University of Tennessee, Knoxville
TRACE: Tennessee Research and Creative
Exchange
Masters Theses
Graduate School
5-2016
Using phylogenetics to understand the evolutionary relationships
of Hibiscus section Furcaria
Whitaker Matthew Hoskins
University of Tennessee - Knoxville, whoskins@vols.utk.edu
Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes
Part of the Evolution Commons
Recommended Citation
Hoskins, Whitaker Matthew, "Using phylogenetics to understand the evolutionary relationships of Hibiscus
section Furcaria. " Master's Thesis, University of Tennessee, 2016.
https://trace.tennessee.edu/utk_gradthes/3745
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To the Graduate Council:
I am submitting herewith a thesis written by Whitaker Matthew Hoskins entitled "Using
phylogenetics to understand the evolutionary relationships of Hibiscus section Furcaria." I have
examined the final electronic copy of this thesis for form and content and recommend that it be
accepted in partial fulfillment of the requirements for the degree of Master of Science, with a
major in Ecology and Evolutionary Biology.
Randall Small, Major Professor
We have read this thesis and recommend its acceptance:
Brian O'Meara, Edward Schilling
Accepted for the Council:
Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
Using phylogenetics to understand the evolutionary relationships of polyploids in Hibiscus
section Furcaria
A Thesis Presented for the
Master of Science
Degree
The University of Tennessee, Knoxville
Whitaker Matthew Hoskins
May 2016
Acknowledgments
I would like to thank several people that made this work possible in terms of academic and
personal support. First, I want to thank Randy Small for being a flexible, supportive advisor
throughout my undergraduate and graduate career. I would also like to thank my committee
members Brian O’Meara and Ed Schilling for giving me great ideas on how to expand my
project and encouraging my learning process. Additional thanks need to go to several professors
that taught me a myriad of skills and tidbits along the way: Karen Hughes, Ken McFarland,
Brandon Matheny, Meg Staton, Joe Williams, Phillip Li, and many others. Also, thank you to
Justin Hendy, Cassie Dresser, John Reese, Jayne Lampley, and all the graduate students in the
EEB department for being great friends and colleagues. Additionally, I would like to thank the
Department of Ecology and Evolutionary Biology for providing funding to complete this work.
Lastly, thank you to my lovely wife, PJ Hoskins for supporting the decision to come back to
school and live in Knoxville once again.
ii
Abstract
Neopolyploids constitute at least 35% of known species of angiosperms, and because
polyploidization is a pertinent process in plant diversification and domestication, it is a thriving
field of study. Hibiscus section Furcaria includes several groups of polyploids in addition to ten
known diploid species. In previous studies genome groups for Hibiscus section Furcaria were
determined through artificial hybridization experiments and patterns of biogeography were
elucidated based on the distribution of diploids and polyploids. For instance, the Australian
hexaploids contain 3 genomes (designated G, J, and V) and are thought to have developed from a
polyploidization event between an African diploid relative (G) and two unknown donors (J and
V). This study seeks to perform phylogenetic analysis using a suite of chloroplast and nuclear
regions to determine the maternal genetic relationships between the diploids and the Australian
hexaploid lineage, and to reconstruct the origin of this group in order to determine if any
surviving diploid donors exist related to the unknown J and V genomes. Four chloroplast regions
and two nuclear regions were explored to determine genome relationships. Results showed the
Australian hexaploid species form a well-supported clade using chloroplast genes (ndhCtrnV,ndhF-rpl32R,rpl32F-trnL,rps16-trnK) and ITS with Hibiscus sudanensis as the maternal
donor of the G genome group. Possible donors to the J and V genome groups are proposed based
on the phylogenies, morphology, and biogeography but more sampling of clones is needed to
ensure the identity of possible donors lineages.
keywords: polyploidy, Hibiscus section Furcaria, phylogenetics, chloroplast DNA, nuclear
DNA, genome donor
iii
Table of Contents
Chapter 1: Introduction.....………………………..…..................……………………………..… 1
Chapter 2: Methods.…….....……………………………………....................….….......................8
Chapter 3: Results.……..…..……………………………………....................…………...…......13
Chapter 4: Discussion..……………..……………………...……...............…………......……....18
References……….…………………………………...……………………...…...…....................22
Appendices.....................................................................................................................................27
Appendix A: Tables.......................................................................................................................28
Appendix B: Figures......................................................................................................................32
Vita.....................................………….………………….………………………....…………..…45
iv
List of Tables
1. Plant materials used in this study. Voucher specimens at TENN..............................................29
2. Best fit models for cpDNA and nDNA......................................................................................30
3. Percent of variable and parsimony-informative sites from all sequence analyses.....................31
v
List of Figures
1. Species occurrence maps for each study species in Hibiscus section Furcaria.......................33
2. Total species occurrence map for all study species in Hibiscus section Furcaria....................34
3. Bayesian analysis of 4 cpDNA regions with genome groups, geography, and ploidy level....35
4. Bayesian analysis of GBSSI region..........................................................................................36
5. Bayesian analysis of ITS region...............................................................................................37
6. Maximum likelihood analysis of 4 cpDNA regions..................................................................38
7. Maximum likelihood analysis of GBSSI region........................................................................39
8. Maximum likelihood analysis of ITS region.............................................................................40
9. Parsimony bootstrap analysis of 4 cpDNA regions...................................................................41
10. Parsimony bootstrap analysis of GBSSI region.......................................................................42
11. Parsimony bootstrap analysis of ITS region............................................................................43
12. Probability plot for sampling all genome groups in the Australian hexploids in Hibiscus sect.
Furcaria as a function of the number of clones sampled..............................................................44
vi
Chapter 1: Introduction
What is polyploidy?
Historically, polyploids were defined as organisms containing multiple chromosomes
compared to an ancestral state, and ploidy level was determined by chromosome number (Lutz
1907, Husband 2013). Modern studies emphasize whole genome duplication as the inherent
process leading to polyploidy (Husband 2013). Whole genome duplications can be determined
through the comparison of the number of genes and genome size in terms of megabases (Mb) in
related genomes. Yet, this criterion expands the definition to include chromosome numbers that
do not always show strict doubling and may be the result of a restructured genome and/or loss of
paralogs. Research on polyploidy is increasing as molecular technology reaches a point where
whole genomes can be sequenced more efficiently and less expensively. Polyploids can be
classified as neopolyploids from a recent doubling event, or paleopolyploids from an ancient
duplication that subsequently underwent diploidization, but there is no specific temporal
distinction (Wolfe 2001, Adams & Wendel 2005). Diploidization is an evolutionary process that
converts quadrivalent chromosome pairing in tetraploids to bivalent chromosome pairing and
these species function genetically like diploids while maintaining the same overall chromosome
number (Wolfe 2001). Jiao et al. (2011) highlight the importance of paleopolyploidy in the
development of diverse plant groups by using expressed-sequence-tag (EST) sequences to
estimate ancient duplication events. The authors find one polyploidization event prior to the
diversification of seed plants and one prior to the diversification of angiosperms, thus suggesting
a correlation exists between polyploidy and cladogenesis.
