chloroplast DNA

sequence variation to detect and reconstruct historical evolutionary events, including

population demographics, migration, colonization, and both ancient and contemporary

hybridization events (Rieseberg and Soltis, 1991; McCauley, 1995; Rieseberg et al.,

1996; Ennos et al., 1999). Variation in chloroplast sequence data has been used to

reconstruct phylogenetic relationships among plants as diverse and ancient as land

plants to variation among recently diverged populations within a single species.

Pteridium is a well-defined and evolutionarily isolated genus comprising several closely

related taxa. Examining patterns of chloroplast variation presents an excellent

opportunity to infer patterns of widespread dispersal, colonization, and divergent

evolutionary history on a global scale. This study represents one of the only studies of

worldwide variation in a terrestrial plant.

The objectives of this present study are threefold. First, we examine chloroplast

DNA variation within Pteridium in a global phylogenetic context to ascertain evolutionary

patterns of divergence, dispersal and colonization. Second, we examine maternal

ancestry in the two tetraploid taxa hypothesized to be of hybrid origin. Third, we

establish a clear framework for developing hypotheses of evolutionary history that can

be tested with comparative nuclear sequence data. These results also provide important

information necessary for taxonomic revision of infrageneric taxa in Pteridium.

 12
MATERIALS AND METHODS

Taxonomic sampling—

Sampling was designed to cover the range of morphological and geographical

diversity within Pteridium, representing nearly all currently recognized species, and most

infraspecific taxa, with the exception of P. aquilinum subsp. feei (W. Schaffn. ex Fée) J.

A. Thomson, Mickel & Mehltreter, endemic to central Mexico. Determination of

specimens used in this study follow Thomson and Alonso-Amelot (2002), Thomson

(2004), and Thomson et al. (2005, 2008). In addition to the materials used here, a large

series of specimens from major herbaria has been examined in the course of the

taxonomic revisions listed. We sampled 77 bracken specimens, most of which were

included previously in the taxonomic studies described. Tissue samples were collected

from either wild sources or from sporophytes grown in a common garden derived from

known wild sources and propagated from rhizome segments or mass spore sowings

(Thomson, 2000a). Complete voucher information with geographic sources and

GenBank accession numbers is provided in Appendix B.

DNA extraction, PCR amplification, and sequencing—

Total genomic DNA was extracted from tissue that had been silica-dried, freeze-

dried, or pickled in CTAB/NaCl/ascorbate (Thomson, 2002). The chloroplast markers

trnSGGA–rpS4 spacer+gene and rpL16 intron were amplified in 25 µL polymerase chain

reactions (PCR) using the fern-specific primers published in Small et al. (2005). Each

PCR reaction contained 25–50 ng of template DNA, 1× Promega PCR buffer (Promega,

Madison, Wisconsin, USA), 1.5 mM MgCl2, 0.20 mM of each dNTP, 0.25 µM each of the

forward and reverse primers, and ~12.5 U Taq polymerase. Most PCR reactions were
 13
completed under a standard temperature cycle procedure beginning with a 2 min

denaturation step at 94°C; followed by 30 cycles of 94°C, 48°C and 72°C each for 1 min;

finishing with a 7 min elongation step at 72°C. Problematic samples were amplified using

a slow ramp thermal cycle protocol that began with a 2 min denaturation step at 95°C,

followed by 30 cycles in which the sample was denatured at 95°C for 1 min, dropped to

45°C for 1 min, then slowly raised to 68°C at a rate of 0.2°C/sec and held at 68°C for 4

min. After cycling, reactions were held at 68°C for 10 min to complete elongation of PCR

products. PCR products were directly cycle-sequenced in both directions using the PCR

primers and ABI BigDye Terminator version 3.1 chemistry (Applied Biosystems, Foster

City, California, USA).

Sequence alignment and phylogenetic analyses—

Sequences were checked against electropherograms and manually edited, if

necessary, using the program 4Peaks version 1.7 (Griekspoor and Groothuis, 2006).

Sequences were manually aligned using the program Se-Al version 2.0a11 (Rambaut,

2002). The aligned concatenated data matrix is available in TreeBase (http://

www.treebase.org; study number S2235). Maximum parsimony (MP) analyses were

performed on the two-marker concatenated data set in the program PAUP* version

4.0b10 (Swofford, 2002). All nucleotide site characters were unordered and equally

weighted, treating gaps as “missing” data. Heuristic MP searches used tree-bisection-

reconnection (TBR) branch swapping on starting trees generated from 10,000 random

stepwise addition sequence replicates, holding 10 trees at each addition step. All the

most parsimonious trees were saved, and the strict consensus of these was calculated.

MP bootstrap support was assessed from 10,000 bootstrap replicates using heuristic
 14
searches with TBR branch swapping on starting trees generated from 10 random

stepwise addition sequence replicates and saving all the most parsimonious trees.

Models of DNA sequence evolution used in Bayesian phylogenetic inference (BI)

were selected for each chloroplast marker using the second order Akaike information

criterion (AICc) implemented in the program MrModelTest version 2.2 (Nylander, 2004),

using likelihood scores estimated in PAUP* for the neighbor-joining tree under

alternative models of evolution. The total number of alignment sites in each data partition

was used as the sample size for AICc calculations (Posada and Buckley, 2004). Akaike

weights and evidence ratios were examined to help guide selection of the best-fit model

of molecular evolution for each partition (Burnham and Anderson, 2002).

Bayesian analysis was performed in the parallel version of the program MrBayes

version 3.1.2 (Ronquist and Huelsenbeck, 2003; Altekar et al., 2004). Data were

partitioned for the two chloroplast markers, allowing model parameters for each partition

to vary independently while linking topology and branch lengths across partitions. Two

independent runs, with six chains each, were conducted simultaneously for 10,000,000

generations. Parameter estimates were sampled every 1000 generations. The average

standard deviation of split frequencies and the potential scale reduction factor (PSRF)

were calculated after discarding the first 25 percent of the samples (2.5 million

generations) as burn-in to assess topological and parameter convergence (respectively)

between the two runs. BI clade credibility values (i.e., posterior probabilities for clades)

and tree posterior probabilities were also calculated after the first 2.5 million generations

were discarded as burn-in.

Alignment of two outgroup taxa (Dennstaedtia davalloides (R. Br.) T. Moore and

Hypolepis muelleri N. A. Wakef.) with the Pteridium sequences was not possible due to a
 15
high level of divergence (both sequence substitution saturation and ambiguous indels).

Inclusion of these taxa in preliminary phylogenetic analyses contributed to the

destabilization of clades within Pteridium. Because of this, the midpoint rooting method

implemented in PAUP* was used to root the Pteridium phylogeny in both MP and BI

analyses. While this approach is not ideal, it has yielded acceptable results in other taxa

when appropriate data are not available (Schuettpelz and Hoot, 2006). The monophyly

of Pteridium and the midpoint root was confirmed by outgroup phylogenetic analysis of

rbcL sequences from a subset of our sampled Pteridium taxa aligned with additional

outgroup taxa from Dennstaedtiaceae (data not shown).

Biogeographic analysis—

Broad geographic regions were mapped onto the phylogeny using the parsimony

criterion to elucidate major historical biogeographic events. Geographic areas were

coded as six unordered character states (Hawaii, North/South America, Europe, Asia/

India, Africa, Austral) and traced onto the tree using the program Mesquite version 2.5

(Maddison and Maddison, 2008). Specimens in cultivation were coded from the

geographic area of their source.

RESULTS

Sequence characteristics—

The aligned trnS–rpS4 spacer+gene and rpL16 intron data matrices included

1018 and 753 nucleotide sites with 29 and 25 variable sites, respectively. Of those

variable sites, 22 were parsimony-informative in trnS–rpS4 spacer+gene and 21 sites

were parsimony-informative in rpL16 intron. There were two 5-bp repeat sequence

indels located 32 nucleotides apart in the trnS–rpS4 spacer. Individuals either lacked
 16
one or both of the repeat sequences, resulting in three observed indel haplotypes. These

haplotypes have been reported previously (Thomson et al., 2008), and our study is

consistent with those findings, so we adopt their nomenclature here (haplotypes A, B,

and C). This study newly establishes the haplotypes for Pteridium aquilinum subsp.

decompositum (Gaudich.) Lamoureux ex J. A. Thomson, P. caudatum (L.) Maxon, and P.

semihastatum (Wall. ex J. Agardh) S. B. Andrews, and extends coverage of P. a. subsp.

pinetorum (C. N. Page & R. R. Mill) J. A. Thomson to eastern Europe. Haplotype B (the

presence of the first repeat sequence, GTTTT) was observed in P. a. subsp. aquilinum

from Europe and both P. a. subsp. centrali-africanum Hieron. and P. a. subsp. capense

(Thunb.) C. Chr. in Africa, while haplotype C (the presence of the second repeat

sequence, AGTCT) was observed in P. arachnoideum (Kaulf.) Maxon in Central and

South America, P. esculentum (G. Forst.) Cockayne in Australia, New Zealand, and New

Caledonia, P. semihastatum in Australia and Malaysia, and a single accession of P.

caudatum from Costa Rica. Haplotype A (the absence of both repeat sequences) was

observed in the remaining taxa (i.e., P. aquilinum from Asia and North America and the

remaining P. caudatum accessions). Comparison of bracken haplotypes with those

found in Dennstaedtia davalliodes and Hypolepis muelleri reveals that haplotype C is

likely to be plesiomorphic. Parsimony character reconstruction of these indels on our

phylogeny indicates that the second repeat was lost in the common ancestor of P.

aquilinum, and the first repeat was gained in the common ancestor of the African and

European P. aquilinum subspecies. The evolution of these indels is mapped on the

Bayesian phylogeny (Figure 2-1).


Loss of second repeat
-> Haplotype A
100/1.0
Gain of first repeat
-> Haplotype B
100/0.9
Ancestral haplotype
(presence of second repeat,
absense of first repeat)
-> Haplotype C
0.3 substitutions/site
Figure 2-1. Bayesian phylogeny. Phylogram inferred from the concatenated trnS-rpS4
spacer+gene and rpL16 intron alignment. Branch lengths are proportional to the number
of nucleotide substitutions/site. Support values from maximum parsimony bootstrap
analysis (BS) and Bayesian posterior probabilities for clades (PP) are given above
branches (BS/PP). Clades receiving less than 50% BS or 0.5 PP are indicated with a
dash (—).
17
63/0.99 P. caudatum, 238 HECR, Costa Rica
P. caudatum, 274 QCOL, Colombia
P. caudatum, MD2-2VENZ, Venezuela
64/0.98 P. a. ssp. latiusculum, 147 WMCH, USA, Michigan
P. a. ssp. pseudocaudatum, 169 FLUS, USA, Florida
P. a. ssp. pseudocaudatum, 203 HFLA, USA, Florida
P. a. ssp. pseudocaudatum, Der 68, USA, Florida
P. a. ssp. latiusculum, Wolf 921, USA, Connecticut
P. a. ssp. latiusculum, Barrington 2287, USA, Vermont
P. a. ssp. latiusculum, 148 BRMN, USA, Maine
P. a. ssp. pubescens, Der 67, USA, California
P. a. ssp. pubescens, Der 66, Canada, British Columbia
86/1.0 P. a. ssp. pubescens, 325 OWUS, USA, Washington
P. a. ssp. pubescens, 100 AOUS, USA, Oregon
P. a. ssp. latiusculum, 143 YCCM, USA, Massachusetts
P. a. spp. decompositum, 292 MAHI, USA, Hawaii
P. a. spp. pinetorum, 164 KUKR, Ukraine
P. a. spp. pinetorum, N6nR, Siberia
P. a. ssp. japonicum, 029 TTWN, Taiwan
87/1.0 P. a. ssp. japonicum, 071 AIJP, Japan
P. a. ssp. japonicum, 085 YSCH, Taiwan
P. a. ssp. japonicum, 096 GNCH, China
P. a. ssp. japonicum, 104 MOGJ, Japan
P. a. ssp. japonicum, 280 CHJP, Japan
63/0.98 P. a. ssp. japonicum, 316 SHJP, Japan
P. a. ssp. pinetorum, N2nR, Russia, Siberia
P. a. ssp. pinetorum, RU4K, Russia, Siberia
P. a. ssp. japonicum, 113 YOJP, Japan
P. a. ssp. wightianum, 001 AMSL, Sri Lanka
P. a. ssp. wightianum, 007 KLPH, Philippines
87/1.0
P. a. ssp. wightianum, 068 SERI, Indonesia
P. a. ssp. wightianum, 182 PMAL, Indonesia
P. a. ssp. wightianum, 354 WFNQ, Australia, Queensland
P. a. ssp. wightianum, 362 HKSL, Sri Lanka
P. a. ssp. wightianum, 416 TREV, Taiwan
P. a. ssp. wightianum, 305 YGIN, India
— /0.96 P. a. ssp. aquilinum, 103 ASSP, Spain
P. a. ssp. aquilinum, 106 OXLN, UK, Great Britain
P. a. ssp. aquilinum, 017 FZSW, Switzerland
P. a. ssp. aquilinum, 065 CLYW, UK, Wales
P. a. ssp. aquilinum, 099 EDSC, UK, Scotland
P. a. ssp. aquilinum, 188 CORF, France
P. a. ssp. aquilinum, 194 AMTK, Turkey
51/0.92 P. a. ssp. aquilinum, 202 RVIT, Italy
P. a. ssp. aquilinum, 218 ACAW, UK, Wales
P. a. ssp. aquilinum, 226 LBSP, Spain
P. a. ssp. aquilinum, 256 AZOR, Portugal, Azores
62/1.0 P. a. ssp. aquilinum, 293 CPHW, UK, Wales
P. a. ssp. aquilinum, 307 BRGW, UK, Wales
P. a. ssp. aquilinum, 336 RAMD, Portugal, Madeira
72/1.0 P. a. ssp. aquilinum, 350 NIFR, France
P. a. ssp. aquilinum, TAUR, Ukraine
P. a. ssp. aquilinum, 031 BRFR, France
61/0.97 P. a. ssp. capense, 110 KUFZ, Zambia
P. a. ssp. capense, 503 CAPM/ZOMA, Malawi
P. a. ssp. capense, 191 GSAF, South Africa
—/0.97 P. a. ssp. capense, 228 MCAM, Cameroon
P. a. ssp. capense, 353 BBSA, South Africa
P. a. ssp. capense, 368 NDNG, Nigeria
87/1 P. a. ssp. centrali-africanum, 112 CH3Z/ZAMB, Zambia
P. a. ssp. centrali-africanum, AKFZ, Zambia
P. a. ssp. centrali-africanum, CAF4, Zambia
P. arachnoideum, ME2-1VNZA, Venezuela
P. esculentum, 387 CRDQ, Australia, Queensland
P. esculentum, 401 RYNA, Australia, New South Wales
85/1.0 P. caudatum, 323 COCR, Costa Rica
55/0.99 P. semihastatum, 251 PFNT, Australia, Northern Territory
P. semihastatum, 278 KMAL, Malaysia
P. esculentum, 275 KONC, New Caledonia
P. esculentum, Wolf 638, New Zealand
P. esculentum, 332 SVNZ, New Zealand
—/0.87 63/0.99 P. esculentum, 324 HNNZ, New Zealand
P. esculentum, 213 CRAN, Australia, New South Wales
63/1.0 P. esculentum, 083 WAWA, Australia, Western Australia
P. esculentum, 127 KEWA, Australia, Western Australia
P. arachnoideum, 144 RMEX, Mexico
P. arachnoideum, 317 SPBR, Brazil, São Paulo
 18
Phylogenetic analyses—

