Professional Documents
Culture Documents
by
Joshua P. Der
A Thesis
Submitted in Partial Fulfillment of the Requirements for the
Master of Science Degree
JOSHUA P. DER, for the Master of Science degree in Plant Biology, presented on 2 July
2005 at Southern Illinois University Carbondale.
Santalaceae are a diverse group of root and stem hemiparasitic plants in the
sandalwood order (Santalales), which occur worldwide in both tropical and temperate
genera) and was expanded to include these taxa in the recent APGII classification
(Santalaceae sensu lato). Phylogenetic analyses were performed using DNA sequence
data from three genes (nuclear small-subunit ribosomal DNA and chloroplast rbcL and
matK) and nearly complete generic-level sampling was achieved (44 of 45 total genera).
Sequence data for each gene were analyzed separately and combined using maximum
separate gene partitions are largely congruent, but differ in their level of resolution. Eight
distinct and highly supported clades are recovered in combined three-gene analyses. A
revised classification based on this phylogeny is proposed which recognizes these eight
clades at the family level. Viscaceae is monophyletic and is retained unchanged from
intact, but its taxonomic rank is raised to family. Anthobolus (tribe Anthoboleae) is
excluded from Santalaceae sensu lato and allied with Opiliaceae (outgroup). The
remaining two genera in tribe Anthoboleae (Exocarpos and Omphacomeria) are well
i
supported as sister to some members of a polyphyletic tribe Santaleae +
Eremolepidaceae. This clade, which contains the type species of Santalaceae (Santalum
album), is recognized here as Santalaceae sensu stricto and includes all three genera of
Eremolepidaceae, Exocarpos and Omphacomeria, and six genera from tribe Santaleae.
from the polyphyletic tribe Santaleae and Buckleya and Kunkeliella are members of
Thesiaceae. Arjona and Quinchamalium (Thesieae) form the eighth well-supported clade
ii
ACKNOWLEDGEMENTS
This research was made possible with financial support from the National Science
I wish to express my sincere gratitude to the many people who helped to make
First, I want to thank Daniel Nickrent for presenting me with the opportunity to
work on this interesting group of plants in his lab. He was a source of encouragement
and an invaluable resource on the biology and taxonomy of Santalaceae both in personal
Parasitic Plant Connection. He graciously granted me access to his DNA and tissue
collection, lab equipment, reagents, and provided financial support in the form of a
Research Assistantship. Without him, I would not have embarked on this project.
I would also like to thank my thesis committee members: Sedonia Sipes – who
generously allowed me to use the automated sequencer in her lab and with whom I had
the pleasure of working with as a teaching assistant; and Andy Anderson – who
introduced me to the basics of maximum likelihood and Bayesian analyses and assisted
Table 3 lists the names of the collectors of plant material used in this study.
Several additional people who have worked in the Nickrent lab in the past generated
some of the sequences I used in my analyses. These people include Valéry Malécot,
Miguel García García, María-Paz Martín Esteban, and Erica Nicholson. Photo credits for
images used in Figure 2 are given in that legend. I would like to acknowledge the
supported my use of the College of Education iMac G5 microcomputer lab and Anil
Mehta, who let me into the lab after-hours to check on my analyses. Thanks also to Gary
iii
Kolb, who authorized after-hours access to the College of Mass Communication/
Information Technology’s New Media Center. A special thanks also to Eric Rowan,
whose cooperation and flexibility made it possible to simultaneously use twelve of the
2.0 GHz dual processor PowerMac G5 computers in New Media Center. Without these
Romina Vidal Russell has been an inspiration to me from the day I met her. She
has been my teacher and mentor in the lab. She patiently tutored me in molecular
techniques (and Spanish), helped me trouble-shoot problems in the lab and has kept me
company on late Friday nights at school when Guille and Kristal were far from Illinois.
She has been a wonderful friend and comrade these past two years and I thank her for all
Cordoba Granada, Liz Saunders, Natalie West, Beckie Mooneyhan, Laura Forest and
many others for kind friendship, Latin music, dancing, coffee, maté, the occasional beer
And finally, I want to thank Kristal Watrous for her love and support. She
bravely came here to be with me, so far from everything familiar. She had confidence in
me when I wasn’t so sure of myself. She listened patiently when I rambled on about my
work, read drafts of my writing and helped me keep the details of life from piling up in
the sink or the laundry basket. For these things and so much more I want to thank her,
iv
TABLE OF CONTENTS
ABSTRACT.....................................................................................................................i
ACKNOWLEDGEMENTS............................................................................................iii
LIST OF FIGURES......................................................................................................viii
INTRODUCTION
Biology of Santalaceae ................................................................................................1
Historical Classification of Santalaceae .......................................................................5
Phylogeny of Santalales...............................................................................................7
Modern Molecular Phylogenetics, Previous Work, and Gene Selection .......................9
Objectives .................................................................................................................11
RESULTS
Nuclear SSU rDNA ...................................................................................................18
Chloroplast rbcL........................................................................................................18
Chloroplast matK.......................................................................................................19
Combined Gene Analyses..........................................................................................20
Phylogenetic Relationships........................................................................................20
DISCUSSION
Molecular Phylogenetics of Santalaceae.................................................................... 24
Phylogenetic Classification of Santalaceae sensu lato
Opiliaceae............................................................................................................. 27
Comandraceae ...................................................................................................... 27
Thesiaceae ............................................................................................................ 28
Arjonaceae ........................................................................................................... 30
Nanodeaceae......................................................................................................... 32
Pyrulariaceae ........................................................................................................ 33
Santalaceae sensu stricto....................................................................................... 34
Amphorogynaceae ................................................................................................ 35
Viscaceae ............................................................................................................. 38
Evolution of aerial parasitism in Santalaceae............................................................. 39
Conclusion................................................................................................................ 39
LITERATURE CITED.................................................................................................. 60
v
APPENDIX I
Primer sequences, PCR reagents, and Thermal Cycle Parameters .............................. 73
APPENDIX II
Example PAUP* and MrBayes Command Blocks..................................................... 75
APPENDIX III
Log Likelihood Plots from Bayesian Analyses .......................................................... 78
APPENDIX IV
Supplemental Phylogenetic Trees.............................................................................. 79
Supplemental tree IV-1: Nuclear SSU rDNA BI majority rule consensus............. 80
Supplemental tree IV-2: rbcL BI majority rule consensus .................................... 81
Supplemental tree IV-3: matK MP strict consensus with Phacellaria ................... 82
Supplemental tree IV-4: matK ML phylogram with Phacellaria .......................... 83
Supplemental tree IV-5: matK BI majority rule consensus with Phacellaria ........ 84
Supplemental tree IV-6: matK BI majority rule consensus without Phacellaria ... 85
Supplemental tree IV-7: Three-gene MP strict consensus with Phacellaria.......... 86
Supplemental tree IV-8: Three-gene ML phylogram with Phacellaria .................. 87
Supplemental tree IV-9: Three-gene BI majority rule consensus without
Phacellaria, partitioned by gene ....................................................................... 88
Supplemental tree IV-10: Three-gene BI majority rule consensus with Phacellaria,
partitioned by gene and codon .......................................................................... 89
Supplemental tree IV-11: Three-gene BI majority rule consensus without
Phacellaria, partitioned by gene and codon ...................................................... 90
VITAE .......................................................................................................................... 91
vi
LIST OF TABLES
Table Page
Table 3: Voucher information and Genbank accession numbers for the taxa sampled in
this study......................................................................................................45
Table 4: Models of molecular evolution chosen and used in maximum likelihood and
vii
LIST OF FIGURES
Figure Page
Figure 3: Nuclear SSU rDNA maximum parsimony strict consensus tree with bootstrap
Figure 5: rbcL maximum parsimony strict consensus tree with bootstrap support values
Figure 7: matK maximum parsimony strict consensus tree with bootstrap support values
Figure 9: Three-gene maximum parsimony strict consensus tree with bootstrap support
viii
INTRODUCTION
Biology of Santalaceae
sandalwood order (Santalales Dumort.) that occur worldwide in both temperate and
characters, the family has no clear synapomorphies and is difficult to distinguish from
other families in Santalales. Most species are small woody shrubs and herbaceous
perennials, but a few are trees (notably, Santalum, Scleropyrum and Okoubaka). Leaves
are simple and usually entire, but may be reduced to scales (e.g. in many xerophytic
(Acanthosyris) (Kuijt, 1969). Tropical and subtropical species often have thick leathery
leaves, but thin deciduous leaves are common in temperate representatives (e.g.
branches, which may consist of short and long shoots (Acanthosyris) or an alternating
Exocarpos) growth patterns. Many species are able to regenerate vegetatively from
underground organs when aboveground portions are killed (Kuijt, 1965; Lepschi, 1999).
Some authors suggest that this may represent an adaptation to fire-prone habitats in some
species (Kuijt, 1969; Bean, 1990). Additionally, Arjona and Comandra both have
underground rhizomes and/or tubers that form temporary storage organs from which new
growth begins in the following growing season (Kuijt, 1969; Bean, 1990).
1
Figure 2 shows some of the range in floral diversity in the group. Flowers are
typically small and inconspicuous, often green or pale colored (yellow and orange
flowers are monochlamydous and have undifferentiated perianth parts, which are free in
most genera, but may be fused at the base to form a campanulate or tubular perigone (e.g.