Hybridization is another fundamental distinction in the origin of polyploids.
Autopolyploids represent whole genome duplications occurring within a species and perhaps just
1
one individual, while allopolyploids result from hybridization between different species.
However, it may be difficult to categorize a species as autopolyploid or allopolyploid due to
recurring introgression or paralog loss over time (Mason-Gamer 2013). Therefore the distinction
between autopolyploids and allopolyploids is not always clear within certain taxa (Stebbins
1970, Ramsey & Schemske 1998, Tate et al. 2005).
Two main mechanisms can form polyploids- somatic doubling and gametic nonreduction. Somatic doubling is a result of the failure of mitosis and cytokinesis in a zygote or
meristematic shoot. Gametic non-reduction is a result of a failure of one of the meiotic divisions
to halve the chromosome number. Gametic non-reduction is expected to form more viable
hybrids by using a ‘triploid bridge’ to allow polyploids to form offspring and populate an area.
The triploid bridge involves the backcrossing of triploids with diploids or a self-fertilizing
triploid to form fertile tetraploid individuals (Ramsey & Schemske 1998). An interesting
component to the development of polyploidy in plants is the conspicuous absence of a
centrosome for the microtubule-organizing center (Marshall 2009). Andreuzza & Siddiqi (2008)
provide a perspective piece on d-Erfurth et. al (2008) that explains how a mutant AtPS1 gene
specific to plants can produce a parallel spindle in Arabidopsis which leads to gametic nonreduction. The creation of non-reduced gametes leads to polyploidization in plants and may
explain why plants develop polyploid individuals at a higher rate than other kingdoms of life.
Cellular mechanisms like these, along with phylogenetic history and ecology, are key to the
understanding and utilization of polyploid plants in biotechnology. Resurgence occurring in
polyploid research is built around trying to understand ecological implications, genetic
complexity, and possible innovations in agricultural production (Glennon et al. 2014, Soltis et al.
2010, Sattler et al. 2016).
2
Importance of Polyploidy in Plants
As human population continues to increase crop production must keep pace in order to
supply adequate food for future generations (Hooke et al. 2012). This means the knowledge of
how to maximize sustainable production of plants is paramount and this is applied to the
increasingly inter-related fields of biotechnology and agriculture. Many of our existing crop
plants are polyploid (Renny-Byfield & Wendel 2014, Blanc & Wolfe 2004) which has
implications for understanding how crops and plants in general respond to selection.
On an organismal level polyploids may have certain advantages over diploids due to their
multiple gene copies. One advantage is the ability of polyploids to mask recessive alleles that are
harmful (Comai 2005). Also, polyploid plants can self-fertilize which allows polyploids to
overcome the reproductive barrier of minority cytotype exclusion. Minority cytotype exclusion is
the process by which most gametes of polyploids (2x) are lost due to their incompatibility with
the more abundant (1x) gametes in their environment (Levin 1975). Apomixis, or the formation
of embryos asexually, is another way polyploids may overcome reproductive barriers and
continue to proliferate. It has even been suggested these apomictic species are competitive but
reach a dead-end in terms of their evolutionary history (Stebbins 1970). Lastly, polyploidization
can lead to new functions or sub-functionalization of homeologs for adaptation (Adams &
Wendel 2005). Disadvantages also exist but mostly apply to cellular machinery and epigenetic
consequences (Comai 2005). These advantages stem from the complex molecular consequences
of polyploidization. Unfortunately, these complexities can also make it difficult to study
polyploid plants both in situ and ex situ.
3
Issues with the study of polyploids
Polyploids are organisms with homeologs, a term for paralogous chromosomes
developing from a genome duplication event (Wolfe 2001). The effect of polyploidization on the
phylogenetics of plant populations is increasingly studied as genetic technology enables the
assessment of these inherently difficult study systems. An example of these challenges includes
possible introgression among a polyploid and diploid that may generate polyphyletic clades and
incongruent gene trees (Mason-Gamer 2013). Additionally, the extinction of a diploid progenitor
of polyploid species may lead to no matching in a particular homeolog at all (Mason-Gamer
2013).
In addition to the genetic conundrums that polyploids present, there is a challenge to
ecologists because few universal ecological differences have been demonstrated between
diploids and polyploids (Soltis et al. 2010). Even when the duplication event is intraspecific
(autopolyploid) the ecological consequences are not necessarily predictable. In other words, even
species developing from autopolyploidization show little predictable ecological effects between
ploidy levels. Potential issues to explain the ecology of polyploids versus their diploid
counterparts include: climatic tolerance, secondary chemistry, invasiveness, flowering
phenology, and mating system (Hahn et al. 2012, Hull-Sanders et al. 2009, Pandit et al. 2014,
Robertson et al. 2011, Segraves & Thompson 1999).
Hibiscus Taxonomy
The traditional recognition of Hibiscus L. (Malvaceae) includes 250-350+ species (Pfeil
2005) however this group is not monophyletic and contains species within several tribes of
Hibisceae (Pfeil & Crisp 2005). Hibiscus section Furcaria is a group of ca. 200 species that
4
occurs around the globe and is prevalent in tropical and sub-tropical zones, especially in the
Southern Hemisphere. The section name Furcaria comes from the bifurcating bracteoles that
emerge below the calyx, sometimes referred to as an epicalyx. The distinguishing characters of
this section are the thickened midribs and marginal ribs of the calyx and presence or absence of
nectaries along these ribs. This group also includes two important crop species, H. sabdariffa or
roselle, used in making tea and jellies, and H. cannabinus, or kenaf, used in fiber and paper
production.
Several mating studies by Margaret Menzel and colleagues determined species within
Furcaria belong to distinct “genome groups” (Menzel & Martin 1971, Menzel & Wilson 1963,
Menzel & Martin 1980, Wilson 1993) and these genome groups are designated by letters (A, B,
C, D, E, G, H, J, P, R, V, X, Y). Hibiscus section Furcaria contains only 10 known diploids.