Maximum parsimony (MP) analysis of the concatenated data set resulted in 12

most parsimonious trees with a length of 59 (consistency index, CI = 0.9153; retention

index, RI = 0.9892). The best fit model used in Bayesian Inference (BI) was GTR+G and

HKY+G for trnS–rpS4 spacer+gene and rpL16 intron, respectively. The AICc weight of

the best fit model was 1.4 times greater than the next-best model for trnS–rpS4 spacer

+gene and 1.3 times greater for rpL16 intron. The topology of the MP strict consensus

tree (Figure 2-2A) is congruent with the BI phylogeny (Figure 2-1). MP bootstrap support

(BS) and BI posterior probabilities of clades (PP, i.e., clade credibility values) are

reported on the tree for supported nodes (Figure 2-1). Two fully supported clades are

resolved at the base of the phylogeny, separating P. arachnoideum, P. esculentum, and

P. semihastatum from P. aquilinum. There is a basal polytomy within the P. aquilinum

clade. A single accession of the tetraploid species P. caudatum is grouped with P.

semihastatum in the former clade, while the remaining P. caudatum accessions are

grouped with subspecies pseudocaudatum (Clute) Hultén in the P. aquilinum clade.

Biogeographic analysis—

Mapping geographic areas onto the phylogeny revealed a number of

biogeographic patterns (Figure 2-2A). The Austral taxon P. esculentum is found in a

clade with P. arachnoideum from South and Central America and P. semihastatum from

Southeast Asia and Australia, forming a group with a post-Gondwanan (after Africa split

away) southern continental distribution referred to here as the Austral/South American

clade. Within the P. aquilinum clade, basal biogeographic patterns are unresolved, but

the European P. aquilinum subsp. aquilinum accessions emerge from within the African

brackens and P. aquilinum subsp. decompositum in Hawaii is in a clade with all of the
 19

Figure 2-2. Biogeography of bracken. (A) Broad geographic distribution of bracken

specimens traced onto the maximum parsimony phylogeny using parsimony-based
character reconstruction. Geographic areas were coded as six unordered character
states: Hawaii, North/South America, Europe, Asia/India, Africa, Austral. (B) Global map
of sampled bracken specimens, color-coded consistently with the geographic area
character states in (A).
 20
sampled accessions of P. aquilinum subsp. pubescens (Underw.) J. A. Thomson, Mickel

& Mehltreter from western North America. The locations of specimens included in this

study are indicated on a global map (Figure 2-2B).

DISCUSSION

Phylogenetic relationships among bracken taxa—

Pteridium species form two distinct and fully supported basal clades (100%

maximum parsimony bootstrap—BS, 1.0 Bayesian posterior probability—PP). The first

clade contains P. esculentum from Australia, New Caledonia, and New Zealand and P.

arachnoideum from the neotropics, but neither of these species form distinct

monophyletic groups. The tetraploid species P. semihastatum and a single accession of

the tetraploid species P. caudatum from Costa Rica are included in this first main clade

and together are monophyletic (85% BS, 1.0 PP). This tetraploid group is derived from a

clade containing an accession of P. esculentum from New Caledonia (55% BS, 0.99 PP).

The second main clade in Pteridium includes all of the P. aquilinum accessions we

sampled and three of the four P. caudatum accessions we included in our analysis

(Costa Rica and northern South America). This second main Pteridium clade (the P.

aquilinum clade) is fully supported as monophyletic (100% BS, 1.0 PP), but phylogenetic

relationships of lineages within this group are not resolved. The three P. caudatum

accessions in the P. aquilinum clade are supported as monophyletic with 63% BS and

0.99 PP and are included in a polytomy with all of the P. aquilinum subsp.

pseudocaudatum accessions we sampled (Florida, USA) and a P. a. subsp. latiusculum

(Desv.) Hultén accession from Michigan, USA (64% BS, 0.98 PP). Pteridium a. subsp.

latiusculum accessions are scattered throughout the P. aquilinum clade and therefore is
 21
not monophyletic in our analyses. This phylogenetic pattern in P. a. subsp. latiusculum

may be explained if this taxon is primarily defined by plesiomorphies (morphologically)

and/or if it is susceptible to widespread hybridization (Speer et al., 1998). Together, P. a.

subsp. japonicum (Nakai) Á. Löve & D. Löve from coastal Asia and P. a. subsp.

pinetorum from the boreal Asian interior form a clade supported by 63% BS and 0.98 PP,

but neither subspecies forms a monophyletic group. Pteridium a. subsp. decompositum

(Gaudich.) Lamoureux ex J. A. Thomson from Hawaii and P. a. subsp. pubescens from

western North America and a single P. a. subsp. latiusculum accession from

northeastern North America form a polytomy supported by 86% BS and 1.0 PP. This

pattern suggests a trade wind dispersal route from North America to Hawaii consistent

with other wind-dispersed fern taxa (Geiger et al., 2007). Pteridium a. subsp. wightianum

(J. Agardh) W. C. Shieh from the Himalayas of northern India (isolate YGIN) emerges

from the aquilinum clade polytomy, while the remaining P. a. subsp. wightianum

accessions from Sri Lanka and Southeast Asia form a monophyletic group supported by

87% BS and 1.0 PP. Isolate YGIN was also distinguished from other P. a. subsp.

wightianum by both a distinctive nuclear genome marker and morphology in an earlier

study (Thomson, 2000a). All the P. a. subsp. aquilinum accessions we sampled (Europe)

are monophyletic (62% BS, 1.0 PP), but emerge from a paraphyletic grade of African P.

aquilinum subsp. capense. Also emerging from P. a. subsp. capense is P. a. subsp.

centrali-africanum, which is supported as monophyletic with 87% BS and 1.0 PP.

We emphasize here that apparent paraphyly at the level of species or below in

our data set should not be used to recircumscribe taxa for two reasons. First, we have

examined only variation in chloroplast DNA. Because of lineage sorting and

recombination, we have no reason to expect exactly the same patterns for nuclear
 22
genes (Soltis et al., 1992). Second, some models of speciation predict a high frequency

of paraphyly among recently diverged species (Rieseberg and Brouillet, 1994; Funk and

Omland, 2003).

Hybridization and the origin of the tetraploid species—

Although the majority of Pteridium taxa are at the same ploidy level (2N = 104;

Page, 1976), recent evidence suggests that P. semihastatum and P. caudatum are

tetraploid taxa (4N = 208; Tan and Thomson, 1990b; Thomson, 2000a, b; Thomson and

Alonso-Amelot, 2002). Allopolyploidy appears to be a common speciation process in

plants, and it often involves a triploid intermediate (Ramsey and Schemske, 1998). Two

main types of evidence for allopolyploid speciation can be gathered. Nuclear markers,

preferably fixed for different alleles in the different parents, can show additive effects in

allotetraploids, and simultaneously reveal both parents, whereas uniparentally inherited

markers will reveal one parent and whether there have been multiple origins (Soltis and

Soltis, 1993). Although we have no evidence for the mechanism of inheritance of

chloroplast DNA in Pteridium, several studies have demonstrated maternal inheritance in

other species of ferns (Gastony and Yatskievych, 1992; Vogel et al., 1998).

Pteridium caudatum and P. semihastatum have been shown to be tetraploid

bracken species based on DNA c-values, spore size, guard cell length, and morphology

of the cells in the false indusium (Thomson, 2000a, b; Thomson and Alonso-Amelot,

2002). Hypothesized allopolyploid origins have been inferred from intermediate

morphology and additive AP-PCR DNA genotypes of these two species (Thomson,

2000a; Thomson and Alonso-Amelot, 2002). These studies have suggested putative

progenitors of P. caudatum to be P. aquilinum subsp. pubescens in the north and P.

arachnoideum in the south (Thomson and Alonso-Amelot, 2002). These data have
 23
similarly been used to infer the putative progenitors of P. semihastatum in Southeast

Asia and northern Australasia as P. aquilinum subsp. wightianum (=P. revolutum sensu

Brownsey, 1989) and P. esculentum (Thomson, 2000a; Thomson and Alonso-Amelot,

2002). Our data based on the phylogeny of chloroplast sequences support the maternal

parentage of P. semihastatum to be P. esculentum, while P. caudatum emerges from two

parts of the phylogeny (with North American P. aquilinum subsp. semicaudatum/

latiusculum and embedded in the Austral/South American clade, which includes P.

arachnoideum, P. esculentum, and P. semihastatum). Two possible explanations for the

phylogenetic placement of our P. caudatum accessions seem plausible: (1) reciprocal

and multiple hybrid origins of this species or (2) a single hybrid origin of P. caudatum

with the maternal parent as P. a. subsp. pseudocaudatum/latiusculum with subsequent

introgression of the tetraploid P. semihastatum chloroplast genomes. Additionally, the

Austral/South American parent of P. caudatum may be an unsampled haplotype of the

co-occurring P. arachnoideum from South America or long-distance hybridization from an

Austral member of that clade. Long-distance hybridization in bracken has been

documented between eastern North America and Scotland (Rumsey et al., 1991). To

evaluate these hypotheses, we need nuclear genetic data to determine parentage and

additivity of genomic elements in the allotetraploid species. Nuclear sequence data

promise to be especially useful in elucidating specific phylogenetic relationships among

the various bracken taxa (Schuettpelz et al., 2008).

Phylogeography of bracken—

Pteridium occurs throughout the world, except in hot and cold desert regions.

Range-wide sampling is necessary to adequately assess variation within the genus and

delineate taxa. Only one such study has been accomplished in Pteridium: that of Tryon
 24
(1941). While the varieties in Tryonʼs treatment are often geographically based, he only

briefly discussed the geographic ranges of each taxon and instead focused on the

morphological differences between the varieties. Page (1976) expanded and updated

the ecological and phytogeographic information known for each of Tryonʼs varieties, but

emphasized that the adoption of Tryonʼs nomenclature was done for practical reasons

rather than validation of these taxa, and that an updated worldwide revision of the genus

was needed. While our sampling strategy attempts to cover the range of variation in

Pteridium worldwide, our study would benefit from additional sampling from Madagascar,

South and Central America, the Caribbean, and parts of North America.

Recent phylogenetic analyses of ferns, which have included extensive taxonomic

sampling and have used fossils for molecular date calibrations, have estimated

divergence dates between Pteridium and Dennstaedtia to be about 114 mya (Schneider

et al., 2004) and 92 mya (Pryer et al., 2004). These estimates represent upper ages for

the evolution of Pteridium and correspond to the Cretaceous period. Fragments of fossils

attributable to Pteridium are known from the Tertiary period as far back as the Oligocene

(33.7–23.8 mya) from Europe to Australia, suggesting that even by this time, bracken

may have achieved a widespread distribution (Page, 1976). During the time when

bracken likely originated and the earliest divergences may have occurred (from 100 to

30 mya), Africa and India separated from a southern landmass that included Australia,

Antarctica, and South America (Scotese, 2001). This separation may correspond to the

basal divergence among brackens, splitting the Austral/South American clade from the P.

aquilinum clade of African and Laurasian affinity. The polytomy in the P. aquilinum clade

may represent a rapid radiation of African and Indian bracken as they moved north,

colliding with a warm temperate and paratropical Laurasia by the end of the Paleocene
 25
(about 50 mya). Alternatively, the polytomy in the P. aquilinum clade may result from

insufficient data to distinguish phylogenetic relationships among lineages in the clade.

As continents have come to their modern global locations, and with the onset of

the current ice age, the Quaternary glaciation beginning 2.5 mya, there have been

periods of extensive ice sheets and cold, dry steppe coming into Europe and North

America and likely driving bracken into southern refugia. These repeated glacial periods

may be invoked to explain the phylogeographic pattern whereby the European brackens

are derived from within the African taxa if Africa served as a source for colonization of

Europe during recent geological times. The strong geographic structure observed at

large (continental) scales among bracken taxa is consistent with patterns expected

under a vicariance model, with range expansion across land bridges and only occasional

long-distance dispersal resulting in hybrid forms. This evidence contradicts the common

assumption in ferns that repeated long-distance dispersal of spores will erode

biogeographic patterns resulting from vicariance in widespread species via constant

gene flow (Tryon, 1985; Wolf et al., 2001). Recent empirical evidence in Ceratopteris

Brongn. shows that genetic reproductive barriers can accumulate quickly in allopatry,

thereby restricting gene flow and potentially maintaining the signature of vicariance

(Nakazato et al., 2007).