Arjona and Quinchamalium). The precise ontology and nature of the perianth has been
variously reported as petals, tepals, perigone and sepals. The latter view seems to
predominate in the literature (Smith and Smith, 1943; Kuijt, 1969; Malécot et al., 2004),
but this interpretation requires a loss of the corolla and the redevelopment of an expanded
calyx from ancestors that possessed a calyx. For example, Olacaceae have a calyx, but
reversal, where a calyx is reinvented, isn’t parsimonious and doesn’t seem likely. In his
perianth probably represents the corolla, in line with the view taken here. Most species
have bisexual flowers, but monoecious, dioecious and polygamous members do occur
bisexual and male flowers, stamens are equal in number to and inserted opposite each
perianth lobe. A tuft of hair is commonly found on the perigone immediately behind the
point of stamen insertion. Flowers have a single style at the apex of an inferior, half-
inferior or superior ovary. Distyly is found only in the South American genera Arjona
and Quinchamalium (Dawson, 1944; Riveros, Arroyo, and Humana, 1987). A superior
remainder of the family has an inferior, or partially inferior ovary (Pilger, 1935; Smith
2
and Smith, 1943; Stauffer, 1959). Ovaries are unilocular, but the chamber may be
partially divided at the base, with a short free-central placental stalk bearing one to three
(rarely four or five) pendulous ovules (Smith and Smith, 1943), only one of which
develops into a seed. One of the most striking features of santalaceous flowers is the
presence of a conspicuous lobed floral disc at the base of the style, which often produces
copious amounts of nectar (Macklin and Parnell, 2002). The lobes of the disc alternate
with the perigone lobes (Smith and Smith, 1943). The homology of this structure is
unclear, but Kuijt (1969) suggests that it may represent extreme reduction of the corolla,
though this interpretation seems unlikely due to the interior position of the disk relative to
the stamens. The seed lacks a seed coat and is difficult to distinguish from carpellary
many species, which in turn is surrounded by a thin, often bright-colored, fleshy exocarp
(Kuijt, 1969) forming drupes or nuts (Macklin and Parnell, 2002). In Anthoboleae, the
peduncle subtending the fruit enlarges and aids in dispersal (Stauffer, 1959; Kuijt, 1969).
In Arjona and Quinchamalium, the fruit is a small achene (Dawson, 1944; Johri and
Agarwal, 1965).
evidence suggests that all members of the family are hemiparasitic (Herbert, 1920; Kuijt,
1969; Malécot et al., 2004). See Table 1, where all but five genera have positive
although stem parasitism (including mistletoes and dendroparasites) has been derived in
seven genera (Kuijt, 1969, 1988; Macklin and Parnell, 2002). Santalaceae are typically
quite generalized with respect to host range and a single individual may parasitize hosts
3
in numerous families, themselves (i.e. autoparasitism) or other individuals of the same
species (Herbert, 1920; Rao, 1942b; Fineran, 1965b; Musselman and W. F. Mann, 1979;
Leopold and Muller, 1983; Hewson and George, 1984; Fineran, 1991; Lepschi, 1999).
Given that most Santalaceae are generalized parasites, species that do show host
specificity are remarkable. For example, Leptomeria pauciflora has only one known
host, Eremaea pilosa (Herbert, 1920; Lepschi, 1999), and Phacellaria species are
1969; Macklin and Parnell, 2002). Haustorial morphology and anatomy has been studied
in detail for several species (Rao, 1942b; Fineran, 1962, 1963b, d, e, 1965a, b;
Warrington, 1970; Weber, 1977; Fineran, Juniper, and Bulluck, 1978; Fineran, 1979;
Fineran and Bulluck, 1979; Nietfeld, Weber, and Weberling, 1983) and falls within the
range of haustorial diversity in other families of Santalales (Kuijt, 1969; Fineran, 1991).
Members of Santalaceae are found in all parts of the world, from Alaska through
the neotropics to Tierra del Fuego, from Europe to East Asia and South Africa, Malaysia
to Australia and Hawaii (Table 1). Most genera are restricted to either the New World or
the Old World, but a few notable exceptions exist (Kuijt, 1969). Both Pyrularia and
Buckleya have a disjunct distribution between Asia and eastern North America (Kuijt,
1969; Li, Boufford, and Donoghue, 2001), Mida occurs in New Zealand, the Juan
Fernandez Islands and southern South America, and Comandra is found in North
America and Europe. At least one species of Thesium has invaded and naturalized in the
United States (Musselman and Haynes, 1996), the remainder of Thesium occur the in Old
Eubrachion and Lepidoceras) are restricted to the New World (Kuijt, 1988) and the aerial
4
parasites in Amphorogyneae are found in Southeast Asia and New Guinea (Danser, 1940,
Santalaceae was first described by Robert Brown in his Prodromus Florae Novae
1802 to 1805 as the naturalist on Matthew Flinder’s voyage around Australia, together
with specimens collected by Joseph Banks and Daniel Solander on Captain James Cook’s
first voyage around the world in 1768-1771 (Stearn, 1960). This work represents the first
Jussieu’s Genera Plantarum (1774). Brown included Thesium L., Leptomeria R. Br.,
Choretrum R. Br., Fusanus R. Br. and Santalum L. in Santalaceae and allied the genera
The next treatment of Santalaceae was that of Heironymus (1889) in Engler and
circumscribed within three tribes (Anthoboleae Bartl., Osyrideae Rchb., and Thesieae
considered at that time to be allied with Santalaceae) and Thesianthium Conw. (a fossil
Santalaceae). Hieronymus’ classification was adopted by Rendle (1925) but the family
classification was updated and revised in Pilger’s treatment ten years later (1935). Pilger
retained Hieronymus’ three tribes, but Champereria Griff. had previously been removed
from Santalaceae (Anthoboleae) and placed in Opiliaceae by Engler (1897). Pilger also
reorganized the two sections of Fusanus (sections Eufusanus Benth. & Hook. and Mida
Benth. & Hook.) at the generic level (Eucarya T. Mitch. and Mida A. Cunn. ex Endl.)
5
and included three additional genera in Osyrideae (syn. Santaleae A. DC.) for a total of
29 genera in the family. Although subsequent authors have made significant changes,
Pilger’s work represents the most recent generic-level systematic and taxonomic
treatment for the family worldwide. Danser published a complete revision of Phacellaria
Benth. (Danser, 1939). This led him to subsequent work on the closely related and
complex mistletoe genus Henslowia Blume. Danser (1940) segregated some Henslowia
Dendrotrophe, and described 19 new species (Danser, 1940, 1955). Stauffer revised
Daenikera Hürl. & Stauffer, Amphorogyne Stauffer & Hürl. and the Indomalayan
dendroparasites and mistletoes, including the taxa Danser split from Henslowia (Stauffer,
1969; Stearn, 1972). The original tribal designation for Amphorogyneae lacked a Latin
diagnosis, which Stearn validly published (1972) when he described the genus
Thailand in which she synonymized Hylomyza Danser with Dufrenoya Chatin and where
she included all species of Cladomyza Danser in Dendromyza Danser (Macklin, 2000;
Macklin and Parnell, 2000, 2002). The classification of Santalaceae and related families
(Antidaphne Poepp. & Endl., Eubrachion Hook., and Lepidoceras Hook.) restricted to the
6
New World. The rank and systematic placement of this group has been controversial
(Kuijt, 1988). Eremolepidaceae was first proposed as a family by Van Tieghem (1910),
but was not generally accepted until Kuijt validated the family in 1968 and monographed
it in 1988. He recognized though that the placement of this family within Santalales was
uncertain (Kuijt, 1968, 1982, 1988) and it has been variously allied with Olacaceae via
Opilia (Opiliaceae) (Kuijt, 1968), Loranthaceae (Kuijt, 1988), Santalaceae (Barlow and
Wiens, 1971), and Viscaceae (Barlow, 1964; Bhandari and Vohra, 1983). The
monophyly of Eremolepidaceae has more recently come into question following results
from molecular phylogenetic investigations (Nickrent and Duff, 1996; Nickrent et al.,
1998; Nickrent and Malécot, 2001). These studies placed the eremolepidaceous genera
within tribe Santaleae. Current concepts of Santalaceae include 38 genera and ca. 450
The related family of mistletoes, Viscaceae Miers, has recently been included in
Santalaceae by the Angiosperm Phylogeny Group (APG, 2003). This family was
Bhatnagar, 1960; Barlow and Wiens, 1971; Nickrent and Franchina, 1990; Nickrent and
Malécot, 2001; Malécot et al., 2004). See Kuijt (1969) and Calder (1983) for discussions
Phylogeny of Santalales
varied substantially over time (Johri and Bhatnagar, 1960; Kuijt, 1968), current
7
Opiliaceae, “Santalaceae” (including Eremolepidaceae), and Viscaceae (families in
Nickrent et al., 1998; Nickrent and Malécot, 2000, 2001; Malécot et al., 2004).
“Olacaceae” sensu lato (s. lat.) is the basal-most member of the order and contains both
autotrophic and hemiparasitic members (Kuijt, 1969; Nickrent et al., 1998; Nickrent and
Malécot, 2001; Malécot et al., 2004). Evidence supports the evolution of parasitism only
once in Santalales, within “Olacaceae” (Nickrent and Duff, 1996; Nickrent et al., 1998;
Nickrent and Malécot, 2001; Malécot et al., 2004). Loranthaceae and Misodendraceae
are both mistletoe families and, together with Schoepfia (a root parasite), form a
monophyletic group sister to the remaining members of the order (Figure 1). Schoepfia
Olacaceae. Opiliaceae represents the next family to diverge within the order, and is quite
(Nickrent and Malécot, 2001). Santalaceae are currently considered paraphyletic with
respect to Viscaceae (Nickrent and Duff, 1996; Nickrent et al., 1998; Nickrent and
monophyletic group of economic importance (Calder, 1983), and the major relationships
within the Santalaceae were not resolved, some authors have retained Viscaceae and
8
Modern Molecular Phylogenetics, Previous Work, and Gene Selection
relationships. “Natural groups”, those based on overall similarity, have long been the
basis for plant classifications (de Jussieu, 1774). With the introduction of evolutionary
theory (Darwin, 1859) and the advent of phylogenetic classifications (Hennig, 1966),
monophyly has become the standard criterion by which to define taxa (Stace, 1989;
analysis are currently considered the most rigorous methods for assessing phylogeny
(Hillis, Moritz, and Mable, 1996; Soltis, Soltis, and Doyle, 1998; Judd et al., 2002).