Several tetraploid lineages also exist (AB, AX, AY, BY, GH, GP) as well as species with higher
levels of polyploidy (BG _ _, CDEG, CDEGR, GJV). These studies used the compatibility of
chromosome pairing in pollen mother cells (PMCs) of hybrid individuals to determine the gene
affinity index (GAI) for each pair of species crossed. GAI ranges from 0 to 1 and is determined
based on the number of chromosomes that aligned during meiosis, higher scores means more
compatibility. In these studies evidence was found for a group of hexaploid Australian species
that were mostly incompatible with all other species in section Furcaria except those of the G
genome group.
The Australian hexaploid group, designated by the letters GJV, has been the focus of
recent taxonomic update. Craven (2003) revised the taxonomy of the Australian species and
produced an identification key and distribution maps for 23 species within Northern and Western
Australia but in total 29 species of Furcaria are present on the continent. These species are
5
shrubs and small trees found within the coastlands of tropical and subtropical regions of northern
Australia (Kimberley, Northern Territory, and Queensland). Fig. 1 and 2 show the known ranges
of the 4 Australian hexaploids used in this study. Some species are even found on other islands
such as Indonesia/Papua New Guinea.
Objectives
The following research seeks to add to the body of knowledge on the Australian
hexaploid species of section Furcaria in terms of their phylogenetic relatedness to other species
within Furcaria and add to the growing literature on polyploid phylogenetics. The research
objectives start in a broad sense on polyploids and narrow in on the study group- Hibiscus
section Furcaria.
1. Determine the phylogenetic relationships of diploid species to polyploid species using
chloroplast and nuclear DNA.
2. Infer the possible diploid donors for the J and V genomes found in the Australian
hexaploids.
3. Discuss the known ranges of the possible diploid donors and the biogeography of
hybridization events that allowed for the allohexaploids to develop in Australia.
Predictions
Since chloroplasts are typically inherited maternally (Greiner et al. 2015) the cpDNA
phylogeny will only show maternal inheritance relationships. The G genome species are known
to be compatible with the Australian hexaploids, represented as GJV, (Menzel & Martin 1980) so
6
we expect the cpDNA phylogeny to show close relationships with any species that contains a G
genome. Species used for this study in the G genome group are Hibiscus sudanensis, a diploid
representing the G genome, Hibiscus rostellatus, a tetraploid representing the GH genomes, and
Hibsicus furcellatus, a tetraploid representing the GP genomes. Nuclear data was also included
in this study to address the final objective of the origin of the J and V genome groups. Three
possible outcomes for the origin of the J and V genome groups could occur. (1) Cloned copies of
the J and V group may be located within a clade of extant species in section Furcaria. For
example, a genome copy, perhaps J, aligns with the A group while the other copy V aligns with
Y. (2) Cloned copies of the J and V groups may be located in the clade with other G species as
well because they align closely with another group, either the H or P, that is contained within a
tetraploid. This could mean the J, V, or both are modified H and P genomes, which in turn could
be modified from the G group. (3) Cloned copies of the J and V groups will not follow a
phylogenetic pattern because there are no extant progenitors of these groups, these progenitors
have not been found and sequenced, or the clones for the Australian hexaploid species were not
adequately sampled.
7
Chapter 2: Methods
Samples
Seeds of 28 species were obtained from the USDA National Seed Storage Laboratory in
Dry Branch, GA or the Southern Regional Plant Introduction Station at the University of Georgia
Griffin Campus. Table 1 shows all samples used in this study, several are from previous work
done by Randy Small on the diploid and tetraploid species of this group. These species were
selected to fully sample the potential diploid genome groups and sample some tetraploid species
of interest. Eleven species of the Australian hexaploids in section Furcaria were selected to
represent the Australian hexaploid group.
Table 2 shows the eleven Australian species, 2 of which are varieties, attempted for
growth and sampling in this study. These seeds were soaked in a mix of 10% bleach treatment
for an hour then rinsed to prevent fungal contamination. Seeds were then nicked and soaked in
water for several days. Eight of the twelve species germinated and were planted (see Table 2) in
soil saturated with water and ZeroTol (algaecide/fungicide) in order to prevent colonization of
foreign contaminants. Seedlings were watered every five days with low concentration liquid
fertilizer (Dyna-Gro), and ZeroTol and grown in controlled light conditions. Three plant species
within Hibiscus section Furcaria were successfully grown from seed (H. marenitensis, H.
meraukensis, H. splendens) and one previously extracted (H. arnhemensis). For each
successfully grown specimen, a voucher specimen was placed in the University of Tennessee
Herbarium. Collection numbers and voucher information are available in Table 2. The intent of
this study is not to fully sample the Australian group but use representatives to determine the
phylogenetic relationships of the Australian group to other genome groups.
8
Molecular methods
Total genomic DNA was isolated using standard protocols with the DNeasy Plant Mini
Kit (Qiagen) by extracting from young leaves of lab grown plants. PCR amplifications followed
published reaction conditions (Small 2004). Seven cpDNA regions from Shaw et al. (2005) were
screened. Four regions were used (ndhC-trnV,ndhF-rpl32R,rpl32F-trnL,rps16-trnK) that showed
the greatest variability and thus potential phylogenetic utility. The nuclear ribosomal internal
transcribed spacer (ITS) region was amplified using ITS4 and ITS5 primers (White et. al 1990).
Reaction conditions were adapted from White et al. (1990). GBSSI (granule-bound starch
synthase I), a protein-coding, single copy nuclear gene was amplified using primers 2B-9R* (5’CCAATGAACCCAATCAAGGGAG-3’) and 2-X2F (5’-TGACNGTGTCTCCTCGCTATGA-3’).
The GBSSI reaction conditions were 95°C for 3 minutes, 40 cycles of 95°C for 30 seconds,
66°C for 30 seconds, 72°C for 2 minutes. DNA sequencing was performed on column purified
(QIAquick PCR Purification Kit) or ExoSAP-IT (USB) cleaned PCR products using the ABI
Prism Big Dye Terminator Cycle Sequencing kits v. 3.1 (Applied Biosystems), and read on an
ABI 3100 automated sequencer at The University of Tennessee Molecular Biology Resource
Facility, Knoxville.
Cloning
Since multiple copies of each nuclear region were expected in the Australian hexaploid
species (Menzel & Martin 1980) based on hybridization experiments, and multiple bands were
shown through gel electrophoresis, the PCR products were cloned prior to sequencing.