Conclusions—

This study highlights the importance of range-wide perspectives when

undertaking evolutionary and monographic studies. Any taxonomic revision of Pteridium

must reflect a global perspective and should include information from diverse areas of

research including morphology, cytology, genetics, and reproductive biology. To evaluate

the biogeographic and hybrid origin hypotheses presented here, we will need a better-
 26
resolved species phylogeny with increased population-level sampling that incorporates

nuclear data and fossils for date calibration. These data might also allow us to examine

additional reticulation and lineage sorting in the evolutionary history of Pteridium as well

as evaluate contemporary and historical gene flow.

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 32
CHAPTER 3

COMPLETE CHLOROPLAST GENOME SEQUENCE AND HIGH LEVELS OF RNA

EDITING IN THE FERN PTERIDIUM AQUILINUM DETERMINED BY MASSIVELY

PARALLEL PYROSEQUENCING 1

ABSTRACT

RNA editing is the post-transcriptional modification of RNA molecules relative to

their encoding genomic DNA sequences. RNA editing in land plant organelles occurs in

the form of pyrimidine exchanges. In general, levels of RNA editing are higher in

mitochondrial genomes than in chloroplast genomes, and in seed-free vascular plants

and hornworts relative to other lineages of land plants (i.e. liverworts, mosses, and seed

plants). To date, genome-wide chloroplast RNA editing has been systematically

examined in only seed-free plants: a fern (Adiantum capillis-veneris) and a hornwort

(Anthoceros formosae). The extremely high levels of RNA editing observed in

chloroplast transcripts of ferns and hornworts relative to seed plants (up to 25 times

higher) raises a number of interesting questions on the evolution of RNA editing in

plastomes. We present a genome-wide analysis of chloroplast RNA editing in a second

fern species. We use a novel, rapid method to determine the complete chloroplast

genome sequence and identify RNA editing sites using second-generation high-

throughput shotgun DNA sequencing technologies. This study also represents the first

application of RNA-seq to examine RNA editing in a chloroplast transcriptome. The

complete circular mapping chloroplast genome sequence of Pteridium aquilinum is

152,362 bp long and contains a gene set and gene order identical to Adiantum capillis-

1 Coauthors: Aaron M. Duffy, Matt Kusner, Chen Gu, Paul Overvoorde, and Paul G. Wolf.
 33
veneris. There are 117 different genes, including 84 protein coding genes, 4 ribosomal

RNA genes, and 29 transfer RNA genes. We experimentally detected 551 unique C to U

RNA editing sites and 300 U to C RNA editing sites in the chloroplast genome, 2.5 times

that detected in Adiantum and approaching the level of RNA editing observed in

Anthoceros. RNA editing events have been observed in protein coding genes, tRNA,

rRNA, introns, and intergenic regions. This work will enable a reexamination of the

evolution of chloroplast RNA editing by surveying RNA editing in the complete plastid

transcriptome of a second fern. Our approach should also be widely applicable to

studying RNA editing in the chloroplasts of other plant lineages.

INTRODUCTION

Chloroplasts play a critical role in the function of plant cells as the center for

photosynthesis and starch metabolism. Derived from a cyanobacterial-like ancestor and

incorporated into a eukaryotic genetic environment, the chloroplast genome has evolved

a complex system of RNA metabolism that combines features of the ancestral

endosymbiont with those of its eukaryotic host (1). Such a complex system of RNA

processing may function to regulate plastid gene expression (2, 3), to increase molecular

diversity (4, 5), and to functionally preserve the photosynthetic machinery (6). One of the

most striking components of chloroplast RNA metabolism is the occurrence of RNA

editing to alter the nucleotide sequence of mature RNA molecules relative to their

encoding genomic DNA, thus providing an exception to the “central dogma” of molecular

biology (7, 8).

RNA editing has evolved independently in at least seven lineages of eukaryotes,

including animals, fungi, plasmodial slime molds, and land plants, among others (9). The
 34
evolution of RNA editing in plants apparently occurred during the transition to land and is

observed within both organellar genomes (10-12) and the nuclear genome (13),

although evidence suggests that it has been secondarily lost in marchantiid liverworts

(14-17). Rates of RNA editing are typically higher in mitochondria than in chloroplasts

(14, 18), with the extreme case observed in Isoetes, where 1,420 RNA editing sites have

been identified in the mitochondrial genome (19). The chloroplast genomes of hornworts

and ferns also experience high levels of RNA editing, on the order of several hundred

sites (20, 21), whereas only 30-40 RNA editing sites typically occur in the chloroplast

genomes of seed plants (22). The distribution of RNA editing sites (both phylogenetic

and specific sites) is highly dynamic (23), suggesting that RNA editing sites evolve

rapidly. The exceptionally low rates of RNA editing in seed plant plastomes relative to

bryophytes and seed-free vascular plants is generally attributed to the steady

independent loss of ancestral RNA editing sites in different lineages (24, 25).

Given the importance of chloroplasts in plant cell function, the global

identification of RNA editing sites in chloroplast transcripts is required to understand the

precise coding potential of the chloroplast genome and will substantially enhance our

understanding of gene expression and function in this important organelle. Because

RNA editing has been systematically examined in the chloroplast genome of Adiantum

capillis-veneris (21), by examining RNA editing in a second fern species, we can

examine evolutionary patterns of RNA editing in ferns relative to seed plants, where a

dramatic reduction in the level of RNA editing has occurred. These data can be

examined in light of several hypotheses regarding the evolution of RNA editing in plant

organellar genomes (6, 25-27).

 35
The classic approach to surveying RNA editing in complete organellar genomes

involves laborious PCR amplification and sequencing of cDNAs for each gene. This

requires a priori sequence information about each predicted transcript to develop

primers and is dependent on an accurate annotation of the genome. Additionally, without

cloning and sequencing multiple amplicons for each target transcript, it is impossible to

determine the extent of incomplete RNA editing.

Recent developments in high throughput sequencing technologies have

revolutionized the collection and analysis of genome-scale sequence data. Two

independent research groups have reported alternative approaches using second-

generation sequencing technologies for the rapid determination of complete chloroplast

genome sequences (28, 29). Additionally, several recent studies have used deep

sequencing of cDNA to study RNA editing (13, 30-33), but only one of them

systematically identified novel RNA editing sites in a plant organellar genome (32). We

utilized Roche 454 pyrosequencing to determine the complete chloroplast genome

sequence of Pteridium aquilinum and identify novel RNA editing sites. Our approach

differs from previous studies using second-generation sequencing to determin

chloroplast genome sequences in that we did not first isolate chloroplast DNA from

nuclear and mitochondrial DNA. Alternatively, we used bioinformatic methods to extract

the chloroplast DNA sequence in silico. This approach allowed us to avoid the difficult

and laborious sample preparation protocols needed to isolate chloroplast DNA or to PCR

amplify the complete genome in overlapping fragments. At the same time, we obtained

valuable sequence information from the nuclear and mitochondrial genomes in this non-

model organism. By also sequencing cDNA, we were able to obtain expressed

sequence information for the chloroplast transcriptome without relying on any prior
 36
information about the chloroplast genome. These data, used in conjunction with the

plastome sequence, enabled not only an analysis of RNA editing but also provided

experimental evidence for intron splice sites and exon boundaries that were used in

annotation of the genome sequence.

MATERIALS AND METHODS

Chloroplast genome sequencing, assembly, and annotation

Total genomic DNA was extracted from fronds of Pteridium aquilinum ssp.

aquilinum (source: Sheffield 48, Manchester, UK) using a bulk CTAB protocol. Genomic

DNA was further purified by centrifugation on a CsCl gradient, where two nucleic acid

fractions were isolated separately (34). Purified DNA from each fraction was sequenced

separately and pooled on the Roche 454 GS-FLX platform using standard and Titanium

chemistries. In total, 711,178 reads were obtained that represented 216.19 Mbp of

genomic sequence data. Whole genome shotgun DNA sequence reads collected in the

course of this study have been deposited in the NCBI Sequence Read Archive

(submission no. SRA020058). The complete chloroplast genome sequence was

extracted and assembled using a combination of de novo and reference-guided

bioinformatic approaches. Reads were assembled de novo using MIRA (35). Putative

chloroplast contigs were identified with NCBI blastn by querying the complete chloroplast

genome sequences of Adiantum capillus-veneris (Genbank accession: AY178864),

Alsophila spinulosa (Genbank accession: FJ556581), and Angiopteris evecta (Genbank

accession: DQ821119) against the assembly, using an e-value threshold of 1e-4. Contigs

returning significant BLAST hits were parsed out of the complete de novo assembly

using a custom Perl script. Additionally, several reference-guided assemblies were

 37
generated with the YASRA pipeline (36) using the chloroplast genomes of Adiantum

capillus-veneris, Alsophila spinulosa, and Angiopteris evecta as reference sequences at

various levels of sequence similarity stringency. Putative chloroplast contigs from both

de novo and reference-guided approaches were collected and assembled to generate a

version 1 draft Pteridium chloroplast genome sequence. The original DNA sequence

reads were then mapped to the draft genome sequence in Consed v.19 (37) and

DOGMA (38) was used to identify potentially erroneous frame shifts in protein coding

sequences. A corrected version 2 draft sequence (with and without the second inverted

repeat) was then used as the scaffold for a new reference-guided assembly using the

Roche GS Mapper v.2.3 software. The resulting high quality, read-supported contigs

were used to produce the final complete chloroplast genome sequence in the standard

circular orientation typically presented for chloroplast genomes, beginning with the large

single copy region (LSC) followed by the first inverted repeat (IRB), the small single copy

region (SSC), and the second inverted repeat (IRA). The Mauve v.2.2.0 plugin (39) for

Geneious Pro v.5.0.1 (40) was used to generate a whole genome alignment for the

complete Adiantum, Alsophila, and Pteridium chloroplast genomes. This alignment with

annotations for Adiantum and Alsophila was used to annotate the Pteridium chloroplast

genome sequence. A circular gene map for the annotated sequence was generated in

OGDRAW (41).

Transcriptome sequencing and analysis

Total RNA was isolated from Pteridium aquilinum ssp. aquilinum gametophytes

(source: Wolf 84, Norwich, UK) grown in culture on an agarose mineral nutrient media

using the Spectrum Plant Total RNA kit (Sigma), incorporating an on-column DNase

treatment (Qiagen) during extraction. Total RNA was sent to the Center for Genomics
 38
and Bioinformatics at Indiana University (IU CGB) where a normalized transcriptome

(cDNA) library optimized for Roche 454 GS-FLX Titanium sequencing was prepared (K

Mockaitis, unpublished, IU CGB). This library was sequenced using the standard GS-

FLX Titanium protocol on 3 regions of a 4-region PicoTitrePlate. Transcriptome

sequence data are available in the NCBI Sequence Read Archive (SRA012887).

Transcriptome reads were mapped to the chloroplast genome using the Roche GS

Mapper v2.3 software specifying cDNA reads and a genomic reference sequence. RNA

editing sites were identified by parsing the results file reporting differences between

reads and the reference sequence. Additional predicted RNA editing sites were added to

protein coding genes during annotation to correct start and stop codons necessary for

proper translation of the gene. Intron splice sites were verified by mapping transcriptome

reads to the chloroplast genome using GMAP to report intron splice donor and acceptor

positions (42, 43).

RESULTS

Complete chloroplast genome

The complete circular-mapping chloroplast genome sequence of Pteridium

aquilinum is 152,362 bp (Figure 3-1) and arranged in the typical quadripartite plant

plastid genome structure consisting of a large single copy region (LSC; 84,335 bp) and a

small single copy region (SSC; 21,259 bp), separated by two copies of a large inverted

repeat sequence (IRA and IRB; 23,384 bp). The complete annotated nucleotide

sequence has been deposited in Genbank (accession no. HM535629). In mapping

whole genome shotgun sequence reads to the final chloroplast sequence, there was an

average of 34.5x read support for each position (Figure 3-2), with the coverage at
 39

trnS-GCU
trnL-UA
trnF-GA
trnM

A*
A

A
-UG
-CA

trnS
U
trn
R- ccD

C
rbc

GU
UC

trn M
L

ycf3*
a

b
rps4

psaA

D-
G

ps
psaB
ndhJ
ps f4
yc
aI

D
trnV
ce

C
psb
atp
atp

psb
m

-UA
E
A

ndhK
B

trnfM rps14
pe

ndhC

trnT -CAU
U
p

C*
t
pe etL

-GG
p tG

trn G-U sbZ

C- C C
r sa

p CA
rpspl33 J

trn nE-U etN

trn p

oB
G

GU C
18

rp
A
Y- U
LSC
1*
oC

tr
p rp
p sb
p sb J
ps sbF L
rpl t r Eb C2
rps
20
trn nW- rpo
12 P- C C
,5 UG A
psb clp !e G 2
B P* nd rps I
ps atp
psbbT H
H atp
petB *
* psb
N atpF CT
trnR-T CC*
petD* atpA trnG-U
ycf12

rpoA psbI
U
rps11 trnS-GC psbK
rpl36
infA
Pteridium aquilinum trnQ-UUG
rps8 chlB
rpl14
rpl16*
rps3
chloroplast genome rps16*

rpl22
rps19 matK
rpl2*
rpl23 U 152,362 bp
trnI-CA ndhB*
trnT-U
-ACG GU*
n2 trnR
n exo rrn5 .5
orpha GU*
ndhB trnT-U rrn4

3 trnR
rrn2 rrn5 -ACG
* rrn4
GC .5
IR

A
A-U * rrn2

IR
trn GAU
B

3
nI -
tr
1 6 trn
rrn rp A-U
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2, I-G *
3! AU
e *
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ps s7 16

trn
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d
en
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r ps
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-GG
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chlL
trn
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chlN
ndh
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photosystem I
trn

trnP

ndhD

ndhA*

rps15

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ndhH

photosystem II
f2
yc

cytochrome b/f complex

ATP synthase
trn

NADH dehydrogenase
N
-GU

RubisCO large subunit

AG
ccsA

RNA polymerase
trnL-U

ribosomal proteins (SSU)

rpl3 1
2
rpl2

ribosomal proteins (LSU)

clpP, matK
other genes
hypothetical chloroplast reading frames (ycf)
transfer RNAs
ribosomal RNAs

Figure 3-1: Gene map of the Pteridium aquilinum chloroplast genome. Genes on
the inside of the circle are transcribed clockwise and those on the outside are
transcribed counterclockwise. Genes with introns are indicated with an asterisk. Genes
are color coded according to protein complexes or functional categories indicated in the
legend. Note that rps12 is trans-spliced with two copies of the 3ʼ end and that exon 2 of
ndhB is located in the inverted repeat resulting in an orphan copy of this exon.
 40
individual sites ranging from 4 to 88 reads. When the IR is considered only once, a total

of 117 different genes have been detected and annotated, including 84 protein coding

genes, 4 ribosomal RNAs, and 29 transfer RNAs (Figure 3-1). The full gene complement

and gene order observed in the Pteridium chloroplast genome is identical to Adiantum

capillus-veneris, but differs from Alsophila spinulosa in the absence of psaM. There are

63,270 G or C nucleotides in the sequence, representing 41.5% nucleotide GC content

across the whole genome. Nucleotide GC content is positively correlated with sequence

coverage and reaches a local maxima in the ribosomal RNA genes located in the IR

(Figure 3-2). There are 17 genes with introns and one gene is trans-spliced (rps12). An

orphan copy of ndhB exon 2 is located in the inverted repeat. GMAP identified 51 intron

splice-junction site pairs, which were supported by 83 transcript reads.