DNA sequence data for four genes and ca. 56 genera in Santalales were generated in the
Santalales and Olacaceae (Nickrent and Franchina, 1990; Nickrent and Malécot, 2000,
2001). These data suggested that Santalaceae are not monophyletic, but the phylogenetic
structure of the family was weak (Nickrent and Malécot, 2001). These data sets first
included nuclear small-subunit ribosomal DNA (SSU rDNA); then the chloroplast gene
rbcL was added (Nickrent and Malécot, 2000, 2001). These data were collected to
examine family relationships within Santalales and taxon sampling within Santalaceae
but incomplete resolution of deeper nodes precluded any revision of its classification
(Nickrent and Malécot, 2001). Additional sequence data for nuclear large-subunit (LSU)
rDNA and chloroplast matK were obtained (mostly from taxa in Olacaceae, but some
9
Santalaceae as well). These data increased resolution and showed promise for resolving
The phylogenetic utility of nuclear SSU rDNA has been recognized for some time
and has proven to be especially useful at higher taxonomic categories (Nickrent and
Soltis, 1995; Soltis et al., 1997; Soltis et al., 1999). Likewise, rbcL has emerged as one
of the most widely used genes for plant phylogenetic inference as evidenced by more
than 25,000 sequences deposited in Genbank. This protein coding chloroplast gene is
green plants, making PCR amplification and alignment straightforward, yet still evolves
at a sufficiently high rate that it can be used to examine relationships at many taxonomic
levels from all land plants to angiosperm orders, families and sometimes genera (Martin
and Dowd, 1991; Bousquet et al., 1992; Albert et al., 1994; Haufler and Ranker, 1995;
Ueda and Yoshinaga, 1996; Chase and Albert, 1998). MatK has emerged more recently
as a molecular marker useful in angiosperm phylogeny (Hilu and Liang, 1997; Hilu et al.,
2003), and has been used successfully to examine relationships at the family level
(Xiang, Soltis, and Soltis, 1998; Bell and Patterson, 2000; Cabrera, 2002;
Wojciechowski, Lavin, and Sanderson, 2004; Samuel et al., 2005). The use of these
genes in combination has the potential to increase phylogenetic resolution and statistical
support of clades (de Queiroz, Donoghue, and Kim, 1995; Nickrent and Soltis, 1995;
This study nearly completes generic-level sampling of these three genes (nuclear
SSU and chloroplast rbcL and matK) for Santalaceae, and includes two representatives
from each of the six largest genera to test monophyly (Table 3). Due to the paraphyletic
10
nature of the family with respect to Viscaceae and its similarity to Opiliaceae,
representatives of these two families were also sequenced and included in these analyses.
Objectives
phylogenetic relationships.
investigation of the phylogenetic placement of this group. Molecular work using nuclear
SSU rDNA and rbcL placed Eremolepidaceae within tribe Santaleae (Nickrent and
between studies with different taxon and gene sampling (Nickrent and Soltis, 1995;
parasites) that has experienced extensive generic-level revision (Danser, 1940, 1955;
Stauffer and Hürlimann, 1957; Stauffer, 1969; Macklin and Parnell, 2000, 2002).
Examining these taxa in a phylogenetic context would help our understanding of the
taxonomy and biology of this group. Past molecular analyses have provided weak
11
resolution of relationships within Amphorogyneae, but show strong support for the
(Nickrent and Malécot, 2001). Increased taxon sampling and sequence data provide the
that is derived from root parasitism (Kuijt, 1969). Phylogenetic work in Santalales
(Nickrent and Duff, 1996; Nickrent and Malécot, 2001) shows that mistletoes evolved
independently at least five times in the order. Santalaceae includes seven mistletoe
genera (the three New World eremolepidaceous genera and the Southeast Asian genera
Santalaceae.
morphology and anatomy may operate, and which also serves as the basis for the
12
MATERIALS AND METHODS
Taxon Sampling
An extensive collection of Santalales plant material and genomic DNA has been
assembled by D. L. Nickrent (DLN) and has been archived in the Department of Plant
and colleagues or came from plants in cultivation derived from field-collected stock.
This collection served as the source material for the present study. Representatives of 34
representatives in each of the six largest genera (those containing 17 or more species) to
assess their monophyly (Table 3). The only genus in Santalaceae not represented in this
which we have not been able to obtain plant material. Additionally, representatives of all
seven genera in Viscaceae, three genera in Eremolepidaceae and five of the ten genera in
Opiliaceae were sampled for a total of 49 genera and 55 species. Voucher information
Genomic DNA was isolated from herbarium, silica dried or fresh frozen plant
tissue using a modified CTAB method (Nickrent, 1994). Genomic DNA was diluted (to
approximately 5-10 ng/L) for working solutions and these were used as DNA templates
in polymerase chain reaction (PCR) amplifications. Chloroplast rbcL and matK and
nuclear SSU rDNA (small-subunit ribosomal DNA) genes were PCR amplified in an
reactions (see Appendix I for primer sequences, PCR reagents and thermal cycle
13
parameters). To check and quantify PCR reactions, 2-4 µL each was electrophoresed for
using the Omega E.Z.N.A. CyclePur kit if a single band was seen in the agarose check
gels. Alternatively, if multiple bands were seen, the band of correct size was gel purified
using the Sigma GenEluteTM MINUS EtBr spin columns or the Quiagen Quiaquick® gel
extraction kit. In all purification methods the standard manufacturers protocols were
followed with minor modifications. Low yield PCR products were TA cloned using the
Promega pGEM®-T Easy Vector system. Purified PCR products or cloned plasmids
(with inserts) were used directly as templates in cycle sequencing reactions using the ABI
BigDye® v3.1 Terminator cycle sequencing mix, diluted 1/8 with The Gel Company’s
BetterBuffer®. In all cases, the sequencing primers matched the primers used in the
original PCR amplifications, but sometimes an additional primer was used if the PCR
product exceeded 1400 bp long. Cycle sequencing reactions were cleaned using a 3M
Lane tracking and nucleotide basecalling of DNA sequences was performed with
Sequencing Analysis software (ABI). Basecalling was visually checked against the
electropherogram and edited if necessary. Approximately 700 basepair read lengths were
achieved and contigs were assembled manually. Sequences were manually aligned by
eye using Se-Al v2.0a11 (Rambaut, 1996-2004). Alignment was straightforward for
14
nuclear SSU and chloroplast rbcL and required few gaps. For the protein coding genes
(rbcL and matK), alignment was informed by also examining the translation of triplet
Phylogenetic Analysis
Datasets for all three genes were analyzed separately using maximum parsimony
2002) and Bayesian Inference (BI) using pMrBayes v3.0b4 (Ronquist and Huelsenbeck,
2003; Altekar et al., 2004) run in parallel using POOCH (Dauger, 2001). Example
command blocks for analyses performed in this study are given in Appendix II.
Topological congruence among data partitions was visually assessed and DNA sequences
for all three genes were combined following the conditional combination approach, in
which data partitions were combined when substantial differences were not found (de
Queiroz, Donoghue, and Kim, 1995; Huelsenbeck, Bull, and Cunningham, 1996; Johnson
and Soltis, 1998). Only ca. 500 bp of matK sequence data were obtained for Phacellaria,
phylogenetic relationships within this tribe; subsequently, the matK and combined three-
gene datasets were also analyzed without Phacellaria to examine relationships among the
All heuristic MP searches were performed coding gaps as “missing” data, using
starting trees from 100 random addition sequence replicates holding two trees at each
parsimonious trees (MPTs) were saved to files. The strict consensus was computed from
these trees and rooted using Opiliaceae as outgroup. Bootstrap (BS) analysis was
15
performed on all datasets (1000 BS replicates using TBR branch swapping on starting
trees generated by simple stepwise addition sequences holding one tree at each step) to
assess statistical support for clades recovered in the heuristic searches. BS support values
are reported for clades found in greater than 50% of the BS replicates. There was no
limit to the number of trees saved in each bootstrap replicate, except in the SSU rDNA
dataset, where a “MaxTrees” limit of 100 was imposed to shorten the run time; this
procedure is justified because the SSU rDNA dataset contained relatively little
Models of DNA sequence evolution used in ML analyses were evaluated for all
data partitions (i.e. each gene, codon positions in matK and rbcL, and the combined
dataset) using hierarchical Likelihood Ratio Tests (hLRT) and the second order Akaike
Information Criterion (AICc) in Modeltest v3.6 (Posada and Crandall, 1998) using
likelihood scores estimated from a neighbor joining tree. Both the total number of
characters and the number of variable characters in the partition were used as sample
sizes in AICc comparisons (Posada and Buckley, 2004). When hLRTs and AICc selected
different models, the simpler model was chosen to reduce run times in these analyses
(Table 4). When both hLRT and AICc selected the most complex model tested (i.e.