Additionally, tetraploids and some heterozygous diploid species were cloned in previous work
for the ITS region. All cloning used the vector pGEM-T (Promega) system as described in Small
9
et al. (1998). Cloning plates using E. coli were grown overnight in a Luria broth and ampicillin
mixture and plasmid DNA was purified as described in Small et al. (1998). 10 colonies were
picked per plate. The insert DNA was PCR amplified and sequenced to obtain clean nuclear
sequence copies. An analysis of the probability of detecting all gene copies in sampling but this
was done post-cloning (see Fig. 12).
Alignment and phylogenetic analyses
Pre-alignment sequence editing (base correction using chromatograms, forward and
reverse sequence overlap) was performed in Sequencher v. 5.1. Sequence alignment was
performed using Muscle v. 3.7. Post-alignment sequence editing was performed in MacClade v.
4.08 and Gblocks (Castresana 2000). An unaligned NBRF file was uploaded to the Gblocks
server and all 3 options for less stringent selection were selected; then the resulting file was
downloaded, converted to a fasta file, and used in subsequent analyses. Indels in the cpDNA
dataset were coded as binary characters at the end of alignments but no indels were present in the
GBSSI or ITS regions. The four cpDNA regions were edited separately, concatenated, then
partitioned into their 4 regions. The GBSSI region was edited as a whole then partitioned into an
exon region and an intron region (sequences represent a small portion of exon 2, all of exon 3-9
and introns 2,3, 5-8). The ITS region was edited whole and not partitioned.
Phylogenetic model testing in MEGA v. 6 (Tamura et al. 2013) was performed using
discrete characters and lowest BIC scores were used for the maximum likelihood and Bayesian
analyses (see Supplementary Table 1). Each of the four chloroplast regions showed T92 or
T92+G as the best model. The exons of GBSSI were best modeled by K2+G and the introns
were best modeled by T92 but GTR+G+I was employed due to low phylogenetic signal in
10
preliminary tree formation. The ITS region showed K2+G as the best model but GTR+G+I was
employed due to low phylogenetic signal in preliminary tree formation. The Tamura 3-parameter
model (T92) adds a G-C content bias for the sequence data to the Kimura 2-parameter model
(Tamura 1992). The Kimura 2-parameter model uses equal base frequencies, one transition rate,
and one transversion rate. The Generalized Time Reversible (GTR) model uses 6 substitution
rates and is a more neutral model (Yang 1994). The gamma distribution represents rate variation
among sites (Kimura 1980). The invariable sites model (I) allows for a proportion of sites to be
invariable.
Phylogenetic analyses were conducted using parsimony (PAUP*4.0, Swofford 2003),
maximum likelihood (MEGA v. 6, Tamura et al. 2013), and Bayesian methods (Mr. Bayes v.
3.2.6, Ronquist & Huelsenbeck 2003, or under the CIPRES Gateway, Miller et al. 2010).
Parsimony analyses were conducted using a “fast”-stepwise stepwise addition with 10,000
replicates and retaining groups with greater than 50% frequency. Maximum likelihood
parameters were set to the appropriate substitution rate in MEGA v. 6 and 200 bootstrap
replicates were used in order to maintain adequate sampling (Pattengale 2009). A uniform rate
was used for variation among sites and the nearest-neighbor-interchange (NNI) heuristic was
employed with a very strong branch swap filter.
Mr. Bayes parameters were set to the appropriate modeled rates of likelihood. Model
partitioning was used for the Bayesian analyses based on differences in appropriate substitution
models for the 4 cpDNA regions and the exon and intron region of GBSSI. Indels were coded as
0 (absence) and 1 (presence) at the end of the concatenated cpDNA sequence. The MKv model
was used to analyze this portion of sequence (Lewis 2001). The Bayesian analyses were
conducted in two independent runs with four chains each (3 heated, 1 cold, temp=0.2) and a
11
sample was taken every 1,000 generations with an initial 1,000,000 generations used. The first
25% of generations were used as burn-in. The ITS analysis required an additional 4,000,000 runs
due to high standard deviation of split frequencies. Runs reached stationarity after burn-in and
continued until the standard deviation of the split frequencies was lower than 0.01 and a visual
examination of likelihood scores showed equal mixing of the two independent runs.
12
Chapter 3: Results
cpDNA analysis
Each cpDNA region amplified from 1000-1400 bp with a concatenated sequence of 5333
bp, and contained 6.5-9.5% variable sites with 2.1-3.6% parsimony-informative sites. A basic
local alignment search in GenBank showed the chloroplast sequences were highly similar to
sequences from Shaw et al. (2007), with the highest match equal to 97% for H. cannabinus.
All three phylogenetic analyses show similar patterns of evolution within section Furcaria but
the Bayesian analyses will be discussed due to stronger clade support using posterior probability
distributions. (See Figures 6-8 for maximum likelihood analyses and Figures 9-11 for parsimony
analyses.)
Overall, the genome groups discovered through previous hybridization studies (Menzel &
Martin 1971, Menzel & Wilson 1963, Menzel & Martin 1980) form well-supported clades (Fig.
3). The B genome group forms a clade with H. acetosella and H. radiatus (tetraploids) and H.
surattensis- (diploid). The two specimens used for H. surattensis show an unusual relationship
because they are not sister to one another but this relationship has low support (0.55). The Y
genome forms a clade with H. mechowii (diploid) and H. sabdariffa (tetraploid). The G genome
forms a clade with the Australian hexaploid group (H. arnhemensis, H. marenitensis, H.
meraukensis, and 2 H. splendens), H. sudanensis (diploid), H. rostellatus (GH tetraploid), and H.
furcellatus (GP tetraploid). This confirms the G genome lineage as the maternal donor for the
Australian hexaploids. Hibiscus splendens specimen 4-3BP shows a long branch from the other
H. splendens specimen 1423. This is due to a tandem repeat of a single insertion of thymine
throughout each cpDNA sequence. The A genome with all A diploid species (H. asper, H.
cannabinus, H. greenwayi) also forms a clade. The X genome forms a clade with two diploid
13
species (H. hiernianus, H. mastersianus) and two tetraploid species (H. meeusei, H. nigricaulis).
1 outgroup was used for this phylogeny- Urena procumbens. Additionally, there is split between
the B, Y species and the A, G, X species towards the base of the tree. However, the A genome
exists in tetraploid species of the B and Y genomes as well (Fig.3). H. costatus shows as a sister
species to the B and Y genome group but with low support and a long branch. This is consistent
with previous findings on H. costatus in an unknown genome group.
GBSSI analysis
The exon regions of GBSSI amplified a total of approximately 800 bp, and contained
13.9% variable sites with 5.1% parsimony-informative sites. The intron regions of GBSSI
amplified a total of approximately 546 bp, and contained 26.7%, variable sites with 9.7%
parsimony-informative sites. A basic local alignment search in GenBank showed the GBSSI
sequences were much different than most of the sequences on GenBank and the highest match
was equal to 78% for H. moschuetos subsp. moschuetos.