Analysis of RNA editing

Of 725,312 transcriptome reads totaling 260 Mbp of sequence data, 20,291

reads (2.8%) mapped uniquely to the chloroplast genome, covering 80.67% of the

sequence. We experimentally detected 851 unique RNA editing sites (counting the

inverted repeat only once), including 551 C to U sites and 300 U to C sites. There are

233 editing sites located in the inverted repeat region resulting in a total of 1,114 sites in

the 152,362 bp genome (0.73%). The density of these RNA editing sites along the length

of the chloroplast genome is shown in Figure 3-3. There are 660 editing sites in protein

coding sequence. Among these, RNA editing modifies the start codon of 26 proteins,

removes 37 premature stop codons, and generates 6 stop codons. We detected 15

editing sites in tRNA genes and 168 editing sites in rRNA genes: 111 in rrn23, 52 in

rrn16, 5 in rrn4.5, 0 in rrn5. There are 12 RNA editing sites in the introns of 6 genes and

80 editing sites in intergenic regions.

 41

80
60
Coverage

40

1.0
Percent GC content
20

0.5
LSC IRB SSC IRA

0.0
0

0 50000 100000 150000

Sequence position (bp)

Figure 3-2: Chloroplast genome sequence coverage. The per-base read depth
coverage of Roche 454 DNA sequence reads mapped to the chloroplast genome is
indicated by the filled-line graph in red. Mean x-fold coverage across the complete
genome is 34.54x. To summarize rapid changes in coverage for adjacent regions of the
chloroplast genome, the average local coverage for each site was calculated using a 5
kb sliding window (black line, left axis). Average percent GC content was also calculated
on a 5 kb sliding window and overlaid on the figure (blue line, right axis). Overall, 41.5%
of the genome is comprised of G or C nucleotides. The location of the large single copy
region (LSC), small single copy region (SSC), and inverted repeat regions (IRB and IRA)
have been mapped across the length of the sequence.
 42
DISCUSSION

The nucleotide sequence and gene map for the Pteridium chloroplast genome

presented in this study is consistent with the coarse physical restriction and gene map

produced by Tan (44) and Stein et al. (45). By sequencing the chloroplast genome of a

basal polypod (Pteridium), we are able to resolve the evolution of several structural

differences between the Adiantum and Alsophila chloroplast genomes. For example, the

inverted repeat region in both Adiantum and Pteridium has expanded into the SSC to

include functional fragments of the 5ʼ end of chlL and the 3ʼ end of ndhF in a short

overlapping sequence that is 24 to 34 bp long. Additionally, the 565 bp repetitive region

in Alsophila reported by Gao (46) is also absent from Pteridium. Other features are

unique to only one of the polypod species, such as a 2 kb long segment between ndhC

and trnV-UAC that is only present in Pteridium. The clade derived from the common

ancestor of Pteridium and Adiantum includes over 9,000 species and represents

approximately 80% of fern species diversity (47, 48). This fact makes the chloroplast

genome sequence of Pteridium a valuable resource for developing chloroplast DNA

sequence markers and studying chloroplast evolution in the vast majority of ferns.

A previous study of RNA editing in the transcript of accD in Pteridium identified

14 RNA editing sites, including the creation of a functional start and stop codon, the

repair of a stop codon, and two U to C edits (49). Our data confirm all but two of the C to

U edits they report. Based on the nucleotide mismatches in our chloroplast transcript

assembly, we were able to predict the nature (C to U or U to C) and orientation (forward

or reverse strand) of each putative RNA editing site. However, in a small number of

cases, our strand predictions did not match the orientation of predicted transcripts based

on the annotation. This could result from four possible scenarios: first, a pyrimidine
 43

2.0e-05
1.5e-05
Density of RNA editing sites

1.0e-05
5.0e-06
0.0e+00

LSC IRB SSC IRA

0 50000 100000 150000

Sequence position (bp)

Figure 3-3: Density of RNA editing sites. The density probability function of RNA
editing sites across the length of the chloroplast genome sequence using a 2.5 kb
smoothing bandwidth for the kernel density estimator. The area under the curve sums to
one. Individual RNA editing sites are shown as a rug along the bottom of the plot and the
location of the LSC, SSC, and IR regions is indicated by short vertical bars.
 44
exchange in an antisense RNA translated from the opposite DNA strand; second, we

observe a low frequency of non-standard RNA editing events in the chloroplast

transcriptome (e.g. G to A); third, transcript paralogs from the nuclear or mitochondrial

genomes are incorrectly mapping to the chloroplast genome; and fourth, we may be

detecting SNP mismatches between the strain used to generate the chloroplast genome

and that used for transcriptome sequencing (both individuals are sourced from Great

Britain, approximately 200 km apart).

This study is the first to use a bioinformatic approach to isolate the chloroplast

genome from a sample of whole genome shotgun DNA sequencing on a second-

generation high-throughput sequencing platform. We are also the first to use second-

generation sequencing to survey RNA editing across the complete plastid genome of

any species. These approaches should be broadly applicable to rapid plastid genome

sequencing and analyses of RNA editing in other species. In this study, we report the

second complete chloroplast genome sequence for a polypodiaceous fern and identified

the highest level of RNA editing in a vascular plant chloroplast genome. We also report

an exceptional level of RNA editing in ribosomal RNA genes and intergenic regions,

previously unobserved in any plant chloroplast genome.

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 49
CHAPTER 4

DE NOVO CHARACTERIZATION OF THE GAMETOPHYTE TRANSCRIPTOME IN

BRACKEN FERN, PTERIDIUM AQUILINUM1

ABSTRACT

Background

Because of their phylogenetic position and unique characteristics of their biology

and life cycle, ferns represent an important lineage for studying the evolution of land

plants. Large and complex genomes in ferns combined with the absence of economically

important species have been a barrier to the development of genomic resources.

However, high throughput sequencing technologies are now being widely applied to non-

model species. We leveraged the Roche 454 GS-FLX Titanium pyrosequencing platform

to sequence the gametophyte transcriptome of bracken fern (Pteridium aquilinum) to

develop genomic resources for evolutionary studies.

Results

Quality and adapter trimmed reads totaling 254 Mbp were assembled de novo

into 38,889 unigenes with a mean length of 685.8 bp and a total assembly size of 26.7

Mbp with an average read-depth coverage of 8.5x. Estimates of transcriptome coverage

suggest that these sequences cover 87% of the complete transcriptome and have

tagged all of the transcripts. Of the 38,889 unigenes in this data set, 53.6% had blastx

hits in the NCBI non-redundant protein database (nr) and the longest open reading

frame in 30.7% of the unigenes had positive domain matches in InterProScan searches.

We were able to assign 41.4% of the unigenes with a GO functional annotation and

1 Coauthors: Michael S. Barker, Norman J. Wickett, Claude W. dePamphilis, and Paul G. Wolf.
 50
14.2% with an enzyme code annotation. Enzyme codes were used to retrieve and color

KEGG pathway maps. A comparative genomics approach revealed a substantial

proportion of genes expressed in bracken gametophytes to be shared across the

genomes of Arabidopsis, Selaginella and Physcomitrella, as well as identifying a

substantial number of potentially novel genes. We identified 2,320 perfect SSR loci,

providing an extensive resource that can be explored and developed for population

genomics and genetic mapping studies.

Conclusions

This study is the first comprehensive transcriptome analysis for a fern and

represents an important scientific resource for comparative evolutionary and functional

genomics studies in land plants. We demonstrate the utility of high-throughput

sequencing of a normalized cDNA library for de novo transcriptome characterization and

gene discovery in a non-model organism.

BACKGROUND

As the sister lineage to seed plants, ferns (i.e. monilophytes) represent a critical

clade for comparative evolutionary studies in land plants [1, 2]. In contrast to seed

plants, ferns typically retain the ancestral condition for a suite of life history traits (e.g.

the lack of secondary growth, homospory, motile sperm, and independent free-living

gametophyte and sporophyte generations). Ferns are thus an important outgroup for

studying the evolution of wood, seeds, pollen, flowers, and fruit among other

economically important characteristics of seed plants, as well as the evolution of

development in these complex structures and the expansion of gene families associated

with seed plant evolution (e.g. transcription associated proteins). For reasons not yet
 51
fully understood, ferns typically have much higher chromosome numbers and larger

genomes than seed plants [1, 3, 4]. Understanding the factors that influence these

differences and their evolutionary consequences will require developing genomic

resources in ferns to provide comparative context to understand the evolution of these

traits [3-5]. Additionally, because ferns have evolved and maintained free-living and

photosynthetic gametophyte and sporophyte life stages, they are an ideal group for

studies of both life cycle evolution in land plants and genome function in separate

haploid and diploid phases.

While taxonomic sampling of plants in genome-scale projects has expanded

dramatically with a decrease in the cost of DNA sequencing, the development of

genomic resources in ferns has lagged far behind those of other plant groups. This

deficit has primarily been attributed to technical and economic barriers associated with

the complex and large genomes in ferns, and is compounded by the limited agronomic

value of most ferns [4]. To date, genomic information in ferns is limited to a genetic

linkage map [6] and a modest expressed sequence tag (EST) data set comprised of

about 5,000 Sanger sequences [7] for Ceratopteris richardii, a data set for Adiantum

capillus-veneris which includes just over 30,500 ESTs [8, 9], and fewer than 500 ESTs

for Pteridium aquilinum [9].

With the introduction of cost efficient and massively-parallel high-throughput

sequencing technologies, genome scale studies in non-model organisms are being

actively pursued for gene discovery, expression profiling, SNP and SSR marker

development, and studies in functional, comparative, and evolutionary genomics in taxa

where little or no previous genomic information exists [10-24]. We chose the Roche 454
 52
Life Sciences GS-FLX Titanium technology to sequence a normalized cDNA library for

the gametophyte generation of the bracken fern, Pteridium aquilinum (L.) Kuhn.

Pteridium (family: Dennstaedtiaceae) is a cosmopolitan fern genus comprised of

several closely related species which are well differentiated from other genera in the

family. Pteridium aquilinum is the most widespread of the bracken species and is

distributed throughout the northern hemisphere and Africa [25]. Bracken is notorious as

a weed in open fields and is toxic to people and livestock. Despite its toxicity, bracken is

eaten as a delicacy in several parts of the world, and due to its often high local

abundance and large coarse stature, is sometimes used as thatching or packing

material. Because bracken is common, easily cultured and manipulated, and can have a

major economic impact, it has become one of the most intensively studied fern species.

Bracken has been used as a model system for the study of the fern life cycle [26-34],

gametophyte development and the pheromonal mechanism of sex determination

[35-42], cyanogenesis [43], carcinogenesis [44-46], invasion ecology [47-49], and

climate change [50].

This study was conceived to develop an extensive expressed sequence resource

in ferns for evolutionary and functional genomics, with the long term objective of

examining the role that alternation of generations plays in genome evolution. We present

the first comprehensive transcriptome characterization for a fern gametophyte, including

an assessment of transcriptome coverage, gene family and functional classification,

SSR identification, and a comparative analysis of gene sets across land plants.
 53
RESULTS

Sequencing and de novo assembly

Raw Roche 454 GS-FLX Titanium reads were quality- and adapter-trimmed and

size-selected to yield 681,722 cleaned reads with a mean length of 372.6 bp and 254

Mbp of total sequence data (Table 4-1, Figure 4-1A). Reads were first assembled in

MIRA v2.9.46 [51] and the resulting assembly was passed through a second assembly

step in CAP3 [52] to join additional contigs (Table 4-2). The resulting final assembly

consisted of 38,889 unigenes (i.e. unique gene sequences; assembled contigs +

singletons) with a mean length of 685.8 bp, which summed to a total assembly length of

26.7 Mbp (Table 4-1, Figure 4-1B). The average read-depth coverage for the final

unigene assembly was 8.50x (Table 4-2). The distribution of unigene coverage was

highly left-skewed toward low coverage with an extremely long tail (maximum coverage

just under 1800x; Figure 1C,D). The steep decline in read-depth coverage suggests that

cDNA normalization was effective and is typical for a normalized library [15].

Table 4-1 - Transcriptome sequence statistics

Raw reads Cleaned reads Unigenes
Number of sequences 730577 681722 38889
Mean length (bp) 363.55 372.60 685.76
Standard deviation (bp) 118.60 96.36 390.60
Mode length (bp) 416 383 467
Median length (bp) 398 394 554
Range in length (bp) 2 – 624 78 – 624 86 – 4897
Total length (Mbp) 265.60 254.01 26.67
Summary statistics for sequence data at different stages of processing.
 54

A Cleaned reads B Unigene assembly

10000
Number of sequences

Number of sequences
50000

6000
20000

0 2000
0

0 100 300 500 700 0 500 1500 2500

Read length (bp) Unigene length (bp)

C Unigene coverage D Unigene length x coverage

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0

1 4 7 10 14 18 22 26 >30 0 1000 2000 3000 4000 5000

Unigene coverage Unigene length

Figure 4-1 - Overview of P. aquilinum transcriptome sequencing and assembly.