GTR+I+G), this model was used for both ML and BI analyses. Alternatively, when a
simpler model was selected, MrModeltest v.2.0 (Nylander, 2004) was used to reevaluate
the likelihood scores under a limited set of evolutionary models for BI (24 instead of 56
models). Again, hLRT and AICc (for all and variable characters) was used and the
simpler models were selected. Heuristic ML searches were performed in PAUP* using
16
TBR branch swapping on an MP starting tree. Best-fit model parameters were set to the
values estimated during model selection (on the Neighbor-Joining tree) and ML branch
coupled Markov chain Monte Carlo, or “p(MC)3,” algorithm in pMrBayes (Ronquist and
Huelsenbeck, 2003; Altekar et al., 2004). Codon positions were partitioned in rbcL and
matK and the full dataset was partitioned by both gene and by gene + codon. Model
parameters for each data partition were estimated independently as part of the analyses
together with tree topology and branch length (i.e. the parameter estimates for each
partition were unlinked from each other, but topology and branch length were linked
across all parameters in the analyses). A uniform distribution of prior probabilities was
implemented for all parameters. Four p(MC)3 chains were distributed across four G5
Macintosh clusters connected via Ethernet LAN and assembled using POOCH. Each
analysis was run twice for five million generations with trees sampled every 1000
generations. To determine if parameter stationarity was achieved and to delimit the burn-
in, log likelihoods were plotted against generation time. A typical log likelihood plot is
shown in Appendix III. Trees recovered during the first 50 000 generations were
discarded as burn-in and the remaining trees from each run separately and combined
(pooled) were used to compute the 50% majority rule consensus tree. The mean –ln
likelihood of the remaining trees was also calculated. Frequencies of clades in the
consensus tree represent the posterior probability of that clade given the data and model
17
RESULTS
The aligned nuclear SSU rDNA data partition included 1825 nucleotide character
sites for 51 taxa (see Table 3 for complete sampling information). There were 414
(22.7%) variable sites, including 239 (13.1%) parsimony-informative sites. The nuclear
SSU MP heuristic search recovered 3211 most parsimonious trees (MPTs) of length
1135. The strict consensus of these trees is shown in Figure 3. BS support values and
Bayesian posterior probabilities (PP) from the separate runs combined are mapped on the
evolution with parameters set using estimates calculated from the neighbor joining (NJ)
tree. Figure 4 shows the ML phylogram of the single most optimal tree with a negative
natural log likelihood score (–lnL) = 8722.54101. BI results are more resolved, but in
agreement with the ML phylogeny (Appendix IV-1). The mean –lnL of the Bayesian
Chloroplast rbcL
The aligned rbcL data partition included 1428 nucleotide character sites for 50
taxa. There were 447 (31.3%) variable sites, including 276 (19.3%) parsimony-
informative sites. The rbcL MP heuristic search recovered 76 MPTs of length 1053. The
strict consensus of these trees (Figure 5) includes several well-supported clades not
supported in the nuclear SSU phylogeny (for example, Buckleya is included with
members of Thesieae and several members of Santaleae are grouped in a clade with
partitioned analyses) are mapped on this tree. The ML analysis implemented the
18
GTR+I+ model with parameter estimates calculated from the NJ tree. Figure 6 shows
the ML phylogram of the single most optimal tree (–lnL= 8136.63960). Codon
partitioned Bayesian analyses resulted in a topology nearly identical to ML, and after
burn-in, had a mean –lnL = 8009.1139 (Appendix IV-2). Note that ML reveals the
Chloroplast matK
The aligned matK data partition included 1258 nucleotide character sites for 50
taxa. There were 830 (66.0%) variable sites, including 585 (46.5 %) parsimony-
character to the matK matrix and was excluded from most analyses (see Appendix IV-3,
-4 & -5) for matK analyses which include Phacellaria). The MP heuristic search
recovered 31 MPTs of length 2513. More recent clades are well resolved in the strict
consensus (Figure 7), but this tree lacks resolution of basal relationships within
parameters set using estimates calculated from the NJ tree. Figure 8 shows the ML
phylogram of the single most optimal tree with a –lnL = 13988.94084 (an ML phylogram
Bayesian analyses under the GTR+ model, also lacked support for deeper relationships
but these trees were not incongruent with the ML trees (Appendix IV-5 and -6). After
trees from the first 50 thousand generations were discarded as burn-in, the mean Bayesian
–lnL = 14061.98536 when Phacellaria was included and –lnL = 14028.49919 when
19
Combined Gene Analyses
The aligned three-gene data matrix included 4516 nucleotide character sites for 55
taxa. There were 4516 (37.4%) variable sites, including 1100 (24.4 %) parsimony-
informative sites. The MP heuristic search recovered two MPTs of length 4798 when
Phacellaria was excluded (12 trees, L=4804 with Phacellaria; Appendix IV-7). The
strict consensus tree contains a polytomy near the base of Santalaceae, but several
additional nodes receive low support (Figure 9). ML analyses implemented the
GTR+I+ model of molecular evolution with parameters estimated from the NJ tree.
Figure 10 shows the ML phylogram of the single most optimal tree (–lnL =
32132.07152). When Phacellaria was included in ML analysis, there were two equally
optimal trees (-lnL = 32165.54345), which differed from each other and with the other
IV-8). Fully partitioned Bayesian analyses (partitioned by both gene and codon) were
more resolved than BI analyses partitioned only by gene (Appendix IV-9, 10 & 11).
Phylogenetic Relationships
paraphyletic in all analyses, a result in agreement with previous work (Nickrent and Duff,
1996; Nickrent et al., 1998; Nickrent and Malécot, 2000, 2001). Viscaceae and
Eremolepidaceae are monophyletic, but derived from within the traditional Santalaceae.
Tribe Anthoboleae is polyphyletic and the type genus for the tribe, Anthobolus, is allied
with Opiliaceae (outgroup) in all analyses. The remaining santalaceous taxa (i.e.
20
Amphorogyneae is monophyletic and tribe Santaleae is polyphyletic. Arjona and
Quinchamalium are not included with other members of tribe Thesieae, while Buckleya
Results from all three genes are largely congruent, but differ in the level of
are included in a clade with Eubrachion (Figure 3, 79% BS and 100% PP). This
relationship is not seen in results from any of the other data partitions, including the three
gene analyses. The placement of Arjona in the rbcL dataset varies based on the
optimality criterion used. For example, the rbcL MP strict consensus tree places Arjona
sister to Antidaphne (but with BS support < 50%, Figure 5), whereas ML and BI place
Arjona near the base of Santalaceae associated with Thesium impeditum. This latter
taxon is not sister to the other Thesium species included (T. fruticosum), from which it is
separated by several well-supported nodes (Figure 5). In matK analyses, the varied
with moderate BS support (87%), but not with Ginalloa, its sister taxon in nuclear SSU,
rbcL and combined gene analyses. In ML and BI analyses of matK data, Korthalsella is
separated from Viscaceae by two nodes with 64% and 100% PP. Mida and Nanodea are
well supported sister taxa (100% BS and PP in all analyses), but their position relative to
21
Of the six genera for which I sampled multiple accessions to test for monophyly,
only Quinchamalium was paraphyletic in the three-gene analyses. However, in the single
gene analyses, Exocarpos, Thesium and Quinchamalium were paraphyletic in the nuclear
Despite substantial care during laboratory work and sequence alignment, some of
these anomalous results may have been caused by error introduced during manual
branches in Arjona (rbcL, Figure 6) and Korthalsella (matK, Figure 8) and missing rbcL
artifactual results.
1; 100% BS and PP) and moderately supported as sister (85% BS/100 PP) to a clade
containing all sampled genera in tribe Amphorogyneae (Clade 2; 100% BS and PP).
Clade 3 (100% BS and PP) is comprised of eleven genera, including the type genus of
and are basal in this clade. Eremolepidaceae is also included in Clade 3 as are several
Myoschilos). Clade 4 (100% BS and PP) includes six genera segregated from tribe
22
Santaleae. Taxa in this clade include Acanthosyris, Cervantesia, Jodina, Okoubaka,
Scleropyrum and Pyrularia. Mida + Nanodea are separated from other members of tribe
Santaleae and constitute Clade 5 (100% BS and PP) with an uncertain position relative to
the other clades. Arjona + Quinchamalium are segregated from tribe Thesieae to form
Clade 6 (100% BS and PP). Clade 7 includes the remaining members of tribe Thesieae
with Buckleya and Kunkeliella (98% BS and 100% PP). Comandra and Geocaulon form
Clade 8 (100% BS and PP), which is basal to the other seven clades. Nearly all of the
internal nodes within these eight clades are also resolved with moderate to high support,
23
DISCUSSION
taxonomic treatment in 70 years (Pilger, 1935). Since this treatment, the systematics of
Santalaceae has experienced extensive revision. New species, genera, and tribes have all
been described and included within Santalaceae, dramatically expanding the family.
Several species and genera have been synonymized or included within other taxa. As
new taxa were described, their authors necessarily attempted to place them within the
existing classification, though sometimes could not definitively place them. For example,
when Stearn described the genus Kunkelliella (1972), he noted its remarkable similarity
with Osyricocarpos and Thesium (he even suggested that this new taxon might represent
a new section of Thesium), but from which it differed by having a fleshy fruit (a character
used to distinguish tribes Thesieae and Santaleae (Hill, 1915)). On this difference and on
Rhoiacarpos in tribe Santaleae. Regional floras and systematic works have also
attempted to organize taxa within the existing classifications (Dawson, 1944; Hewson
and George, 1984; Macklin and Parnell, 2000, 2002; Xia and Gilbert, 2003; Hilliard,
unpublished; Polhill, unpublished, 2003), but these have been limited in scope and
Earlier molecular work in the Nickrent lab demonstrated the paraphyletic nature
of the traditional Santalaceae but as mentioned previously, taxon sampling and lack of
sampling for three genes, strong patterns of evolutionary relationships have emerged.