The Bayesian analysis of the GBSSI region (Fig. 4) shows support for the grouping of
species within section Furcaria but not as strongly as the cpDNA analysis. A clade of 8 GJV
copies, including three of the Australian hexaploid species sampled, forms with Hibiscus
sudanensis, confirming the G genome as a donor lineage to the Australian hexaploids. A clade
with low support (0.7 posterior probability) forms with H. mechowii of the Y group, H.
surattensis of the B group, and 4 H. splendens clones from the GJV group. Lastly, a separate
clade of 8 GJV copies, involving all 4 of the Australian hexaploid species sampled forms near
the base of the tree, indicating uncertainty in the genome group it may represent. 1 outgroup was
used for this phylogeny- H. macrophyllus. Interestingly A, G, X genome groups form a well14
supported clade matching the pattern seen in the chloroplast regions. The B and Y genome
groups are once again split from the A/G/X clade but the support is low for the placement of this
division in the GBSSI gene region (0.53 posterior probability). Hibiscus costatus again forms a
polytomy near the base of the phylogeny further indicating an uncertain relationship for this
American diploid species.
More extensive sampling of this gene region in Furcaria is required (only 20 clones
successfully generated) to eliminate GBSSI as a useful region for phylogenetic inference of
donor lineages to the J or V genomes of the Australian species.
ITS analysis
A basic local alignment search in GenBank showed ITS sequences were somewhat
similar to existing sequences and the highest match was equal to 94% for H. sabdariffa. The ITS
regions amplified a total of approximately 750 bp, and contained 54.8% variable sites with
28.4% parsimony-informative sites.
The Bayesian analysis of the ITS region (Fig. 5) also shows support for genome groups
within Furcaria forming clades but some have with low support. The A genome forms two
clades due to strong heterozygosity within the diploids of this group (Small et al. unpub). The
top-most A genome clade contains 1 H. asper copy (diploid), all 3 H. cannabinus copies
(diploid), 5 tetraploid copies (H. acetosella, H. meeusei, H. nigricaulis, H. radiatus, H.
sabdariffa), and 1 GJV copy from H. meraukensis but with low support (0.56 posterior
probability). The lower-most A genome clade contains 2 H. asper copies (diploid), 2 H.
greenwayi copes (diploid), 3 tetraploid copies (H. meeusei, H. nigricaulis, H. sabdariffa), and all
3 copies of the GP tetraploid, H. furcellatus with high support (1 posterior probability). This may
15
indicate the P genome of H. furcellatus is a modified A genome but the posterior probability is
low for this relationship (0.69). The G genome forms a clade with a diploid H. sudanensis
(diploid), 2 copies of the tetraploid H. furcelltatus (GH), and 44 GJV hexaploid copies (23 H.
splendens, 13 H. arnhemensis, 7 H. marenitensis, 2 H. meraukensis). All clones from the
Australian hexaploids form a clade with the G genome species and do not aid in the elucidation
of the J or V genome donor species to these lineages. This is likely due to high concerted
evolution in the G genome species for the ITS region (Small et al. unpub.) Additional studies
have found ITS sometimes does not provides high taxonomic resolution due to non-variable sites
and complex indels (Schilling, Mattson, & Floden 2013). The B genome forms a clade with 2
copies of H. surattensis (diploid), 1 copy of H. acetosella (tetraploid) and 1 copy of H. radiatus
(tetraploid). Hibiscus costatus (diploid) maintains the uncertain phylogenetic pattern seen in the
cpDNA and GBSSI analysis. The X genome forms a clade with two diploid species (H.
hiernianus, H. mastersianus) and two tetraploid species (H. meeusei, H. nigricaulis). The Y
genome forms a clade with 2 individuals of H. mechowii (diploid) and 2 individuals of H.
sabdariffa (tetraploid). 2 outgroups were used for this phylogeny- H. macrophyllus and Urena
procumbens. No split of the B/Y clades from the A/G/X clades is present in the ITS phylogeny.
In total 44 clones were sequenced for the ITS region. Fig. 16 shows this level of sampling would
be adequate for finding all the copies in the Australian hexaploids. However, this assumes the
gene copies are sequenced with equal probability. Therefore, more sampling might be useful in
for the ITS region as well.
16
Chapter 4: Discussion
Implications
Hibiscus section Furcaria is well supported as a distinct clade with multiple genome
groups. The previous morphological and cytological work performed by Menzel and others
(Menzel 1963, Menzel & Martin 1980, Menzel et al. 1983) is consistent with the phylogenetic
relationships found in the present study. The cpDNA phylogeny clearly shows monophyletic
groups for the A, B, X, Y, and G genomes and a potential split in the clades giving rise to the
B,Y genomes and the A,G,X genomes. This split may help explain the current distribution of
species for section Furcaria seen in the world, but no clear patterns are seen in the distribution of
the diploids on the African continent to support this division (Wilson 1993).
The GJV genome group is shown to be monophyletic using maternal cpDNA and
emerges from the G genome group represented by H. furcellatus (GP), H. rostellatus (GH), and
H. sudanensis (G). This also confirms H. furcellatus and H. rostellatus as relatives of the G
genome group. The G copy from H. sudanensis forms an unresolved polytomy within the G/GJV
clade. One possible explanation for this is that the GBSSI and ITS regions may not diverge
enough in the J or V genomes to separate H. sudanensis from the Australian hexaploids. The G
genome may be homogenized through recombination and maintained during hybridization
events. The ITS gene region is known to sometimes provide low phylogenetic resolution due to
the presence of pseudogenes, introgression, and extensive variation after duplication events
(Álvarez & Wendel 2003) so this region may not be suitable for analysis within Furcaria
without additional methods.
The G genome diploid lineage represented by H. sudanensis is located in a specific area
of Central Africa (Zaire, Central African Republic, and South Sudan) but species with the G
17
genome show a widespread distribution across the Southern Hemisphere, suggesting a
Gondwanan distribution. However, the Eumalvoideae clade that contains Hibiscus is much too
young to be influenced by continental divides, with an estimated divergence time of 58-60 mya
(Carvalho et al. 2011). Therefore long-distance dispersal coupled with vicariance within multiple
continents likely explains the current distribution of this section.