(A) A histogram of the filter passed and adapter/quality trimmed Roche 454 GS-FLX
Titanium read lengths. (B) A histogram of unigene lengths for the final unigene set after
the 2-step assembly. Note that the longest unigene is 4,489 bp and the x-axis has been
truncated at 3 kb. (C) A histogram of the average read-depth coverage for unigenes. The
steep decline in coverage observed here is typical of normalized libraries [15]. Coverage
values between 30x and 1800x have been binned (see the vertical axis in Figure 1D).
(D) A density scatterplot showing the relationship between unigene length and coverage.
Points with a higher local density are darker.
 55
Table 4-2 - Assembly summary statistics
Primary Secondary
assembly assembly
(MIRA) (CAP3)
Number of reads assembled 622074 622074
Number of reads discarded 59648 0
Number of singletons 638 183
Number of primary contigs 50020 32801
Number of secondary contigs 0 5905
Number of unigenes 50658 38889
Mean unigene length (bp) 637.70 685.76
Largest unigene length (bp) 4489 4897
Total assembly length (Mbp) 32.30 26.67
Average unigene coverage 5.49 5.99
Average assembly coverage 7.02 8.50
A comparison of the primary and secondary assemblies. Secondary assembly was used
to join additional contigs and reduce redundancy in the final unigene set. The number of
singletons and primary contigs in the secondary assembly are the number of sequences
for each class retained from the primary assembly. Two methods were used to calculate
coverage for each assembly. “Average unigene coverage” is the arithmetic mean of the
average read-depth coverage for each unigene. “Average assembly coverage” is the
weighted mean of the coverage for each unigene, weighted by unigene length
(equivalent to the sum of the read lengths divided by the sum of the unigene lengths).
 56
Transcriptome coverage and data quality

Because information about the actual size and composition of the transcriptome

is often unknown, we utilized a simulation-based tool, ESTcalc [53], to estimate the

expected depth and breadth of transcriptome coverage for this data set. The model for

transcriptome coverage backing ESTcalc was parameterized using the well

characterized Arabidopsis thaliana transcriptome and several “next-generation”

sequencing runs using both normalized and non-normalized cDNA libraries [53]. Using

the results from their simulations (retrieved using ESTcalc), our dataset is expected to

have sequenced 87% of the transcriptome and to have tagged 100% of the genes, 70%

of which are expected to be sequenced to 90% of their length (Table 4-3). Consistent

with these estimates, we were able to identify 338 of 357 (94.7%) single copy eukaryotic

ultra-conserved orthologs (UCOs [54]) and 155 of 163 (95.1%) shared single copy tribes

from Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, and Oryza sativa [55].

These gene sets represent a conserved subset of genes expected to be present in

eukaryotic and plant transcriptomes, respectively, and can be used as a proxy for overall

gene detection and sampling breadth. As a final measure of gene detection in this data

set, we utilized a bootstrapped data resampling approach using the distribution of reads

in our final assembly (see methods section) to generate a unigene accumulation curve

which plots the number of unigenes detected as a function of sequencing effort (Figure

4-2). Using this method, we estimate that on average 90%, 95% and 99% of the

unigenes were tagged after approximately 323,445; 402,951; and 521,184 reads were

sampled (Figure 4-2). On average, it took 1,357.2 reads to detect each of the last ten

unigenes.
 57
Table 4-3 - Transcriptome coverage estimates: ESTcalc
Input Parameters ESTcalc Actual
estimate
Number of technologies 1 1
Technology 454 GS-FLX 454 GS-FLX
(Titanium)
Library type normalized normalized
MB/Plate 254 254.0076
Reads/Plate 681722 681722
BP/Read (mean) 372.6 372.6
Output Estimates
Total Assembled Sequence (MB) 26.2 26.67
Unigene count 32044 38889
Mean unigene length (bp) 819 685.80
Mean unigene length (longest unigene per gene, bp) 1143 —
Singleton yield (%) 19 0.0047
Percent transcriptome (%) 87 —
Percent of genes tagged (%) 100 —
Percent of genes with 90% coverage (%) 69.8 —
Percent of genes with 90% coverage by largest 56.4 —
unigene (%)
Percent of genes with 100% coverage (%) 23.7 —
Percent of genes with 100% coverage by largest 22.2 —
unigene (%)
Estimates of transcriptome coverage based on simulations modelled using the
Arabidopsis thalliana floral transcriptome.
 58

40000
30000
Number of unigenes

20000
10000
0

0e+00 1e+05 2e+05 3e+05 4e+05 5e+05 6e+05

Number of sequence reads (# ESTs)

Figure 4-2 - Unigene accumulation curve
The mean number of unigenes detected as a function of the number of reads sampled.
The complete set of reads in the 2-step assembly were shuffled and drawn at random for
1000 bootstrap replicates.

To identify potential contaminant sequences in the sample or sequencing library,

we examined the taxonomic distribution of blastx hits for each unigene searched in

theNCBI nr database. We examined both the taxonomic classification of the best hit as

well extracted from the PlantTribes2.0 database ([56] and CWD, unpublished). These

two as the lowest common ancestor (LCA) assignment for each unigene using MEGAN

v.3.7.2 [57]. Of the 22,716 unigenes with a blast hit, only 1% had a best hit to an

organism outside of the green plants and 0.5% of the unigenes with a blast hit received

an LCA assigned taxon which is not within, or a super set of, land plants (Table 4-4). We

also examined the unigene set for potential genomic DNA contamination by screening

unigenes for blastn hits to the complete chloroplast genome sequence of Pteridium

aquilinum (JPD, unpublished). None of the chloroplast sequences identified in the

transcriptome were longer than 5 kb or contained more than five adjacent genes and

thus can reasonably be considered putative transcripts [58, 59]. That we did not detect
 59
Table 4-4 - Taxonomic distribution of unigene blastx hits in the nr database
Lowest common
Best blastx hit ancestor for blastx
hits
Taxonomic category Percent of Percent of
Number of Number of
unigenes unigenes
unigenes unigenes
with hits with hits
Eukaryotes 22,685 99.9% 20,489 90.2%
Green plants 22,468 98.9% 20,296 89.3%
“Green algae” 104 0.5% 90 0.4%
Land plants 22,364 98.5% 20,091 88.4%
“Bryophytes” 375 1.7% 3,160 13.9%
Vascular plants 21,989 96.8% 13,060 57.5%
Lycophytes 60 0.3% 18 0.1%
Ferns 371 1.6% 242 1.1%
Seed plants 21,558 94.9% 12,735 56.1%
Gymnosperms 4,817 21.2% 9,700 42.7%
Angiosperms 16,741 73.7% 1,571 6.9%
Animals 185 0.8% 63 0.3%
Fungi 2 0.0% 10 0.0%
Other eukaryotes 30 0.1% 3 0.0%
Bacteria 22 0.1% 40 0.2%
Artificial sequences, 9 0.0% 2,137 9.4%
hits don’t pass threshold,
or taxon not assigned
Unigenes were searched in the NCBI nr protien database using blastx with an e-value
threshold of 1e-10, keeping the best ten hits. Of the 38,889 unignes, 22716 (58.4%) had
a positive hit. The lowest common ancestor (LCA) assignment for a sequence was
calculated using the LCA algorithm implemented in MEGAN v3.7.2 [57] based on at least
three blastx hits with a bitscore greater than 75 and within 10% of the best bitscore.
Note: the predicted proteins from Selaginella moellendorfii are not currently included in
the nr database and thus are not reflected in these results.
 60
any long fragments of chloroplast DNA in the transcriptome assembly gives us

confidence that our DNase treatment during RNA extraction was effective and the

resulting cDNA library used in sequencing is free of contaminant genomic DNA.

Functional annotation

Unigenes were annotated with gene ontology (GO) terms, enzyme codes, and

conserved protein domain functions using the Blast2GO suite [60-62]. Unigenes were

first interrogated against the NCBI nr protein database using a blastx e-value threshold

of 1e-10, keeping the top 20 high scoring alignments longer than 33 residues, resulting

in 22,716 unigenes (58.4%) with positive blast hits. The longest open reading frame

(ORF) from 11,940 unigenes (30.70%) had positive matches to conserved protein

domains using InterProScan (IPS) searches implemented in Blast2GO. These results (nr

blastx and IPS) were used to assign 64,578 GO terms to 15,855 unigenes (Appendix C).

These GO terms were used to map 6,978 enzyme codes to 5,554 unigenes. Enzyme

codes were then used then to retrieve and color 141 KEGG pathway maps. The full set

of GO terms for each unigene was also mapped to the plant GO-slim vocabulary to

examine the broad classification of gene functions represented in the transcriptome

(Figure 4-3).

Comparative genomics

Unigenes were classified into 7,126 tribe (inflation level 3) and 9,548 orthogroup

MCL clusters (Appendix C) in the PlantTribes2.0 gene family database on the basis of

the best blastx hit to the inferred protein models of the ten complete plant genomes

included in PlantTribes2.0 ([56] and CWD, unpublished). To evaluate the level of gene

overlap between the Pteridium gametophyte transcriptome and other land plants, we
 61
electron transport (458)
transcription (789)
lipid metabolic process cellular amino acid and
(545) derivative metabolic
DNA metabolic process process (1,365)
(295) carbohydrate metabolic
process (870)
catabolic process (882)
secondary metabolic
cellular component process (197)
organization (1,184)
response to stress (728)
translation (769)
signal transduction (700)
multicellular organismal
transport (1,713) development (165)
generation of precursor
metabolites and energy
(785)
photosynthesis (379)
response to abiotic
protein modification stimulus (289)
process (1,226)

Biological Process

transporter
activity (908)
RNA binding hydrolase
(440) activity (2,509)

transcription
factor activity
(228)

receptor
activity (191)
nucleotide
binding (2,614)
kinase activity
(1,179)
translation
factor activity,
nucleic acid structural
binding (183) molecule
activity (542)
protein binding
(2,139)

Molecular Function

endoplasmic extracellular
reticulum (317) region (327)
nucleoplasm cytoskeleton
(376) (238)
vacuole (274) thylakoid (529)
Golgi cell wall (274)
apparatus (212)
nucleolus (197)
ribosome (697)
cytosol (448)

mitochondrion
(1,967)

plastid (3,613)

plasma
membrane
(841)

Cellular Component

Figure 4-3 - Distribution of plant GO-slim functional categories

The relative proportion of plant GO-slim terms represented by more than 150 unigenes
for the three major categories in the GO vocabulary (biological process, molecular
function, and cellular component).
 62
examined overlap in both PlantTribes2.0 orthogroup cluster membership and blastx hits

for predicted proteins in Physcomitrella patens, Selaginella moelendorfii, and

Arabidopsis thaliana (Figure 4-4).

SSR identification

A total of 2,320 perfect di-, tri-, and tetranucleotide simple sequence repeats

(SSRs) longer than 9, 6, and 5 repeats, respectively, were identified in 1,990 unigenes

using the SSR identification tool (SSRIT) [63]. Additionally, primers for 424 potentially

amplifiable non-compound SSRs were chosen using Primer3 [64] as implemented in

msatCommander [65] (Appendix C). Since this RNA was extracted from gametophytes

derived from spores collected from a single diploid sporophyte, we are unable to

determine the level of variation present at these SSR loci in natural populations.

DISCUSSION

We have used high-throughput sequencing data to characterize the gametophyte

transcriptome of Pteridium aquilinum, a species for which very little genomic data are

available. These data also represent an 865-fold increase over the expressed sequence

data previously available for Pteridium in Genbank [9].

Assembly quality

Because contaminant adapter/primer sequences, polyA/T repeats, and low

complexity end sequences can substantially compromise de novo assembly and can be

difficult to completely remove (KM Dlugosch, personal communication), we aggressively

filtered and trimmed the reads beyond the default instrument-level processing routines at

the cost of sequence information loss (approximately 11.6 Mbp were removed,
 63

A Unigene OrthoGroup cluster membership (PlantTribes 2.0)

B Number of unigene BLASTx hits to complete proteomes

Arabidopsis thaliana Physcomitrella patens Arabidopsis thaliana Physcomitrella patens

2285 1274 471 1453 6202 890

16697 14839
1393 881 654 543

514 316

Selaginella moellendorfii 15374 Selaginella moellendorfii 13992

Figure 4-4 - Unigene overlap with Arabidopsis, Physcomitrella, and Selaginella

(A) PlantTribes2.0 Orthogroups: Unigenes were assigned to Tribe- and OrthoMCL
clusters derived from PlantTribes2.0 based on the best blast hit for each unigene. The
presence of genes from Arabidopsis thaliana, Physcomitrella patens, and Selaginella
moellendorfii in each OrthoGroup was evaluated. Of 38,889 total unigenes, 15,374
unigenes did not have a positive blast hit and thus were not assigned to an OrthoGroup
cluster. (B) Blastx: The complete unigene set was queried against the complete set of
predicted proteins in the genomes of Arabidopsis thaliana, Physcomitrella patens, and
Selaginella moellendorfii using an e-value cut off of 1e-5. Unigenes with positive hits in
more than one proteome are shown in the intersect for those species. Of 38,889 total
unigenes, 13,992 unigenes did not have a positive blast hit.
 64
representing 4.4% of the raw data). Considering the sheer quantity and depth of

sequencing produced by next-generation sequencing platforms, we deemed this an

acceptable level of loss to improve accuracy in the assembly. We also used a two-step

assembly strategy to maximize the information content of our final unigene sequence

set. We adopted this approach because MIRA is able to handle the large number of

reads produced by next-generation sequencing technologies and utilizes a multi-pass

strategy to identify and correct sequencing and assembly errors to produce a highly

accurate assembly, but is sensitive to uneven sequencing depth of coverage and allelic

diversity, resulting in a high number of redundant contigs. CAP3 is a proven and efficient

DNA sequence assembler that can be used to join highly similar overlapping sequences,

but is unable to handle the large number of reads produced by these new massively-

parallel sequencing platforms. By combining these two assembly tools, we were able to

join contigs and singletons that failed to assemble in MIRA to reduce sequence-level

redundancy in our final unigene data set.