24
These results provide a much clearer concept of evolutionary and phylogenetic
relationships in the family. As monophyly is the primary criterion on which taxa and
their subsequent hierarchical classification should be based (Stace, 1989; Steussy, 1990;
de Queiroz and Gauthier, 1992), the need for taxonomic revision becomes apparent and a
new classification is justified. Such a classification should serve two purposes. First, it
should reflect evolutionary history (as suggested by monophyly) and, second, it should
aid in organization of biological diversity (Stace, 1989; Steussy, 1990). Therefore, taxa
There are three possible approaches that may be taken to meet the monophyletic
taxon requirement in outlining a new classification. First, one could abandon the
taxon rank, as under the Phylocode (de Queiroz and Cantino, 2001). Second, a broad, all-
inclusive family could be outlined to also include Viscaceae and Eremolepidaceae (i.e.
Santalaceae s. lat.) as APGII has done (2003). If this approach were taken, the sub-
familial/tribal classification would require revision. Alternatively, one could split the
traditional Santalaceae and related groups into well-supported, smaller, more narrowly
defined families.
This latter approach has been chosen for several reasons. While Phylocode
nomenclature and fails to solve other problems inherent to both codes, for example,
25
widespread adoption of Phylocode faces a number of practical obstacles including
challenges in organization of large herbarium collections and floras. With these concerns
in mind, a hybrid approach has been chosen, which recognizes new taxa within the
The traditional Santalaceae are quite diverse and heterogeneous with respect to
ambiguously defined and are difficult to distinguish from other families in Santalales.
Smith (1937) noted this heterogeneity and suggested the family “perhaps should be
divided.” Two closely related families, Viscaceae and Opiliaceae, are well supported as
monophyletic taxa in this and other studies. Viscaceae are also economically important
plants (primarily pathogens) and are easily recognized. Additionally, the well-supported
clades found in this study represent several more restricted groups, which may be much
significance in itself, it is a “major” rank in ICBN and is used throughout the botanical
literature for organization of diversity. A case in point is that families are a primary unit
For these reasons, the eight major clades of Santalaceae s. lat. (Figure 9) are
defined here within a phylogenetic context and recognized at the family level (Table 5).
(Note: publication of taxon names in a thesis does not constitute effective publication
under either ICBN or Phylocode. As such, the new names proposed here are invalid and
authorship has been omitted.) Support from molecular phylogenetic results is discussed
26
for each new family in turn, as well as generic circumscription and relationships within
descriptions and a complete nomenclatural account are beyond the scope of this work, the
phylogeny and classification presented here may illuminate such future work within a
recognized.
OPILIACEAE
monophyly can be made from this study. Opiliaceae, however, was monophyletic and
basal to Santalaceae in previous studies (Nickrent et al., 1998; Nickrent and Malécot,
2000, 2001; Malécot et al., 2004). Anthobolus is segregated from Santalaceae and is
allied with this family. Anthobolus is the type genus for tribe Anthoboleae Stauffer
(1959) and this transfer leaves the other two members of the tribe (Exocarpos and
s. str. Opiliaceae otherwise remains unchanged and classification should follow that of
Heipko (1979; 1982; 1985; 1987) with the inclusion of Anthobolus. A new clade
COMANDRACEAE
Comandra and Geocaulon are strongly supported as sister (100% BS and PP) and
constitute Clade 8 (Figure 9). The position of this clade varies among analyses, but is
27
recovered with high support in all data partitions. This clade tends to hold a basal
position relative to other clades, but its sister cannot be confidently assigned.
Comandra occurs disjunct between North America and Europe. Both genera are
monotypic and quite similar in habit, with short (to 30 cm) herbaceous upright flowering
(1960) suggested that Comandra was distinctive enough from other groups in
embryology and details of the ovary and placenta. Johri and Bhatnagar’s work
This clade is recognized here at the family level and the following clade definition
is given: Comandraceae are the least inclusive clade that contains Comandra umbellata
and Geocalon lividum. The type species is Comandra umbellata (L.) Nutt. (The Genera
THESIACEAE
supported clade (98% BS and 100% PP). The generic relationships within this group are
fully resolved and these five genera constitute Clade 7 (Figure 9). This clade also shows
basal affinities in Santalaceae s. lat. as in Comandraceae, but again, its sister clade cannot
28
be identified. Thesium is moderately supported as monophyletic (89% BS and 85% PP)
based on the two species sampled in this study. Thesidium differs from Thesium by
Thesium is by far the largest genus in the traditional Santalaceae, with more than
200 species in four sections, whose systematics are beyond the scope of this work.
Numerous genera have been described, but are currently considered to be components of
Buckleya is a small tree and represents the basal lineage in this clade.
Osyridocarpos and Kunkeliella are small shrubs while the remaining two genera
(Thesidium and Thesium) show a trend toward the herbaceous habit with many
Kunkeliella with members of this clade. Stearn noted that the habit of Kunkeliella
species of Thesium. The primary difference Stearn used to place this genus near Osyris
was the presence of a fleshy fruit, a feature otherwise absent in Thesium, Osyridocarpos
and the other taxa considered to be included in tribe Thesieae (Arjona and
Quinchamalium). At the time the affinities of Buckleya (which also has a fleshy fruit)
endemic to South Africa and the Canary Islands, respectively. Osyridocarpos is found in
29
tropical and southern Africa and Thesium reaches its peak diversity in southern Africa,
but extends throughout the Old World, Australia and South America. Buckleya has a
distribution unlike all the other members of this clade, and is found disjunct between
North America and China and Japan. There are four recognized species (Carvell and
Eshbaugh, 1982; Li, Boufford, and Donoghue, 2001) which have an interesting
biogeographic relationship. Two species pairs are formed in which the North American
taxon is sister to one of the Chinese species, while the other Chinese species is sister to
the Japanese species (Li, Boufford, and Donoghue, 2001). The affinity with and basal
position of Buckleya in Clade 7 has interesting implications for the biogeography of this
group.
This clade is recognized here at the family level and the following clade definition
is given: Thesiaceae are the least inclusive clade that contains Buckleya distichophylla
and Thesium alpinum. The type species is Thesium alpinum L. (Species Plantarum 1:
207. 1753).
ARJONACEAE
Arjona and Quinchamalium form a well-supported clade (100% BS and PP, Clade
6, Figure 9). These two genera are quite speciose and morphologically similar. The
and Arjona are both distylous (Skottsberg, 1916; Riveros, Arroyo, and Humana, 1987), a
unique condition among Santalaceae. They also share an herbaceous habit, have parallel
venation in the leaves, have a tubular perigone with a long filamentous style and fruits
30
These two genera have long been associated with each other (Bentham and
Hooker, 1862-1883; Hieronymus, 1889; Van Tieghem, 1896; Skottsberg, 1913, 1916;
Pilger, 1935; Skottsberg, 1940; Dawson, 1944; Johri and Agarwal, 1965). Myoschilos
has also been mentioned as an ally of this group (Johri and Bhatnagar, 1960; Johri and
Agarwal, 1965), but this relationship was not directly examined by those authors. The
origin of this relationship in the literature began with Bentham and Hooker (1862-1883)
Pilger (1935) and results here also do not support this relationship.
The distinctiveness of these two genera prompted Van Tieghem to erect a new
family Arionacée which included Arjona and Quinchamalium. He justified this on his
assessment of the carpellary origin of the disc at the base of the style (i.e. it is epigynous,
which he erroneously contrasted to the androecial origin of the disc other Santalaceae);
sepals with a tuft of epidermal hairs above the point of stamen insertion (in contrast to the
hypodermal origin of hairs in other Santalaceae); an ovary that is unilocular above and
plurilocular below with one ovule in each locule (unilocular in other Santalaceae, but not
exclusively: Choretrum, Leptomeria and Osyris are exceptions). This family was
rejected by Pilger (1935), who noted that the disc is always epigynous in Santalaceae and
is usually expanded to the tepals. Johri and Agarwal (1965) also rejected Van Tieghem’s
family on the grounds that Arjona and Quinchamalium resemble other Santalaceae in
several ways (e.g. they have a free central placenta with three hemianatropous subapical
ovules, the chalazal end of the embryo sac extends up to the base of the placenta and they
have seeds which lack a testa). They did, however, suggest that these genera were
31
distinct enough (provisionally along with Myoschilos, unexamined) to be recognized at
the tribal level. They cite the fact that they share a persistent cup-like “calyx” around the
fruit, whose ontogeny suggests is bracteal in origin. Johri and Agarwal (1965) also cite
nine additional characters uniting these two genera. They suggest that prominent
synergid and antipodal haustoria and the persistent bracts, which form mesocarp-like
thickenings to protect the fruit, sufficiently separate these taxa from other Santalaceae to
justify the tribal designation (which they named Arjoneae and allied with Santaleae and
Osyrideae).