Hibiscus tiliaceus provides an example of a species within the genus, but not the section,
that could explain how a genome may have spread and developed through long-distance
dispersal. Hibiscus tiliaceus or the sea hibiscus is a pantropical species that likely underwent
multiple long-distance dispersal events but retains species continuity through gene flow
(Takayama et al. 2008). Tang et al. (2011) show habitat differentiation of estuarine and inland
populations in southern China for this species using retrotransposon markers. Gene flow is
prominent in the estuarine populations of H. tiliaceus and this might explain how polyploid
genome groups could occur in the same area and undergo allopolyploidization. Once a species
reaches the coastline it may become established and could diverge through vicariance, later that
same species or a different one may arrive and hybridize with this diverged species. Cotton
studies provide an example of how species with different genome groups can migrate long
distances and hybridize to form combinations in novel environments (Wendel & Cronn 2003,
Wendel & Grover 2015). Hibiscus tiliaceus is not within section Furcaria but is distributed in a
similar coastline area of northern Australia. No direct conclusions about the biogeography of the
J or V genome donor linegaes are evident from this work, but more integrated methods with in
situ and ex situ components in estuarine plant populations may reveal a connection between
ploidy and biogeography in section Furcaria.
18
The J and V genomes do not show a clear pair of diploid progenitors. However, some
diploids are likely candidates for further study. Hibiscus rostellatus, a GH tetraploid is one such
candidate species. The H genome may be homologous to the J or V genomes (Small et al.
unpub.), as evidence shows H. rostellatus can successfully cross with H. heterophyllus, a GJV
species (Menzel & Martin 1971). H. rostellatus may have developed as an autotetraploid (i.e.
GG) and diverged from the G genome early on in the evolution of section Furcaria to become
GH. This GH tetraploid could then have hybridized with another species in Furcaria with a
different genome group. The ITS phylogeny shows H. rostellatus in a clade with the GJV species
but increased sampling of H. rostellatus is necessary to determine if this grouping is due to the G
or H genome.
Hibiscus mechowii is sister to one of the Australian GJV genome group clades in the
GBSSI phylogeny but with weak support (0.7 posterior probability). Morphologically this
species is an intriguing potential donor for the J or V genome because it is one of only two
species that has both an entire set of bracts and no calyx nectaries, two unique characteristics of
the GJV Australian species (Wilson 1993). Hibiscus australensis, a species with unknown
genome group or chromosome count, occurs on the Austral Islands in French Polynesia and also
shares this unique morphology (Wilson 2006). The distribution of the Y genome is currently
limited to central Sub-Saharan Africa with only one tetraploid species known, H. sabdariffa
(AY). The evolution of the Y genome into the J or V genome may be possible due to the limited
amount of species represented in the Y genome currently but increased sampling of Y genome
species is necessary to provide an adequate result.
19
Future Work
Additional clonal sampling and phylogenetic analyses into other nuclear regions could
promote the understanding of evolution and polyploidy in Hibiscus section Furcaria and lead to
definitive evidence of the biogeography within this group. Emphasis should be placed on
increased clonal sampling for a single, easily-sequenced hexaploid species and determining the
genome groups of species with unknown types- H. costatus, Pacific Island species, and other
Australian species in order to eliminate or confirm these species as being donors or relatives of
the Australian hexaploids.
Next-gen sequencing is key to the study of polyploidy in the future as genomes can be
separated from one another through indices using SNPs (Page et al. 2013). The current study
developed a small picture of what is occurring in the polyploid genomes of Hibiscus section
Furcaria but sequencing larger portions of the genome could explain the story of evolution in
this group more fully. In addition to the development of molecular methods more hybridization
studies could be performed to test the validity of potential diploid donors.
Lastly, more clones are likely needed to ensure full coverage of the gene copies within
the hexaploid of Hibiscus section Furcaria. In order to determine the adequate level of sampling
necessary to obtain all gene copies from the Australian hexaploids a probability function was
created in R by Dr. Brian O’Meara to assess an adequate level of sampling. Assuming all
genome groups are sequenced at the same rate the probability of sampling all genome groups
will increase as more clones are sampled. Fig. 12 shows two lines representing the number of
gene copies. The red line represents 6 copies while the black line represents 3 gene copies. The
number 6 was chosen to represent each copy of a gene that is actually present in the Australian
hexaploids (GGJJVV) and 3 was chosen to represent each copy from one pair of the sister
20
chromosomes in each genome group (G, J, or V) in this polyploid group. The blue line shows a
95% probability of sampling all gene regions. If pairs of sister chromosome copies are the same
for the gene regions used (black line) the level of adequate sampling is quite low (below 10).
However, if each 6 gene copies are needed then the 95% probability requires close to 25 clones
(a level only obtained by ITS copies.) This also assumes each species contains G, J, and V
genomes that are not altered in the nuclear gene regions sampled for this study (ITS, GBSSI).
Lastly, the adaptive radiation of the Australian species likely occurred due to open niche
space after long-distance dispersal of seeds from one continent to the other combined with
vicariance, but the specific geographic or biological factors leading to this speciation are
relatively unknown. Perhaps the release from sugar production in calyx nectaries for the allowed
for an adaptive advantage in the Australian progenitor that led to speciation but this has yet to be
tested. Figures 1 and 2 show these species occur in distinct geographic areas and some have
much wider distributions than others. The reason for this ecological separation is currently
unknown and could warrant further study as well. Further knowledge of the ecological factors
that construct complex polyploid relationships in this group would be greatly beneficial.
21
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26
Appendices
27
Appendix A: Tables
28
Table 1 Plant materials used in this study from Hibiscus section Furcaria. Voucher specimens
deposited at the University of TN Herbarium for Australian species sampled. Adapted from
Wilson (1993) Table 1.
Species
Accession #
Genome Group
Area of origin
H. acetosella
A59-152
AB
Africa
H. apser
A82-1255, A93-1417
A
Africa
H. arnhemensis
A68-754
GJV
Australia
H. cannabinus
74200 I4, 51-7
A
Africa
H. costatus
A60-243
Unknown, but diploid
America
H. furcellatus
A91-1412
GP
Americas
H. greenwayi
A75-1085, A75-1177
A
Africa
H. hiernianus
A69-803
X
Africa
H. marenitensis
A86-1389
GJV
Australia
H. mastersianus
A75-1136
X
Africa
H. mechowii
A70-814
Y
Africa
H. meeusei
A69-805
AX
Africa
H. meraukensis
A82-1288
GJV
Australia
H. nigricaulis
PI 196180
AX
Africa
H. radiatus
A59-53
AB
India
H. rostellatus
A60-211
GH
Africa
H. sabdariffa
A59-56
AY
Africa
H. splendens
A94-1423, 4-3 BP
GJV
Australia
H. sudanensis
PI 267679
G
Africa
H. surattensis
A75-1139, A59-151
B
Africa, Asia
29
Table 2 Australian plants grown from seed in Hibiscus section Furcaria. The 4 specimens used
for this study deposited at the University of TN Herbarium have assigned collection numbers.