In examining the taxonomic distribution of nr blastx hits for the unigenes, we

identified only a small proportion of sequences with best blast hits or LCA assignments

outside of the green plants. When we examine these hits in greater detail, we find that

many of them only align to short conserved domains, are hypothetical proteins of

unknown function from model organisms, or are genes which are conserved across

broad taxonomic levels, such as cytochrome P450, alpha-tubulin and dynein proteins.

Additionally, because no other fern genomes have been sequenced, some of these

sequences may represent novel fern genes. Thus the evidence indicates that there is

very little contamination in these data.

 65
Transcriptome coverage

While the simulations that underlay ESTcalc are based on the well characterized

Arabidopsis thaliana floral transcriptome (approximately 18,000 genes with transcripts

averaging 1,500 bp long) and assume perfect cDNA normalization and sequence

assembly, Wall et al. [53] show that their results were highly predictive for empirical

datasets from diverse eukaryotic species and tissues, making their simulations useful as

a null model for predicting transcriptome coverage in other organisms. However, the

predicted singleton rate for the level of sequencing we achieved is much higher than

observed in our two-step assembly (19% versus 0.005%). This is likely an artifact of our

primary assembly, in which MIRA discarded reads that were not assembled into any

contig and thus were not carried forward into our final assembly. This default behavior of

MIRA (-OUT:sssip=no), can be switched on in future unigene builds if singletons are

desired. Other possible explanations for this deviation are that the cDNA library

preparation [17, 66] or the longer read lengths obtained in this study resulted in a read to

transcript profile that doese not fit the ESTcalc models well. Finally, the transcriptome of

Pteridium aquilinum may encompass fewer genes than Arabidopsis, resulting in a higher

than predicted level of transcriptome coverage for our sequencing level and

subsequently more efficient recruitment of singletons into contigs during assembly.

Possible over-assembly seems an unlikely explanation for the deficit of singletons we

observed, because of the stringent percent similarity parameters used for both stages of

our two-step assembly (94% and 95% for MIRA and CAP3, respectively). Furthermore,

the final number of unigenes in our final assembly remains higher, and the mean

unigene length is shorter, than predicted by ESTcalc. Consistent with the ESTcalc

estimate that we have tagged 100% of the transcripts present in this library, and based
 66
on our unigene accumulation curve, the rate of new unigene detection for this cDNA

library has declined to the point that any additional sequencing is unlikely to detect new

genes, but may however serve to condense and join non-overlapping contigs in our

assembly.

Functional annotation

The distribution of GO functional categories represented in the gametophyte

transcriptome reflects the typical distribution of expressed plant genes for the biological

process, cellular component and molecular function GO categories (Figure 4-3). Most of

the unigenes annotated with a cellular component are localized to plastids or

mitochondria, but a large number of them are also targeted for ribosomes or the plasma

membrane. The molecular function of unigenes is heavily dominated by binding nucleic

acids or proteins and metabolic activity, including hydrolase and kinase activity. The

biological processes represented include all of the major cellular processes from

transport and cellular organization to transcription, translation, and metabolism. Visual

examination of the annotated/colored KEGG maps (not shown) indicates that we have

captured all of the genes required for glycolysis and the citrate cycle, a large number of

genes involved in nucleic and amino acid metabolism, chlorophyll biosynthesis, and

plant hormone biosynthesis, including gibberellin, abscisic acid, strigolactone, cytokinin,

brassinosteroid, and auxin.

Comparative genomics

The PlantTribes2.0 database contains an objective classification system for

plant genes and gene families [56]. By identifying similar sequences in this classification

system, we assigned unigene sequences with putative gene family identities. The most
 67
abundant of these gene families present in the unigene set was the pentatricopeptide

repeat protein (PPR) family, with over 900 unigenes classified as PPR proteins. We were

also able to identify 65 unigenes classified in the MADS-box transcription factor family.

Using this classification, gene sequences from Pteridium can be extracted for gene

families of interest for use in studies of gene family evolution or phylogenomics. The

overlap in orthogroup membership and blast hits for proteins in Arabidopsis thaliana,

Selaginella moellendorffii, and Physcomitrella patens is similar (Figure 4A/B), but some

striking differences can be observed. In both the PlantTribes and blast-based venn

diagrams, most of the unigenes which were identified in Arabidopsis, Selaginella, or

Physcomitrella are also shared across all three species. In the PlantTribes classification,

most of the genes are shared with Arabidopsis (21,649 unigenes), which is the most

closely related species included in this comparison, while slightly less and approximately

equal gene representation is shared with Selaginella and Physcomitrella (19,649 and

19,485 unigenes, respectively). This is in contrast to the blast-based examination of

gene set overlap in which Arabidopsis again has the greatest number of unigenes with

hits (23,148 unigenes), but Physcomitrella has hits with 6,122 more unigenes than

Selaginella (Figure 4-4). At first this seems counterintuitive because Pteridium shares a

more recent common ancestor with Selaginella than with Physcomitrella, but both

Pteridium and Physcomitrella have maintained a homosporous life cycle with a large

independent gametophyte stage. If genes required in a free living gametophyte are

maintained in Pteridium and Physcomitrella, but are free to be lost or evolve new

functions in Selaginella, we expect to see a higher degree of similarity between

Pteridium and Physcomitrella for a certain subset of genes.

 68
CONCLUSIONS

This study is the first comprehensive sequencing effort and analysis of gene

function in the transcriptome of a fern and represents the most extensive expressed

sequence resource available in ferns to date, nearly 16 times more data than exists for

Adiantum capillus-veneris. These data are an important new scientific resource for

comparative evolutionary studies in land plants and will be of great value for studies of

genome structure and function in ferns. These data can be used to develop microarrays

for gene expression assays or serve as a reference transcriptome for RNA-seq

experiments in Pteridium. As additional genome-scale projects in diverse plants are

undertaken, these data will be of immense value in representing ferns, the sister clade to

seed plants, in comparative genomic analyses.

METHODS

Gametophyte culture, library preparation, and sequencing

Pteridium aquilinum ssp. aquilinum spores (collection number: Wolf 83; sourced

from a single sporophyte individual collected in Norwich, UK) were sown onto sterile

agar nutrient media containing Boldʼs macronutrients and Nitchʼs micronutrients

(prepared as described by [67]) and grown under white light. Whole gametophytes

including both vegetative and sexually mature male, female, and hermaphoditic

individuals of various ages (up to 9 months from germination) were flash frozen in liquid

nitrogen and ground to a fine powder. Total RNA was isolated using the Sigma Spectrum

Plant Total RNA Kit, incorporating on-column DNase I (Qiagen) digestion during

extraction to remove traces of genomic DNA. Total RNA was concentrated by

precipitating in 2.5 M ammonium acetate and 70 % ethanol, then resolubilizing the RNA
 69
pellet in RNase-free water to approximately 500 ng/µL. Total RNA was quantified and its

quality verified using an Agilent Bioanalyzer 2100. Total RNA was sent to the Center for

Genomics and Bioinformatics at Indiana University, Bloomington (IU CGB), where a

normalized transcriptome (cDNA) library optimized for Roche 454 GS-FLX Titanium

sequencing was prepared using custom methods (K Mockaitis, unpublished, available

upon request). This library was sequenced using the GS-FLX Titanium process on 3

regions of a 4-region PicoTitre plate, according to manufacturer instructions.

Sequence preprocessing, transcriptome assembly,

and coverage assessment

Sequence reads generated in this study were deposited in the NCBI sequence

read archive (SRA012887). Raw sequence reads that passed instrument software

quality filters were trimmed of custom oligonucleotide adapter sequences (Justin Choi,

unpublished, IU CGB). The resulting sequences were further processed with SeqClean

[68] and SnoWhite v1.0.3 [69] to remove low quality, short, and contaminant sequences,

and to aggressively trim polyA/T sequences. Cleaned reads were assembled de novo in

MIRA v2.9.46 [51, 70] using a minimum percent identity of 94% to align reads. This

primary assembly was passed through a secondary assembly step in CAP3 (95%

identity, 25 bp overlap) [52] to reduce redundancy in the final assembly and join

additional contigs. The final set of unigene sequences analyzed in this paper is included

with the reads in the Sequence Read Archive. Custom perl scripts were used to extract

summary information about the reads and assemblies (Tables 4-1 and 4-2; scripts

available by request from JPD). The average unigene coverage was calculated as the

arithmetic mean of the average read-depth coverage for each unigene, whereas the

average read-depth coverage for the assembly as a whole takes into account read and
 70
contig lengths, and is equivalent to the per-base read-depth coverage across all of the

unigenes.

We utilized a web-based tool, ESTcalc [53], to estimate the predicted level of

transcriptome coverage for our data set. Input parameters to ESTcalc require that we

specify the sequencing technology used (or a combination of technologies) and either

the total sequencing level (Mbp), or the number of reads and an estimate of read

lengths. We used the best approximation for sequencing technology available (454 GS-

FLX) and the empirical values observed for the cleaned sequence data (254 Mbp or

681,722 reads with an average of 372.6 bp/read) to obtain our estimates. The estimates

reported were identical whether we parameterized on total sequence or supplied read

length information as well.

To determine the number of eukaryotic ultra-conserved orthologs (UCOs [54]) we

captured in the Pteridium transcriptome data set, we queried a list of 357 UCO coding

sequences from Arabidopsis (A Kozik, unpublished) into the unigene set with an e-value

threshold of 1e-10 using NCBI tblastx. These blast results were then parsed to

determine then number of UCOs with a positive hit that returned an amino acid

alignment greater than 30 residues long.

We assessed the changing rate of new gene detection as a function of sampling

effort (unigene accumulation curve, Figure 4-2) using a bootstrapped random sampling

protocol implemented in a custom perl script (available from JPD on request). This script

uses the empirical distribution of read number per unigene in our final assembly to

randomly sample reads one at a time and tracks the total number of unigenes detected

at each step. Because the order of sampling can impact the shape of this curve, we

computed 1,000 replicate random sample orders and calculated the mean number of
 71
unigenes detected after each draw. To evaluate the level of variation in the number of

unigenes detected, we also calculated the 95% confidence interval on the number of

unigenes. Using this curve, we then estimated the number of reads it took to capture an

average of 90%, 95%, and 99% of the unigenes and the average number of additional

reads required to detect each of the last 10 unigenes.

To evaluate for the presence of potential contaminating sequences, we examined

the taxonomic distribution of blastx hits for the unigene set in the NCBI nr protein

database using an e-value threshold of 1e-10. The top 10 blast hits for each unigene

were kept and examined in MEGAN v.3.7.2 [57]. MEGAN is a tool built for the

examination of metagenomic data sets and provides a number of useful functions to

explore the information content of large blast results. The blast results for each unigene

were mapped onto the NCBI taxonomy tree by examining just the best hit (lowest e-

value) or by using the lowest common ancestor (LCA) algorithm [57]. LCA was

determined using at least three blast hits with a bitscore greater than 75 and within 10%

of the top bitscore for that unigene.

Functional annotation

The same blast search used to examine the taxonomic distribution of blast hits

was used to identify putative homologous proteins and annotate each sequence with

gene ontology (GO) terms using Blast2GO [60-62]. Blast2GO was also used to

automatically handle InterProScan (IPS) searches to identify conserved protein domains

in translations of the longest ORF in each unigene. Any GO terms associated with IPS

hits were then merged into the blast-based GO annotation. GO terms were then used to

map enzyme codes to each sequence. Enzyme codes were then used to automatically

color and retrieve KEGG pathway maps [71, 72]. As a final step in examining a broad
 72
functional representation of the gametophyte transcriptome, GO terms were mapped to

the reduced GO-slim ontology and visualized and explored with directed acyclic graphs

(not shown) and summarized with filtered pie charts including GO categories

represented by at least 150 sequences (Figure 4-3)

Comparative genomics

Unigenes were classified into tribe- and orthoMCL clusters in the PlantTribes2.0

database using a custom perl pipeline (dePamphilis lab, unpublished) which queries

each unigene against the complete inferred protein set from ten plant species that have

complete sequenced genomes, using a blastx e-value threshold of 1e-10. Unigenes

were assigned to clusters based on the best blast hit. Species used for blast searches

and gene clustering in the PlantTribes2.0 database include: Chlamydomonas reinhardtii,

Physcomitrella patens, Selaginella moellendorffii, Oryza sativa, Sorghum bicolor, Vitis

vinifera, Populus trichocarpa, Medicago truncatula, Carica papaya, and Arabidopsis

thaliana. Meta-information about each assigned cluster was extracted from the database

for each unigene and was included in the pipeline output file. Simple text-based

searches examined this information to retrieve gene family names and putative gene

family functional data. The shared single copy tribes for Arabidopsis, Vitis, Populus, and

Oryza were identified in the PlantTribes2.0 database and the number of these tribes

detected in the unigene set was determined by examining the pipeline output file.

Orthogroup assignments for Pteridium unigenes were examined for cluster membership

by Selaginella, Physcomitrella, and Arabidopsis to generate a venn diagram showing

putative gene level overlap (Figure 4-4A). Unigenes were also directly queried against

each of these proteomes using a blastx e-value threshold of 1e-10 to examine the
 73
distribution of similar proteins in these three species. Venn diagrams were generated to

graphically illustrate the overlap of unigenes for each proteome (Figure 4-4B).

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 79
CHAPTER 5

CONCLUSIONS

This dissertation takes a multifaceted approach to examine evolution in bracken,

from a classic systematics and biogeographic study, through complete sequencing of the

chloroplast genome, to surveying genes expressed in the reproductive life stage of ferns.

I examine evolutionary events at scales from species divergence and hybridization within

subspecies all the way to comparisons across major lineages of land plants.