Myoschilos) is recognized here at the family level. The following clade definition is
given: Arjonaceae are the least inclusive clade that contains Arjona tuberosa and
NANODEACEAE
Mida and Nanodea form Clade 5 with 100% BS and PP (Figure 9). The position
of this clade within Santalaceae s. lat. is not certain. Both genera are monotypic and have
been classified in tribe Santaleae. These genera are both woody, but Mida is a tree to 8
meters high, while Nanodea is a diminutive subshrub with a much branched creeping and
cushion-like growth form. Mida is disjuct between New Zealand and the Juan Fernandez
Islands while Nanodea is found in far southern South America on Tierra del Fuego, the
Malvinas Islands and in Andean Patagonia. The diminutive habit of Nanodea may be
related to the extreme cold environment it lives in. Hieronymous placed these taxa near
each other in his treatment and separated them by only one key step. Mida was
32
recognized as a section of Fusanus in this treatment, but was later elevated to genus. The
Santalum was recognized by Bhatnagar (1960), but an examination of Nanodea was not
Department of Botany at the University of Delhi (Ram, 1957; Bhatnagar, 1959; Ram,
1959a, b; Bhatnagar, 1960; Johri and Bhatnagar, 1960; Agarwal, 1962a, b; Johri and
This clade is recognized here at the family level and the following clade definition
is given: Nanodeaceae are the least inclusive clade that contains Nanodea muscosa and
Mida salicifolia. The type species is Nanodea muscosa Banks ex C. F. Gaertn. (De
PYRULARIACEAE
Clade 4 is well supported (100% BS and PP, Figure 9) and includes two distinct
subclades, each including three genera. The first sub-clade includes Acanthosyris,
Cervantesia and Jodina, and is monophyletic with 100% BS and PP. The second clade is
also well supported (100% BS and PP) and includes Okoubaka, Scleropyrum and
Pyrularia. These two clades occupy two different biogeographic regions. The
distribution, whereas Okoubaka, Scleropyrum and Pyrularia are all Old World taxa, with
Scleropyrum is found in tropical India, Asia, Madagascar and New Guinea. Pyrularia
includes two species, one (P. pubera) in the Southeastern United States and one in Asia
(P. edulis) from Bhutan, China, India, Myanmar, Nepal, and Sikkim.
33
This whole group is characterized by having a woody habit and a large
drupaceous fruit with a stony pit. Bhatnagar and Sabharwal (1969) noted the mesocarpic
origin of the stony layer in the fruit of Jodina and suggested the term “pseudodrupe” as
the fruit type (in contrast to endocarpic origin in a true drupe). Stauffer clearly
recognized the affinities of the genera in each of these sub-clades in his Santalales
Cervantesia and Jodina together and provided a table to compare and contrast these
and removed it from Oktonemaceae and placed it in Santalaceae. In that paper, he placed
Okoubaka next to the genera Scleropyrum and Pyrularia, which he noted are also trees
This clade is recognized here at the family level and the following clade definition
is given: Pyrulariaceae are the least inclusive clade that contains Pyrularia pubera and
Cervantesia tomentosa. The type species is Pyrularia pubera Michx. (Flora Boreali-
traditional Santalaceae. Stauffer and Hürlimann (1957) alluded to this and stated that the
tribe Osyrideae (=Santaleae) does not represent a natural group and is divided into
several generic groups distinct from Thesieae and Osyrideae. Stauffer began to formally
divide this group when he recognized the tribe Amphorogyneae (Stauffer, 1969). He also
recognized the affinities of genera in the two sub-clades of Pyrulariaceae discussed above
(Stauffer, 1957, 1961). This heterogeneity has been the source of much taxonomic
34
confusion and many authors have reorganized the taxa in this traditional tribe in many
different ways (Van Tieghem, 1896; Pilger, 1935; Rao, 1942a; Smith and Smith, 1943;
Johri and Bhatnagar, 1960). The analyses presented in this study further corroborate the
polyphyletic nature of these taxa. Several generic groups have been segregated out of the
Clade 3 includes the remaining genera in Santaleae still allied with the type genus,
Santalum plus the eremolepidaceous taxa (Antidaphne, Eubrachion and Lepidoceras) and
Exocarpos + Omphacomeria. This clade is sister to the remaining two clades studied
(Clades 1 and 2) with moderate to weak support of 59% BS and 94% PP (Figure 9).
Some of the relationships among the major groups of genera are not well supported
(Figure 9) and have short branch lengths (Figure 10), so the conservative approach to
include these additional groups in Santalaceae s. str. has been taken. This more inclusive
This clade is recognized here at the family level and the following clade definition
is given: Santalaceae s. str. are the least inclusive clade that contains Santalum album,
AMPHOROGYNACEAE
Santalaceae and is sister to Viscaceae with 85% BS and 100% PP. This clade includes all
35
sampled) was outlined and described by Stauffer (1969). He recognized the
distinctiveness of these taxa relative to other members of tribe Santaleae, and could
distinguish them by the presence of peculiar characteristics of the anthers and placenta.
Specifically, anthers are born on short-stout or nearly absent filaments where each
thecum transversely dehisces independently (in contrast to many other Santalaceae where
dehiscence occurs along a single longitudinal slit common to the two locules of a theca).
In addition, the placenta is short-stout to nearly absent and more strongly associated with
the ovary tissue. The ovules are borne in pockets at the base of the ovarian locule (in
contrast to the stalked placental column with or without basal ovarian pockets in other
Santalaceae). This group includes genera with a diversity of growth forms and levels of
parasitism. Genera included in this clade are woody root parasitic shrubs, woody
dendroparasites (sensu Stauffer, i.e. clambering, liana-like shrubs which are obligate root
and opportunistic stem parasites, in contrast to the use of this term by Macklin, 2002,
who uses it as synonymous with mistletoe) and both woody and herbaceous advanced
mistletoes (i.e. exclusively aerial parasitic). The mistletoe habit is considered a highly
derived trait. There is a distinct trend toward increased levels of parasitism and the
associated loss of foliar leaves in this clade (Stauffer and Hürlimann, 1957). This is best
approaches a holoparasitic habit (Hürlimann and Stauffer, 1957), and the ultimate
mistletoes, and has also adopted the squammate reduced leaf form (Danser, 1939).
Members of this clade are found in tropical Southeast Asia, Malaysia, New
Caledonia and New Guinea to tropical and mediterranean Australia (Stauffer, 1969). The
36
genera found in dry climates also tend to show a reduction of the leaves (e.g. Choretrum,
Leptomeria and Spirogardnera), which parallels this reduction of leaves in the advanced
Generic relationships within this clade remain problematic due to incomplete gene
and taxon sampling. In these analyses, there are two well supported groups (Figure 9)
comprised of the Australian root parasites (Choretrum and Leptomeria) and the stem
come out basal to these two sub-clades, but their sister relationship decays when
Amphorogyneae clearly show it taking the mistletoe habit (Stauffer, 1969). The
placement of Phacellaria within this group based on the DNA sequence data obtained in
this study is uncertain because only approximately 500 base pairs were sequenced.
However, Phacellaria is most likely allied with the other stem parasites in this group
from within the mistletoes is interesting, as this genus has been considered “primitive”
among the stem parasites (Danser, 1940, 1955; Stauffer and Hürlimann, 1957).
thought to possibly be extinct until several populations were rediscovered near Perth.
DNA material was not obtained for this genus, thus it wasn’t included in any analyses.
The affinities of this genus were not explicitly mentioned in Stauffer’s posthumously
published description of the genus (1968), but he repeatedly referenced Choretrum and
37
Leptomeria, with which it shares several features including anther morphology,
investigated more thoroughly. These mistletoe genera were synonymized and included
and Parnell, 2000, 2002). Danser separated these genera based on characters of the fruit,
particularly the nature of fibers derived from exo-, meso- and endocarp tissue. Material
for these taxa were not obtained and the taxonomic revision of Macklin was followed.
This clade is recognized here at the family level and the following clade definition
is given: Amphorogynaceae are the least inclusive clade that contains Amphorogyne
spicata, Leptomeria acida and Dendrotrophe varians. The type species is Amphorogyne
spicata Stauffer & Hürl. (Vierteljahrsschr. Naturf. Ges. Zürich 102: 349. 1957).
VISCACEAE
data (Barlow, 1964; Kuijt, 1968, 1969; Wiens and Barlow, 1971; Barlow, 1983; Bhandari
and Vohra, 1983; Nickrent et al., 1998; Kuijt, 2003). This study further corroborates this
conclusion. All of the genera traditionally classified in this family were sampled and
support for phylogenetic relationships within the family not previously achieved
(Nickrent et al., 1998) were found. This family includes a number of important
pathogenic species and well-known species like the Christmas mistletoe (Viscum album
38
family. As such, Viscaceae remains intact and unchanged and classification should
follow that of previous workers. A new clade definition is not given here.
Aerial parasitism has evolved at least three separate times within Santalaceae s.
lat. This lifecycle is considered a highly specialized and derived characteristic and has
taxa examined in this study, aerial parasitism has evolved independently in Viscaceae,
complete taxon and increased gene sampling, a precise statement about the number of
times the mistletoe habit has evolved cannot be made. Additionally, detailed information
intermediate habit in which a plant will parasitize both roots and stems. Stem parasitism
may have evolved once in Amphorogynaceae, with multiple losses of root parasitism, or
stem parasitism may have been reinvented more than once in the family. A confident
Conclusion
hemiparasitic plants which are difficult to differentiate from other families in Santalales.
The family has a cosmopolitan distribution and has primarily been characterized by
results from previous work which show that Santalaceae represent a paraphyletic
39
assemblage relative to Viscaceae and Eremolepidaceae. Phylogenetic analyses of DNA
sequences from three genes and nearly complete taxon sampling within Santalaceae and
related families reveal eight well-supported clades, which represent more discreet and
potentially better diagnosable units. These eight clades are recognized at the family level
40
Table 1: List of genera, species counts, geographical distribution and documented parasitism in Santalaceae and related families.