Species
Germinated
Accesion Number
Collection Number
H. arnhemensis
Yes
A68-754
RSmall (s.n.)
H. fallax
No
LL 1579
H. forsteri
Yes
A94-1428
H. fryxelli var.
fryxellis
H. fryxelli var. mollis
Yes
A86-1398
No
A84-1337
H. marenitensis
Yes
A86-1389
H. menzeliae
Yes
A67-717
H. meraukensis
Yes
A82-1288
H. reflexus
No
A82-1264
H. splendens
Yes
A94-1423
H. superbus
Yes
A86-1400
H. zonatus
No
A84-1369
WHoskins001
WHoskins002
WHoskins003
30
Table 3 Percent of variable and parsimony-informative sites from all sequence analyses.
cpDNA
ndhF-rpl32R
rpl32F-trnL
ndhC-trnV
trnK-rps16
nDNA
GBSSI
exons
introns
ITS
%Variable Sites
% Parsimony-Informative Sites
9.5
6.5
8.3
8.6
2.8
3.0
3.6
2.1
13.9
26.7
54.8
5.1
9.7
28.4
31
Appendix B: Figures
32
Fig. 1 Species occurrence maps for the four studied species in Hibiscus section Furcaria. Maps
constructed on Living Atlas of Australia (http://www.ala.org.au/) for the four species sampled
Australian species in this study. Occurrences recorded by various organizations and government
agencies of Australia. (a) red dots represent H. arnhemensis (b) pink dots represent H.
marenitensis (c) purple dots represent H. meraukensis (d) blue dots represent H. splendens
33
Fig. 2 Total species occurrence map for the four studied species in Hibiscus section Furcaria.
Occurrences recorded by various organizations and government agencies of Australia. (a) red
dots represent H. arnhemensis (b) pink dots represent H. marenitensis (c) purple dots represent
H. meraukensis (d) blue dots represent H. splendens
34
H. acetosella
0.55
H. surattensis041
0.55
H. surattensis004
1
!"#$%&'()*$+%'
,-&'(-.'
H. radiatus
1
H. mechowii
()*$+%',<&'(<.'
1
0.98
H. sabdariffa
H. costatus
(67*$+%0',8"9":;".'
H. arnhemensis
1
H. marenitensis
(/01*%2$%',345.'
1
H. meraukensis
1
H. splendens1423
1
0.96
1
H. splendens4-3BP
H. furcellatus
1
H. rostellatus
()*$+%&'(67*$+%0',3&'3>&'3?.'
1
H. sudanensis
H. asper001
1
H. asper037
0.97
1
H. cannabinus002
1
()*$+%',(.'
H. cannabinus038
1
H. greenwayi015
1
H. greenwayi048
0.97
H. hiernianus
0.99
H. mastersianus
1
H. meeusei
()*$+%',=&'(=.'
H. nigricaulis
Urena084
0.0030
Fig. 3 Bayesian analysis of 4 cpDNA regions with genome groups, geography, and ploidy level
Colored bars represent genome groups and their location. Colored taxa correspond to ploidy
level (black=diploid, blue=tetraploid, red=hexaploid). This Bayesian analysis shows a phylogeny
using 4 regions (ndhF-rpl32R, rpl32F-trnL, ndhC-trnV, rps16-trnK) of Hibiscus L. section
Furcaria with posterior probabilities at nodes. This phylogeny shows support for an association
between genome groups and biogeographic speciation of Hibiscus section Furcaria. The
Australian GJV group nested within the maternal G group. Bar below represents average
substitutions per site.
35
Fig. 4 Bayesian analysis of GBSSI region
This Bayesian analysis of Hibiscus L. section Furcaria uses GBSSI (granule-bound starch
synthase I) and displays posterior probabilities at nodes. This phylogeny shows high support for
H. sudanensis representing a donor lineage to the Australian species and low support for H.
mechowii (Y group) or H. surratensis representing a donor lineage to the Australian species.
Two other groups of Australian species are contained within the phylogeny but it is unclear what
donor diploid species are contributing to their hexaploidy. Black taxa = diploid species, red
taxa= hexaploid species. Bar below represents average substitutions per site.
36
H. macrophyllus
H. acetosella122.1
H. asper001
0.98
H. sabdariffa126.46
H. meeusei129.48
1
0.82
H. nigricaulis124.6
H. radiatus125.40
H. sabdariffa126.43
0.56
0.72 H. cannabinus002
H. cannabinus038
0.86
H. cannabinus043
H. radiatus125.24
H. nigricaulis124.11
0.57
0.75
0.98
1
0.99
0.98
1
()*$+%',('#$012$#3'%"#'./.*%012$#34'
1 H. meraukensis clone
13 H. arnhemensis clones
23 H. splendens clones
7 H. marenitensis/2 H. meraukensis clones
(53.*%1$%'
',678'
9/:%012$#34'
H. rostellatus
1H. rostellatus016
1
()*$+%',6&'6A4'
H. sudanensis
H. acetosella122.6
0.84
!"#$%&'()*$+%',-'%"#'-'
H. surattensis004
1
H. surattensis041
1
./.*%012$#34'
H. radiatus229.11
H. costatus070
1
H. costatus071
(;/*$+%3',<"="2>"4'
1
H. costatus014
H. asper037
0.98
1 H. asper042
H. sabdariffa126.41
0.69
H. furcellatus128.21
1
()*$+%',('#$012$#3'%"#'./.*%012$#34'
H. furcellatus128.21
(;/*$+%3',6B4'
1
H. furcellatus128.35
1
H. greenwayi048
1
H. greenwayi015
H. meeusei129.54
1
1
H. nigricaulis124.4
H. hiernianus039
1
H. mastersianus120.36
1
H. mastersianus047
()*$+%',@'%"#'@'./.*%012$#34'
1
H. mastersianus120.21
0.94
H. meeusei129.42
1
H. nigricaulis124.1
H. sabdariffa
0.99
H. sabdariffa126.48
()*$+%',?'#$012$#3'%"#'./.*%012$#34'
1H. mechowii006
H. mechowii046
Urena procumbens
0.96
1
0.04
Fig. 5 Bayesian analysis of ITS region
This Bayesian analysis of Hibiscus L. section Furcaria uses ITS (internal transcribed spacer)
with posterior probabilities displayed at nodes. Colored bars represent genome groups and their
location. Colored taxa correspond to ploidy level (black=diploid, blue=tetraploid,
red=hexaploid). The phylogeny shows support clades for most genome groups and high support
for H. sudanensis representing a donor lineage to the Australian species but no other lineages.