In chapter 2, I reviewed the state of systematic knowledge within Pteridium and

constructed a comprehensive phylogenetic framework for the genus, sampling bracken

specimens across its geographic range. My results enabled specific biogeographic and

hybrid origin hypotheses to be proposed and set the stage for further work on the

systematics and evolution in bracken. This study also highlighted the importance of

range-wide perspectives when undertaking evolutionary and monographic studies.

In chapter 3, I sequenced the complete chloroplast genome of Pteridium

aquilinum and performed a genome-wide survey of chloroplast RNA editing. This study

is the first reported use of Roche 454 sequencing and bioinformatic extraction/assembly

of a complete chloroplast genome and the first study to utilize second-generation

sequencing technologies to analyze RNA editing in a plastid genome. The chloroplast

genome of bracken contains 117 different genes, including 84 protein coding genes, 4

ribosomal RNA genes, and 29 transfer RNA genes. A total of 551 unique C to U RNA

editing sites and 300 U to C RNA editing sites in the chloroplast genome were

experimentally detected. This is the highest known rate of RNA editing ever detected in a

vascular plant, over 2.5 times that detected in Adiantum capillis-veneris and over 20

times that seen in seed plants.

 80
Chapter 4 leveraged high-throughput transcriptome sequencing to survey genes

expressed in the gametophyte life stage in Pteridium. This study represents the first

comprehensive sequencing effort and analysis of gene function in the transcriptome of a

fern and represents the most extensive expressed sequence resource available in ferns

to date. These data are an important new scientific resource for comparative

evolutionary studies in land plants and will be of great value for studies of genome

structure and function in ferns. These data can be used to develop microarrays for gene

expression assays or serve as a reference transcriptome for RNA-seq experiments in

Pteridium. As additional genome-scale projects in diverse plants are undertaken, these

data will be of immense value in representing ferns, the sister clade to seed plants, in

comparative genomic analyses.

In this dissertation I have presented three studies which examine genome

structure, function, and evolution in bracken. By shifting my sampling strategy dependent

on specific research objectives and questions presented in each study I have been able

to examine evolution in bracken at different scales, from a reconstruction of phylogenetic

relationships and ancient evolutionary events to an examination of chloroplast sequence

composition and chloroplast RNA metabolism, and to a broad survey of expressed

genes in one life stage of the fern life cycle. These studies together form the foundation

for a research program in bracken evolutionary genetics and genomics which shows

potential for a great many projects in the future.

 81

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APPENDIX B

VOUCHER INFORMATION FOR CHAPTER 2

Taxa, vouchers, localities and GenBank accession numbers for bracken specimens used
in Chapter 2. Voucher specimens are deposited in the following herbaria: NSW =
National Herbarium of New South Wales, Australia; UTC = Intermountain Herbarium,
Utah State University, USA.
Taxon — Voucher: Collector (Herbarium accession #) Sample isolate ID; Locality;
Latitude; Longitude; GenBank accesions: trnS–rpS4 spacer+gene; rpL16 intron.

Pteridium aquilinum subsp. aquilinum — T. Reichstein (NSW 420392) 017

FZSW; Switzerland, Filzbach; 47.12; 9.10; FJ177240; FJ177159; R. Prelli (NSW
420391) 031 BRFR; France, Britagne, Erquy; 48.38; 2.27; FJ177241; FJ177160; M. E.
Gillham (NSW 420404) 065 CLYW; United Kingdom, Wales, Neath Valley; 51.65; -3.80;
FJ177242; FJ177161; A. F. Dyer (NSW 420393) 099 EDSC; United Kingdom, Scotland,
Pentland Hills; 55.5; -3.25; FJ177243; FJ177162; R. Viane (NSW 420405) 103ASSP;
Spain, Asturias Province, Villanueva; 43.37; -5.83; FJ177244; FJ177163; M. E. Gillham
(NSW 420396) 106 OXLN; United Kingdom, England, London, Oxshott Common; 51.38;
-0.42; FJ177245; FJ177164; F. Piccoli (NSW 420390) 188 CORF; France, Corsica,
Haute Corse; 42.57; 9.30; FJ177246; FJ177165; E. Sheffield (NSW 420403) 194 AMTK;
Turkey, Amasra, Black Sea Coast; 41.73; 32.40; FJ177247; FJ177166; F. Piccoli (NSW
420394) 202 RVIT; Italy, Ravenna; 44.42; 12.2; FJ177248; FJ177167; C. N. Page (NSW
420408) 218 ACAW; United Kingdom, Wales, Caernarfon; 53.13; -4.27; FJ177249;
FJ177168; B. Capezedo & A. E. Salvo (NSW 420374) 226 LBSP; Spain, Cadiz, Los
Barrios; 36.18; -5.50; FJ177250; FJ177169; A. C. Jermy (NSW 420397) 256 AZOR;
Portugal, Azores, Terceira, Agra; 38.70 -27.20; FJ177251; FJ177170; M. E. Gillham
(NSW 420380) 293 CPHW; United Kingdom, Wales, Caerphilly Mt.; 51.35; 3.12;
FJ177252; FJ177171; M.E. Gillham (NSW 420399) 307 BRGW; United Kingdom, Wales,
Bridgend; 51.52; -3.58; FJ177253; FJ177172; R. Viane (NSW 420388) 336 RAMD;
Portugal, Madeira, Rocha Alta; 32.65; -16.88; FJ177254; FJ177173; R. Prelli (NSW
420376) 350 NIFR; France, Nice, Cagnes-sur-Mer; 43.67; 7.15; FJ177255; FJ177174; V.
Korzhenevsky (NSW 627463) TAUR; Ukraine, Caucasus Mountains, Crimea; 44.98;
34.62; FJ177288; FJ177207. Pteridium aquilinum subsp. capense (Thunb.) C. Chr. —
C. S. McMaster (NSW 617745) 110 KUFZ; Zambia, Kundalila Falls; -12.45; 30.13;
FJ177256; FJ177175; V. Rashbrook (NSW 420332) 191 GSAF; Republic of South
Africa, Grahamstown; -33.19; 26.32; FJ177257; FJ177176; Watling (NSW 420334) 228
MCAM; Cameroon, Mundamba; 4.42; 8.87; FJ177258; FJ177177; P. J. Myerscough
(NSW 420333) 353 BBSA; Republic of South Africa, Bettyʼs Bay, Silver Sands; -34.35;
18.9; FJ177260; FJ177179; B. E. Okoli (NSW 420328) 368 NDNG; Nigeria, Niger Delta;
4.83; 6.25; FJ177262; FJ177181; A. C. Chikuni (NSW 617743) 503CAPM/ZOMA;
Malawi, Zomba Plateau; -15.3; 35.32; FJ177263; FJ177182. Pteridium aquilinum
subsp. centrali-africanum Hieron. — C. S. McMaster (NSW 617746) 112 ZAMB/CH3Z;
Zambia, Mkushi, Chilongoma Hills; -14.38; 29.53; FJ177264; FJ177183; C. S. McMaster
(NSW 617747) AKFZ; Zambia, Kundalila Falls; -12.45; 30.13; FJ177265; FJ177184; C.
S. McMaster (NSW 617757) CAF4; Zambia, Mkushi, Chilongoma Hills; -14.38; 29.53;
FJ177266; FJ177185. Pteridium aquilinum subsp. decompositum (Gaudich.)
 89
Lamoureux ex J. A. Thomson — C. W. Smith (NSW 420357) 292 MAHI; USA, HI, Maui,
Haleakala National Park; 20.75; -156.17 FJ177268; FJ177187. Pteridium aquilinum
subsp. japonicum (Nakai) Á. Löve & D. Löve — S.-M. Chew (NSW 420347) 029
TTWN; Taiwan, Nankang, Taipei; 21.02; 121.02; FJ177269; FJ177188; R. Hirose (NSW
420335) 071 AIJP; Japan, Honshu, Aichi Prefecture; 34.49; 137.12; FJ177270;
FJ177189; K. U. Kramer (NSW 420340) 085 YSCH; China, Kwansi, Guilin, Yao Shan;
25.35; 110.18; FJ177271; FJ177190; Y.-J. Zhang (NSW 420336) 096 GNCH; China,
Gansu Province; 35.00; 105.00; FJ177272; FJ177191; R. Hirose (NSW 420351) 104
MOGJ; Japan, Honshu, Nagano Prefecture, Mt. Ogura; 36.17; 138.25; FJ177273;
FJ177192; T. Kumashiro (NSW 420356) 113 YOJP; Japan, Kyushu, Kagoshima
Prefecture; 31.58; 130.55; FJ177274; FJ177193; R. Hirose (NSW 420348) 280 CHJP;
Japan, Honshu, Chiba Prefecture; 35.60; 140.10; FJ177275; FJ177194; T. Miyata (NSW
420349) 316 SHJP; Japan, Honshu, Shimane Prefecture; 35.47; 133.03; FJ177276;
FJ177195. Pteridium aquilinum subsp. latiusculum (Desv.) Hultén — E. Klekowski
(NSW 420315) 143 YCCM; USA, MA, Yarmouth; 41.70; -70.23; FJ177277; FJ177196;
W. H. Wagner Jr (NSW 420310) 147 WMCH; USA, MI, Waterloo; 42.15; -84.24;
FJ177278; FJ177197; D. Barrington (NSW 420311) 148 BRMN; USA, ME, Bridgton;
44.04; -70.42; FJ177279; FJ177198; D. Barrington (UTC 247719) DSB 2287; USA, VT,
Colchester, Niquette Bay State Park; 44.58; -73.20; FJ177280; FJ177199; P. Wolf (UTC
249671) PGW 921; USA, CT, SE of Storrs, Spring Hill; 41.79; -72.22; FJ177281;
FJ177200. Pteridium aquilinum subsp. pinetorum (C. N. Page & R. R. Mill) J. A.
Thomson — V. Bogatyr (NSW 420402) 164 KUKR; Ukraine, Kiev; 50.43; 30.5;
FJ177235; FJ177154; E. A. Ershova & A. I. Shmakov (NSW 807731) N2nR; Russia,
Siberia, Altai Plain; 53.35; 83.73; FJ177236; FJ177155; E. A. Ershova (NSW 807726)
N6nR; Russia, Siberia, Novosibirsk; 55.03; 82.92; FJ177237; FJ177156; O. N.
Perestoronina (NSW 807736) RU4K; Russia, Kirov Region; 58.60; 49.65; FJ177238;
FJ177157. Pteridium aquilinum subsp. pseudocaudatum (Clute) Hultén — E.
Sheffield (NSW 420317) 169 FLUS; USA, FL, Cape Kennedy; 28.47; -80.47; FJ177282;
FJ177201; P. G. Wolf & M. D. Windham (NSW 420316) 203 HFLA; USA, FL, Hawthorne;
29.55; -82.08; FJ177283; FJ177202; P. Soltis (UTC 249632) Der 68; USA, FL, Paynes
Prairie State Preserve; 29.52; -82.30; FJ177299; FJ177218. Pteridium aquilinum
subsp. pubescens (Underw.) J. A. Thomson, Mickel & Mehltreter — D. Barrington
(NSW 419173) 100 AOUS; USA, OR, Ashland; 42.23; -122.73; FJ177284; FJ177203; J.
Schneller (NSW 420312) 325 OWUS; USA, WA, Olympic Peninsula; 47.55; -124.24;
FJ177285; FJ177204; J. Der (UTC 249629) JPD 66; Canada, British Columbia,
Vancouver; 49.26; -123.26; FJ177297; FJ177216; J. Der (UTC 247718) JPD 67; USA,
CA, Feather River Canyon; 39.74; -120.71; FJ177298; FJ177217. Pteridium aquilinum
subsp. wightianum (J. Agardh) W. C. Shieh — R. D. E Jayesekara (NSW 419562) 001
AMSL; Sri Lanka, Ambewela; 7.03; 80.59; FJ177289; FJ177208; J. V. Pancho (NSW
420371) 007 KLPH; Philippines, Luzon, Kinabuhayan; 14.25; 121.5; FJ177290;
FJ177209; T. Partomihardjo (NSW 419534) 068 SERI; Indonesia, Molucca Island,
Seram; 3.10; 129.05; FJ177291; FJ177210; R. Kiew (NSW 420251) 182 PMAL;
Malaysia, Pahang; 4.00; 105.00; FJ177292; FJ177211; S. P. Khullar (NSW 419570) 305
YGIN; India, Garhwal, Yamunotri Hills ; 30.92; 78.47; FJ177293; FJ177212; J. A.
Thomson (NSW 420257) 354 WFNQ; Australia, N. Queensland, Wallaman Falls; -18.55;
145.80; FJ177294; FJ177213; M. D. Dassayanake (NSW 419571) 362 HKSL; Sri Lanka,
Hakgala; 6.55; 80.48; FJ177295; FJ177214; S. J. Moore (NSW 705077) 416 TREV;
 90
Taiwan, Taitung Hsieh; 23.27; 120.96; FJ177296; FJ177215. Pteridium arachnoideum
(Kaulf.) Maxon — B. Perez-Garcia (NSW 420308) 144 RMEX; Mexico, Molango, Río
Malila; 20.48; -98.44; FJ177221; FJ177140; M. G. E. Noronha & P. G. Windisch (NSW
420303) 317 SPBR; Brazil, Sao Paulo; -22.42; -49.00; FJ177219; FJ177138; M. E.
Alonso-Amelot (NSW 505771) ME2-1VNZA; Venezuela, Mérida, Cerro La Bandera;
8.42; -69.03; FJ177220; FJ177139. Pteridium caudatum (L.) Maxon — J. Villalobos-
Salazar & M. Firenczi (NSW 420247) 238 HECR; Costa Rica, Heredia; 10.00; -84.08;
FJ177222; FJ177141; J. Lenne (NSW 420295) 274 QCOL; Colombia, Valle, Quisquina;
3.60; -76.48; FJ177223; FJ177142; A. C. Jermy & T. G. Walker (NSW 420305) 323
COCR; Costa Rica, Cordillera; 9.15; -83.8; FJ177224; FJ177143; M. E. Alonso-Amelot
(NSW 505770) MD2-2VENZ; Venezuela, Merida, La Hechicera; 8.42; -71.03; FJ177225;
FJ177144. Pteridium esculentum (G. Forst.) Cockayne — J. A. Thomson (NSW
420273) 083 WAWA; Australia, WA, Waroona; -32.51; 115.59; FJ177226; FJ177145; J.
A. Thomson (NSW 420274) 127 KEWA; Australia, WA, Kenton; -34.57; 117.01;
FJ177227; FJ177146; J. A. Thomson (NSW 420848) 213 CRAN; Australia, NSW,
Craven; -32.15; 151.95; FJ177228; FJ177147; J. A. Thomson (NSW 420264) 275
KONC; New Caledonia, Mt. Koghi; -22.17; 166.50; FJ177229; FJ177148; C. Surman
(NSW 420267) 324 HNNZ; New Zealand, South Island, Nelson, Hira Forest; -41.18;
173.17; FJ177230; FJ177149; C. Surman (NSW 420289) 332 SVNZ; New Zealand,
North Island, Wellington, Stokes Valley; -41.07; 175.08; FJ177231; FJ177150; J. A.
Thomson (NSW 420879) 387 CRDQ; Australia, N. Qld, Credition; -21.20; 148.53;
FJ177232; FJ177151; J. A. Thomson (NSW 420269) 401 RYNA; Australia, NSW,
Sydney, Ryde; -33.47; 151.05; FJ177233; FJ177152; P. Wolf (UTC 250545) PGW 638;
New Zealand, North Island, Ruakura; -37.78; 175.31; FJ177234; FJ177153. Pteridium
semihastatum (Wall. ex J. Agardh) S. B. Andrews — P. Brocklehurst & G. Wightman
(NSW 420865) 251 PFNT; Australia, NT, Petherick's Forest Park; -13.06; 130.24;
FJ177286; FJ177205; Mujamil & T. J. Ho (NSW 419554) 278 KMAL; Malaysia, Selangor,
Kapar; 3.07; 101.24; FJ177287; FJ177206.
 91
APPENDIX C