Number
Genus of species Geographic distribution Documented parasitism
SANTALACEAE R.Br. (1810) Total: 446
Acanthosyris (Eichl.) Griseb. (1957) 5 Temperate South America Barroso, 1968
Amphorogyne Stauffer & Hürl. (1957) 3 New Caledonia Stauffer and Hürlimann, 1957
Anthobolus R. Br. (1810) 3 Australia Stauffer, 1959
Arjona Cav. (1798) 10 Tropical & temperate South America Kuijt, 1969; Pilger 1935
Buckleya Torr. (1843) 4 Eastern United States and East Asia Kusano, 1902; Piehl, 1965a
Cervantesia Ruiz & Pav. (1794) 4 Andean South America —
Choretrum R. Br. (1810) 6 Australia Hewson and George, 1984
Colpoon P. J. Bergius (1767)
1 South Africa Bean, 1990
including Fusanus L. (1774)
North America, Europe and the
Comandra Nutt. (1818) 1 Hedgecock, 1915; Piehl, 1965b
Mediterranean
Daenikera Hürl. & Stauffer (1957) 1 New Caledonia Hürlimann and Stauffer, 1957
Dendromyza Danser (1940) Southeast Asia, Indomalaysia, New
21 Danser, 1940
including Cladomyza Danser (1940) Guinea
Dendrotrophe Miq. (1856) Himalayas to the Philippines and
4 Macklin and Parnell, 2000, 2002
including Henslowia Blume Malaysia
Dufrenoya Chatin (1860)
11 Indo-Malaysia Macklin and Parnell, 2000, 2002
including Hylomyza Danser (1940)
Exocarpos Labill. (1800) Benson, 1910; Stauffer, 1959; Fineran,
including Elaphanthera N. Hallé 26 Southeast Asia, Malaysia to Hawaii 1962, 1963a-e, 1965a-b, 1979; Fineran
(1988) and Bulluck, 1979; Philipson, 1959
Geocaulon Fernald (1928) 1 Alaska and Canada Moss, 1926
Jodina Hook. & Arn. ex Meissn. (1837) 1 Southern Brazil, Uruguay, Argentina Bhatnagar and Sabharwal, 1969
Kunkeliella Stearn (1972) 4 Canary Islands Anonymous, 2001
Table 1 (continued)
Number
Genus of species Geographic distribution Documented parasitism
SANTALACEAE (continued)
Leptomeria R. Br. (1810) 17 Australia Herbert, 1920; Lepschi, 1999
Disjunct from New Zealand to Juan
Mida A. Cunn. ex Endl. (1837) 1 Philipson, 1959
Fernandez Islands
Myoschilos Ruiz & Pav. (1794) 1 Chile —
Temperate South America
Nanodea Banks ex C. F. Gaertn. (1807) 1 (Patagonia, Tierra del Fuego, Skottsberg, 1913
Falkland Islands)
Nestronia Raf. (1836)
1 Eastern United States Melvin, 1956
including Darbya A. Gray (1846)
Okoubaka Pellegr. & Normand. (1946) 2 Tropical Africa Stauffer, 1957; Veenendaal et al., 1996
Omphacomeria (Endl.) A. DC. (1857) 1 Southeast Australia Stauffer, 1959
Osyridocarpus A. DC. (1894) 1 Africa —
Europe, Mediterranean, Africa to Ferrarini, 1950; Planchon, 1858; Pizzoni,
Osyris L. (1753) 2
India 1906
Phacellaria Benth. (1880) 5 East India to Southern China Danser, 1939
Southeastern United States, China to
Pyrularia Michx. (1803) 2 Leopold and Muller, 1983
Himalayas
Quinchamalium Molina (1781) 25 Andean South America Ruiz and Roig, 1958
Rhoiacarpos A. DC. (1857) 1 South Africa —
Santalum L. (1753)
including Eucarya T. L. Mitch (1927) 20 Indo-Malaysia to Australia, Hawaii Barber, 1907a, 1907b, 1908
and Fusanus R. Br. (1774)
Scleropyrum Arn. (1838)
Malaysia, New Guinea, Southeast
including Scleromelum 6 —
Asia, India
K. Schum. & Lauterb. (1900)
Table 1 (continued)
Number
Genus of species Geographic distribution Documented parasitism
SANTALACEAE (continued)
Spirogardnera Stauffer (1968) 1 Southwestern Australia (endemic) Stauffer, 1968
Thesidium Sonder (1857) 8 South Africa Hill, 1915; Visser, 1981
Europe, Africa, Asia, Australia,
Thesium L. (1753)
245 South America. Two species Mitten, 1847; Visser, 1981; Weber, 1977
including Austroamericium Hendrych
introduced into North America
EREMOLEPIDACEAE Tiegh. ex Kuijt Total: 12
Central and South America,
Antidaphne Poep & Endl. (1838) 8 Kuijt, 1965, 1988
Caribbean, Mexico
Jamaica, Dominican Republic, Puerto
Eubrachion Hook. (1846) 2 Rico, Brazil, Argentina, Uruguay, Kuijt, 1988
Venezuela
Lepidoceras Hook. (1846) 2 Chile Kuijt, 1988
VISCACEAE Miers 1802 Total: 501
Kuijt, 1960; Scharpf, 1963 ; Hull and
Leonard, 1964; Hawksworth and
Arceuthobium M. Bieb (1819) 26 North America, Europe, Asia, Africa
Wiens, 1972; Alosi and Calvin, 1984;
Rey et al., 1991
Dendrophthora Eichl. (1868) 68 Caribbean and South America Kuijt, 1961
Ginalloa Korth. (1839) 5 Indonesia and Malaysia Mistletoe, no direct reference
Africa, Madagascar, Himalayas to
Korthalsella Tiegh. (1896) 10 Stevenson, 1934 ; Danser, 1937
Japan, Australia, New Zealand
Notothixos Oliv. (1864) 8 Sri Lanka, Southeast Asia, Australia Mistletoe, no direct reference
Hawksworth, 1966 ; Kuijt, 1994; Fineran
Phoradendron Nutt. (1848) 234 North and South America
and Calvin, 2000; Kuijt, 2003
Viscum L. (1753) 150 Temperate and tropical Old World Kuijt, 1986
Table 2: Traditional classification of Santalaceae and related families. This
classification is based on Pilger (1935) with modifications and additions by Danser
(1955), Hewson & George (1984), Macklin (2000), Macklin and Parnel (2002), Stauffer
(1959; 1968; 1969), Stauffer and Hürlimann (1957), and Stearn (1972). Classification of
Viscaceae after Barlow (1964), Eremolepidaceae follows Kuijt (1988) and Opiliaceae
follows Hiepko (Hiepko, 1979, 1982, 1985, 1987).
Santalaceae R. Br.
Tribe Anthoboleae (Dumort.) Spach Tribe Thesieae Rchb.
Anthobolus R. Br. Arjona Cav.
Exocarpos Labill. Osyridocarpus A. DC.
Omphacomeria (Endl.) A. DC. Quinchamalium Molina
Tribe Amphorogyneae Stauffer ex Stearn Thesidium Sonder
Amphorogyne Stauffer & Hürl. Thesium L.
Choretrum R. Br. Eremolepidaceae Tiegh. ex Kuijt
Daenikera Hürl. & Stauffer Antidaphne Poepp. & Endl.
Dendromyza Danser Eubrachion Hook.
Dendrotrophe Miq. Lepidoceras Hook.
Dufrenoya Chatin Viscaceae Miers.
Leptomeria R. Br. Arceuthobium M. Bieb
Phacellaria Benth. Dendrophthora Eichl.
Spirogardnera Stauffer Ginalloa Korth.
Tribe Santaleae A, DC. Korthalsella Tiegh.
(syn. Osyrideae Rchb.) Notothixos Oliv.
Acanthosyris (Eichl.) Grieseb. Phoradendron Nutt.
Buckleya Torr. Viscum L.
Cervantesia Ruiz & Pav. Opiliaceae Valeton
Colpoon P. J. Bergius Agonandra Miers ex Benth.
Comandra Nutt. Cansjera Juss.
Geocaulon Fernald Champereia Griffith
Jodina Hook. & Arn. ex Meissn. Gjellerupia Lauterb.
Kunkeliella Stearn Lepionurus Blume.
Mida A. Cunn. ex Endl. Meliantha Pierre
Myoschilos Ruiz & Pav. Opilia Roxb.
Nanodea Banks ex C. F. Gaertn. Pentarhopalopilia Hiepko.
Nestronia Raf. Rhopalopilia Pierre
Okoubaka Pellegr. & Normand Urobotrya Stapf.
Osyris L.
Pyrularia Michx.
Rhoiacarpos A. DC.
Santalum L.
Scleropyrum Arn.
44
Table 3: Voucher information and Genbank accession numbers (when available) for taxa used in this study. When an herbarium
specimen was not made, the collector and no voucher (N.V.) are noted. New sequences which the author has generated are indicated
with his initials (JPD); other sequences generated in the Nickrent lab are denoted by the initials DLN and were generated by Valéry
Malécot, Miguel García García, María-Paz Martín Esteban or Erica Nicholson. “—” indicates sequences that were combined with
another accession of the same species as placeholders in analyses. Missing data are indicated as not available (N.A.).
49
Figure 1: Summary of phylogenetic relationships among the families of Santalales [after
Nickrent et al. (1998), Nickrent and Malecot (2000; 2001) and Malecot et al. (2004)].
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morphology in Santalaceae s. lat. Species (and photographer) names for each image are
given below. A: Arceuthobium pusillum (D. L. Nickrent); B: Amphorogyne spicata (H.
U. Stauffer); C: Choretrum spicatum (H. U. Stauffer); D: Lepidoceras chilense (G.
Glatzel); E: Osyris alba (T. Zumbrunn, Botanical Images Database); F: Santalum
freycinetianum (G. D. Carr, Hawaiian Native Plant Genera); G: Exocarpos gaudichaudii
(G. D. Carr, Hawaiian Native Plant Genera); H: Cervantesia tomentosa (P. M.
Jørgensen, TROPICOS Image Library, MO); I: Nanodea muscosa (J. Puntieri); J:
Quinchamalium chilense (N. Tercero-Bucardo); K: Thesium bergeri (L. J. Musselman);
L: Comandra umbellata (A. H. Bazell, CalPhotos). All images are used with permission
and their respective owners retain copyright.
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shown below branches for those clades also sampled in the combined Bayesian analyses.