Bar below represents average substitutions per site.
37
H._asper001
1
H._asper037
1
H._cannabinus002
()*$+%',(.'
1
H._cannabinus038
0.96
H._greenwayi015
1
0.62
H._greenwayi048
H._hirenanus
0.97
H._meeusei
()*$+%',=&'(=.'
0.82
H._mastersianus
0.41
0.73
H._nigricaulis
H._rostellatus
()*$+%&'(67*$+%0',3&'3>&'3?.'
0.98
H._sudanensis
0.58
H._furcellatus
0.98
H._splendens1423
0.99
H._splendens43BP
0
0.92
H._meraukensis
1
(/01*%2$%',345.'
H._arnhemensis
0.46
H._marenitensis
H._costatus
(67*$+%0',8"9":;".'
H._mechowii
()*$+%',<&'(<.'
1
0.9
H._sabdariffa
H._acetosella
0.92
0.38
H._surattensis041
1
H._radiatus
!"#$%&'
()*$+%'
,-&'(-.'
0.18
H._surattensis004
Urena
0.0030
Fig. 6 Maximum likelihood analysis of 4 cpDNA regions
The evolutionary history was inferred by using the Maximum Likelihood method based on the
Tamura 3-parameter model. The tree with the highest log likelihood (-11083.9067) is shown.
The percentage of trees in which the associated taxa clustered together is shown next to the
branches. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining
method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood
(MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of
substitutions per site. The analysis involved 26 nucleotide sequences. There were a total of 5407
positions in the final dataset. Evolutionary analyses were conducted in MEGA6. Bar represents
average substitutions per site.
38
Fig.7 Maximum likelihood analysis of GBSSI region
The evolutionary history was inferred by using the Maximum Likelihood method based on the
General Time Reversible mode. The tree with the highest log likelihood (-3788.5858) is shown.
The percentage of trees in which the associated taxa clustered together is shown next to the
branches. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining
method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood
(MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of
substitutions per site. The analysis involved 27 nucleotide sequences. There were a total of 1346
positions in the final dataset. Evolutionary analyses were conducted in MEGA6. Bar represents
average substitutions per site.
39
Fig.8 Maximum likelihood analysis of ITS region
The evolutionary history was inferred by using the Maximum Likelihood method based on the
General Time Reversible model. The tree with the highest log likelihood (-5035.2136) is shown.
The percentage of trees in which the associated taxa clustered together is shown next to the
branches. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining
method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood
(MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of
substitutions per site. The analysis involved 95 nucleotide sequences. There were a total of 639
positions in the final dataset. Evolutionary analyses were conducted in MEGA6. Bar represents
average substitutions per site.
40
H._arnhemensis
99.99
H._marenitensis
(/01*%2$%'
',345.'
H._meraukensis
87.44
H._splendens1423
97.31
H._splendens43BP
93.77
H._furcellatus
()*$+%&'
(67*$+%0'
',3&'3>&'3?.'
H._rostellatus
96.94
H._sudanensis
H._asper001
99.99
69.81
H._asper037
99.94
H._cannabinus002
()*$+%',(.'
99.99
H._cannabinus038
91.15
H._greenwayi015
100
H._greenwayi048
61.88
H._hirenanus
H._mastersianus
98.19
66.23
H._meeusei
()*$+%''
,=&'(=.'
H._nigricaulis
H._costatus
(67*$+%0''
,8"9":;".'
H._mechowii
79.49
()*$+%',<&'(<.'
100
H._sabdariffa
H._acetosella
90.05
H._radiatus
99.97
H._surattensis004
!"#$%&'
()*$+%'
,-&'(-.'
H._surattensis041
Urena
Fig. 9 Parsimony analysis of 4 cpDNA regions using “fast” stepwise addition with 10000
replicates and retaining groups with greater than 50% frequency. Performed in PAUP*4.0
(Swofford 2003).
41
Fig. 10 Parsimony analysis of GBSSI region using “fast” stepwise addition with 10000 replicates
and retaining groups with greater than 50% frequency. Performed in PAUP*4.0 (Swofford
2003).
42
Fig. 11 Parsimony analysis of ITS region using “fast” stepwise addition with 10000 replicates
and retaining groups with greater than 50% frequency. Performed in PAUP*4.0 (Swofford
2003).
43
1.0
0.8
0.6
0.4
0.2
0.0
Probability of sampling all genome groups
0
10
20
30
40
Number of clones
Fig. 12 Probability plot for sampling all genome groups in the Australian hexploids in Hibiscus
sect. Furcaria as a function of the number of clones sampled.
This plot shows two lines representing the number of gene copies. The red line represents 6
copies while the black line represents 3 gene copies. The number 6 was chosen to represent each
copy of a gene that is actually present in a hexaploid (GGJJVV) and 3 was chosen to represent
each copy from one pair of homologous genome groups (G, J, or V) in this polyploid group. The
blue line represents a 95% probability of finding all copies.
44
Vita
Whitaker Matthew Hoskins was born in Nashville, Tennesse to Barbara and Stephen Hoskins.
He attended Granberry Elementary, Glendale Middle, McMurray Junior High, and Hume-Fogg
Academic Magnet in Nashville. During his time at Hume-Fogg he was introduced to the world of
biology, including but not limited to the concept of evolution, naturalist writings, genetics, and
the human body by two wonderful teachers- Barbara Allen and Brenda Royal. After graduation
he attended the University of Tennessee- Knoxville where he received a Bachelor’s of Science
degree in Ecology and Evolutionary Biology. During his time at the University of Tennessee he
delved into mycology and participated in research with Dr. Karen Hughes and Dr. Brandon
Matheny. Learning so much about the biological world inspired him to pursue his teaching
degree so he began the Science Education program at UTK and participated in an internship
where he taught Honors Biology and Physical Science at Farragut High School. Upon receiving
his Master’s in Science Education he worked at Siegel Middle School as a 7th grade science
teacher for 1 year. This was a challenging time for him as he learned much on classroom
management. He then returned as a Master’s degree student and dedicated teaching assistant in
the Ecology and Evolutionary Biology Department and continues his love for learning and all
living things under the advisement of Dr. Randall Small. He now studies the phylogenetic
patterns of polyploid plant groups and hopes to go on to a biological research career at a
university or government organization.
45