ADDITIONAL FILES FOR CHAPTER 4

Additional files for analyses performed in Chapter 4 can be downloaded from:

https://docs.google.com/leaf?
id=0BwOnBtQxjHP0MjhkYjdiZTAtMzYwNC00N2M4LWIyZmEtMGQzYTdlMjBjOWFi&hl=en

Additional file 1 – Unigene functional annotations from Blast2GO

Spreadsheet file (csv) with unigene GO annotations assigned by Blast2GO. Each
annotation is on a line, unigenes with multiple GO terms have more than one line. Five
columns correspond to: unigene ID, functional description based on blast hits, GO ID,
GO term, and GO category (P: biological process; C: cellular component; F: molecular
function).
File available at: https://docs.google.com/leaf?
id=0BwOnBtQxjHP0ZDdhZjI2MTMtODBlZC00MjA1LWJmOTctNmRiMDI5MWI5ZmVi&hl=en

Additional file 2 – PlantTribes2.0 gene family classification

Spreadsheet file (csv) with putative tribe and orthogroup assignments (stringency level
3) for each unigene with cluster membership counts from the ten proteomes included in
PlantTribes2.0. Functional and gene family descriptors for clusters are primarily inherited
from the Arabidopsis thaliana genes included in the cluster.
File available at: https://docs.google.com/leaf?
id=0BwOnBtQxjHP0YTFmMzc1MjAtOTdhZi00NTY4LTlhODktMjZhZmEwZWVmZThj&hl=en

Additional file 3 – Primer sequences and details for SSR loci

Spreadsheet file (csv) with primer sequences for potentially amplifyable SSR loci
selected using MSATCOMMANDER.
File available at: https://docs.google.com/leaf?
id=0BwOnBtQxjHP0ZGJmNzI2ODMtM2U1Ny00ZTRkLTkxODItOGFhYjM4N2M3YmYw&hl=en
 92
CURRICULUM VITAE

Joshua P. Der
(June 2010)

Career objective

Faculty position at a liberal arts or state university that emphasises research and
education.

Professional address

Department of Biology
Utah State University
5305 Old Main Hill
Logan, UT 84322-5305

Education

Ph.D. in Biology. Utah State University. June 2010. Dissertation: Genomic

Perspectives on Evolution in Bracken Fern.
Advisor: Dr. Paul G. Wolf

M.S. in Plant Biology. Southern Illinois University, Carbondale. July 2005. Thesis:
Molecular Phylogenetics and Classification of Santalaceae.1
Advisor: Dr. Daniel L. Nickrent

B.S. in Biology and Botany with a minor in Leadership Studies. Humboldt State
University. May 2003. Senior thesis: Investigation of wind pollination in the
Humboldt Bay wallflower (Erysimum menziesii ssp. eurekense) at the Lanphere-
Christensen Dunes Preserve.
Advisor: Dr. Michael R. Mesler

Peer-Reviewed Publications
Daniel L. Nickrent, Valéry Malécot, Romina Vidal-Russell, and Joshua P. Der. 2010.
A revised classification of Santalales. Taxon, 59 (2): 538-558.

Mark W. Ellis, Jessie M. Roper, Rochelle Gainer, Joshua P. Der and Paul G. Wolf.
2009. The taxonomic designation of Eriogonum corymbosum var. nilesii
(Polygonaceae) is supported by AFLP and cpDNA analyses. Systematic Botany
34 (4): 693-703. doi: 10.1600/036364409790139736

1Recipient of SIUC Alumni Association Outstanding Masters Thesis Award and

Midwestern Association of Graduate Schools Distinguished Masters Thesis Award.
 93
Peer-Reviewed Publications (continued)
Joshua P. Der, John A. Thomson, Jeran K. Stratford and Paul G. Wolf. 2009. Global
chloroplast phylogeny and biogeography of bracken (Pteridium:
Dennstaedtiaceae). American Journal of Botany 96 (5): 1441-1449. doi: 10.3732/
ajb.0800333.

Joshua P. Der and Daniel L. Nickrent. 2008. A molecular phylogeny of Santalaceae

(Santalales). Systematic Botany 33 (1): 107-116. doi:
10.1600/036364408783887438.

Charles D. Miller, R. Child, J. E. Hughes, Mekki Benscai, Joshua P. Der, R. C. Sims,

Anne J. Anderson. 2007. Diversity of soil mycobacterium isolates from three
sites that degrade polycyclic aromatic hydrocarbons. Journal of Applied
Microbiology 102 (6): 1612–1624. doi:10.1111/j.1365-2672.2006.03202.x

Daniel L. Nickrent, Joshua P. Der and Frank E. Anderson. 2005. Discovery of the
photosynthetic relatives of the "Maltese mushroom" Cynomorium*. BMC Evol.
Biol. 5:38. doi:10.1186/1471-2148-5-38. *Highly Accessed.

Grants
2008! Center for Integrated Biosystems Student Research Grant, Utah State
University. “Developing genomic resources for both phases of the plant life
cycle.” $6,948.50.

Awards and Special Recognition

2006! Botanical Society of America. Pteridological Section Student Travel Award.
2006! Midwestern Association of Graduate Schools. Distinguished Masterʼs Thesis
Award.
2005! Southern Illinois University, Carbondale Alumni Association. Outstanding
Masterʼs Thesis Award.
2004! National Academy of Sciences. Travel award to attend the Arthur Sackler
Colloquium on Systematics and the Origin of Species, On Ernst Mayrʼs 100th
Anniversary.
2004! Organization for Tropical Studies. Scholarship to the Tropical Plant
Systematics course in Costa Rica (Additional funding from SIUC).
2003! Humboldt State University. Outstanding Student of the Year: Outstanding
Contribution to a University Organization.

Invited Seminars
Joshua P. Der, M.S. Barker, N.J. Wickett, C.W. dePamphilis, P.G. Wolf. 2010.
Functional Genomics Of Fern Gametophytes: Transcriptome Sequencing In
Pteridium aquilinum. International Plant and Animal Genomes XVIII. San Diego,
CA. http://www.intl-pag.org/18/abstracts/W54_PAGXVIII_396.html
 94
Research Reports
Joshua P. Der, S. Jordan, and V. Vega. 2003. Investigation of wind pollination in
the Humboldt Bay wallflower (Erysimum menziesii ssp. eurekense) at the
Lanphere-Christensen Dunes Preserve. Final report submitted to Humboldt Bay
National Wildlife Refuge, US Fish and Wildlife Service. Available online: http://
www.scribd.com/doc/13741061

Published Abstracts
Joshua P. Der and P.G. Wolf. 2009. Exploratory analyses of genomic sequences in
the bracken fern, Pteridium aquilinum. Oral presentation at Botany 2009,
Snowbird, UT. http://2009.botanyconference.org/engine/search/index.php?
func=detail&aid=912
Joshua P. Der, J.A. Thomson, J.K. Stratford and P.G. Wolf. 2009. Global chloroplast
phylogeny and biogeography of bracken (Pteridium: Dennstaedtiaceae). Poster
at Botany 2009, Snowbird, UT.
http://2009.botanyconference.org/engine/search/index.php?func=detail&aid=209
Joshua P. Der, J.A. Thomson and P.G. Wolf. 2006. A global phylogeographic study
of the chloroplast genome in bracken (Pteridium: Dennstaedtiaceae). Oral
presentation at Botany 2006, Chico, CA.
http://www.2006.botanyconference.org/engine/search/index.php?
func=detail&aid=588
Joshua P. Der and D.L. Nickrent. 2005. Molecular systematics of Santalaceae:
phylogeny and classification of a paraphyletic family of hemiparasitic plants.
Oral presentation at Botany 2005, Austin, TX.
http://www.2005.botanyconference.org/engine/search/index.php?
func=detail&aid=213
D.L. Nickrent, F.E. Anderson, and Joshua P. Der. 2005. Phylogenetic analyses
identify the photosynthetic relatives of Cynomorium and Balanophoraceae. Oral
presentation at Botany 2005, Austin, TX.
http://www.2005.botanyconference.org/engine/search/index.php?
func=detail&aid=120
D.L. Nickrent, K.A. Speicher, and Joshua P. Der. 2005. Phylogenetic utility of
chloroplast acetyl-CoA carboxylase in angiosperms. Oral presentation at
Botany 2005, Austin, TX. http://www.2005.botanyconference.org/engine/search/
index.php?func=detail&aid=103
Joshua P. Der and D.L. Nickrent. 2005. Molecular phylogeny and classification of
Santalaceae. Oral presentation at Midwestern Ecology and Evolution
Conference, Southern Illinois University, Carbondale. http://mypage.siu.edu/
meec2005/Abs_EvolPhylo.html
D.L. Nickrent and Joshua P. Der. 2004. Santalaceae: phylogeny, taxonomy, and
biogeography. Oral presentation at Botany 2004, Snowbird, UT.
http://www.2004.botanyconference.org/engine/search/index.php?
func=detail&aid=645
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Additional Presentations
Joshua P. Der and Paul G. Wolf. 2009. Leveraging massively parallel
pyrosequencing for functional and evolutionary genomics in ferns. Utah Plant
Genetics and Genomics conference, University of Utah. Available at: http://
www.scribd.com/doc/25504380
Joshua P. Der. 2009. Developing genomic resources in the bracken fern, Pteridium
aquilinum. Center for Integrated Biosystems Student Research Program, Utah
State University. Available at: http://www.scribd.com/doc/13651759
Joshua P. Der, John A. Thomson and Paul G. Wolf. 2008. Global chloroplast
phylogeny and biogeography of bracken (Pteridium: Dennstaedtiaceae).
Organization for Tropical Studies, Tropical Ferns and Lycophytes Course,
Wilson Botanical Garden, Costa Rica.
Joshua P. Der and Daniel L. Nickrent. 2004. Phylogeographic investigations of
parasitic plants in Santalaceae. Midwestern Ecology and Evolution Conference,
University of Notre Dame.
Joshua P. Der. 2004. Systematics and phylogeny of “Santalaceae”. Organization
for Tropical Studies, Tropical Plant Systematics Course, Wilson Botanical
Garden, Costa Rica.
Joshua P. Der. 2004. Molecular phylogenetic investigations of Santalaceae.
Department of Plant Biology, Southern Illinois University, Carbondale.

Teaching Experience
Utah State University, 2005-present
Plant Taxonomy (1 semester)
General Biology 1 Lab (1 semester)
General Biology 2 Lab (4 semesters)
Genetics Lab (3 semesters)
Biological Discovery Lab (1 semester)
Genetics Lecture (5 guest lectures)
Southern Illinois University, Carbondale, 2003-2005
Plant Diversity Lecture (2 guest lectures)
Plants and Society Lab (1 semester)
Plant Diversity Lab (1 semester)

Service and Leadership Experience

2005 – 10! Graduate Representative, Curriculum Committee, Department of

Biology, Utah State University.
2004 – 05! President, Plant Biology Graduate Student Organization, Southern
Illinois University, Carbondale.
2004 – 05! Treasurer, Midwestern Ecology and Evolution Conference, Southern
Illinois University, Carbondale.
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Service and Leadership Experience (continued)

2004 – 05! Graduate Representative, Curriculum Committee, Department of

Plant Biology, Southern Illinois University, Carbondale.
2001 – 02! Student Representative, Sexual Assault Advisory Committee,
Humboldt State University.
2000 – 02! Outdoor Youth Counselor, Leadership Education Adventure Program,
Youth Educational Services, Humboldt State University.
2000 – 01! Vice President, National Residence Hall Honorary, Humboldt
Chapter, Humboldt State University.
1999 – 2000! Living Group Advisor / Resident Assistant, Department of Housing,
Humboldt State University.
1998 – 99! President, Residence Hall Association, Humboldt State University.

References

Dr. Paul G. Wolf Dr. Daniel L. Nickrent

Department of Biology Department of Plant Biology
Utah State University Southern Illinois University, Carbondale
5305 Old Main Hill Carbondale, IL 62901-6509
Logan, UT 84322 (618) 453-3223
(435) 797-4034 nickrent@plant.siu.edu
wolf@biology.usu.edu