See Appendix IV-1 for the full Bayesian topology and all posterior probabilities greater
than 50%. Consistency index (CI) = 0.4714, homoplasy index (HI) = 0.5286, CI
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Figure 4: Maximum likelihood (ML) phylogram from the nuclear SSU rDNA data
partition analyzed under the GTR+I+ model of molecular evolution (nucleotide
frequencies of A=0.25210, C=0.19810, G=0.27050 and T=0.27930; substitution rate
matrix of AC: 1.664200, AG: 4.398100, AT: 3.106500, CA: 1.664200, CG:
1.025000, CT: 12.216500, GA: 4.398100, GC: 1.025000, GT: 1.000000, TA:
3.106500, TC: 12.216500, TG: 1.000000; proportion of invariable sites = 0.6182;
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Figure 5: rbcL MP strict consensus of 76 trees (length=1053). BS support values
greater than 50% are shown above branches (1000 replicates) and Bayesian posterior
probabilities (5+5 million generations combined, 50 000 generation burn-in discarded for
each run) greater than 50% are shown below branches for those clades also sampled in
the combined Bayesian analyses. See Appendix IV-2 for the full Bayesian topology with
all posterior probabilities greater than 50%. The arrows highlight Arjona, whose position
varies in other analyses, and Thesium impeditum, who is not sister to its congener with
this data partition. The “*” indicates BI support for clades which did not include Arjona
in BI or ML analyses. CI = 0.5489, HI = 0.4511, CI excluding uninformative characters
= 0.4540, HI excluding uninformative characters = 0.5460, RI = 0.6720, and RC =
0.3688.
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Figure 6: ML phylogram from the chloroplast rbcL data partition analyzed under the
GTR+I+ model of molecular evolution (nucleotide frequencies of A=0.2715, C=0.1917,
G=0.2399 and T=0.29690; substitution rate matrix of AC: 1.402200, AG: 2.389100,
AT: 0.520800, CA: 1.402200, CG: 0.627400, CT: 3.150100, GA: 2.389100,
GC: 0.627400, GT: 1.000000, TA: 0.520800, TC: 3.150100, TG: 1.000000;
proportion of invariable sites = 0.4536; gamma distribution shape parameter (alpha) =
0.7296). The arrows highlight Arjona, whose position varies in other analyses, and
Thesium impeditum, who is not sister to its congener in this data partition. Note the long
branches found in these two taxa. The “*” indicates clades also found in three gene
analyses. The –lnL score of the single most optimal tree was 8136.63960.
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Figure 7: matK MP strict consensus of 31 trees (length=2513). BS support values
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probabilities (5+5 million generations, 50 000 generation burn-in discarded for each run)
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combined Bayesian analyses. See Appendix IV-6 for the full Bayesian topology and all
posterior probabilities greater than 50%. The arrow highlights the position of
Korthalsella, which is not sister to Ginalloa, and varies in other analyses of this data
partition. CI = 0.5312, HI = 0.4688, CI excluding uninformative characters = 0.4653, HI
excluding uninformative characters = 0.5347, RI = 0.6490, and RC = 0.3448.
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Figure 8: ML phylogram from the matK data partition analyzed without Phacellaria
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C=0.15800, G=0.15790 and T=0.37400; substitution rate matrix of AC: 1.375300,
AG: 1.740400, AT: 0.264300, CA: 1.375300, CG: 0.537500, CT: 1.740400,
GA: 1.740400, GC: 0.537500, GT: 1.000000, TA: 0.264300, TC: 1.740400,
TG: 1.000000; shape parameter of the gamma distribution (alpha) =1.1338). The –log
likelihood score of this single most optimal tree was 13988.94084. See Appendix IV-4
for ML analysis with Phacellaria. The arrow highlights the position of Korthalsella,
which varies in other analyses of this data partition. The “*” indicates clades also found
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Figure 9: Three-gene MP strict consensus of 2 trees (length=4798). BS support values
greater than 50% are shown above branches (1000 replicates) and Bayesian posterior
probabilities greater than 50% (5+5 million generations combined, 50 000 generation
burn-in discarded for each run) are shown below branches for those clades also sampled
in the combined Bayesian analyses. The dashed line indicates the position of Phacellaria
from BI and smaller numbers right of the slash indicate reduction in support when
Phacellaria is included. Circled numbers indicate the eight well-supported clades, on
which the revised classification of Santalaceae s. lat. is based. The organization of taxa
in the historical classification is indicated at the branch tips (V=Viscaceae,
Am=Amphorogyneae, E=Eremolepidaceae, S=Santaleae, An=Anthoboleae, T=Thesieae,
O=Opiliaceae). See Appendix IV-9, 10 & 11 for the full Bayesian topology and all
posterior probabilities greater than 50%. CI = 0.5102, HI = 0.4898, CI excluding
uninformative characters = 0.4292, HI excluding uninformative characters = 0.5708, RI =
0.6267, and RC = 0.3198.
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Figure 10: ML phylogram from the full three-gene dataset analyzed without Phacellaria
under the GTR+I+ model of molecular evolution (nucleotide frequencies of A=0.27700,
C=0.18620, G=0.22350 and T=0.31330; substitution rate matrix of AC: 1.519300,
AG: 2.190700, AT: 0.678000, CA: 1.519300, CG: 0.532900, CT: 3.201500,
GA: 2.190700, GC: 0.532900, GT: 1.000000, TA: 0.678000, TC: 3.201500,
TG: 1.000000; proportion of invariable sites = 0.4468; gamma distribution shape
parameter (alpha) = 0.7908). The –log likelihood score of this tree was 32132.07152.
Brackets indicate familial circumscription according to the proposed classification in this
study; “‡” marks eremolepidaceous taxa. See Appendix IV-8 for ML results with
Phacellaria.
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72
APPENDIX I
Primer Sequences
Nucleotide primers used in PCR amplification and cycle sequencing reactions.
Name Length Position Primer Sequence 5'-3'
nuclear SSU
12F 20 12 - 21 TCC TGC CAG TAS TCA TAT GC
1131R 21 1130-1150 CAA TTC CTT TAA GTT TCA GCC
1769R 19 1769-1787 CAC CTA CGG AAA CCT TGT T
rbcL
1F 20 1-20 ATG TCA CCA CAA ACA GAR AC
3'R 20 rbcL-accd spacer TAG TAA AAG ATT GGG CCG AG
matK
78F 20 73-92 CAG GAG TAT ATT TAT GCA CT
834F 20 834-853 GCA TTA TGT TAG GTA TCA AG
833R 21 833-853 CTT GAT ACC TAA CAT AAT GCA
1420R 21 1291-1310 TCG AAG TAT ATA CTT TAT TCG
73
Appendix I (continued)
Thermal Cycle Parameters
Nuclear SSU rDNA
Temperature (°C) 94 94 48 72 94 52 72 72 4
Time (min:sec) 5:00 1:00 1:00 2:00 0:30 0:30 1:30 10:00 continuous
Number of Cycles X5 X 33
Chloroplast rbcL
Temperature (°C) 94 94 52 72 94 48 72 72 4
Time (min:sec) 5:00 0:30 0:30 0:30 0:30 0:30 1:00 10:00 continuous
Number of Cycles X5 X 33
Chloroplast matK
Temperature (°C) 94 94 46 72 94 50 72 72 4
Time (min:sec) 5:00 1:00 1:00 2:00 0:30 0:30 1:30 10:00 continuous
Number of Cycles X5 X 35
74
APPENDIX II
Some representative command blocks used in this study. Phacellaria was both included
and excluded from analyses containing matK sequence data. Models of molecular
evolution used in Bayesian and likelihood analyses varied between data partitions (see
Table 4).
PAUP* command block (used for MP and ML analysis of the three-gene dataset)
BEGIN SETS;
CHARSET 18S = 1-1827;
CHARSET rbcL = 1828-3255;
CHARSET rbcLpos1 = 1828-3253\3;
CHARSET rbcLpos2 = 1829-3254\3;
CHARSET rbcLpos3 = 1830-3255\3;
CHARSET matK = 3256-4516;
CHARSET matKpos1 = 3256-4516\3;
CHARSET matKpos2 = 3257-4514\3;
CHARSET matKpos3 = 3258-4515\3;
TAXSET Opiliaceae = 51-55;
TAXSET missing18S = 20 30 36 40;
TAXSET missingrbcL = 9 30 40 42-43;
TAXSET missingmatK = 1-2 5-6 20;
END;
BEGIN PAUP;
Outgroup Opiliaceae/only;
Delete Phacellaria;
END;
75
Appendix II: PAUP* commands (continued)
76
Appendix II (continued)
MrBayes command block (used for the fully partitioned three-gene analyses)
[Bayesain Inference.]
Begin MrBayes;
Log Start File=3GeneFull.mb.log;
Set Autoclose=yes;
Delete Phacellaria;
Outgroup Opilia;
CharSet 18s = 1-1827;
CharSet rbcl = 1828-3255;
CharSet rbcLpos1 = 1828-3253\3;
CharSet rbcLpos2 = 1829-3254\3;
CharSet rbcLpos3 = 1830-3255\3;
CharSet matk = 3256-4516;
CharSet matKpos1 = 3256-4516\3;
CharSet matKpos2 = 3257-4514\3;
CharSet matKpos3 = 3258-4515\3;
TaxSet viscaceae = 1-7;
TaxSet opiliaceae = 51-55;
TaxSet missing18s = 20 30 36 40;
TaxSet missingrbcl = 9 30 40 42-43;
TaxSet missingmatK = 1-2 5-6 20;
Partition full=7:18S,rbcLpos1,rbcLpos2,rbcLpos3,
matKpos1,matKpos2,matKpos3;
Partition gene=3:18S,rbcL,matK;
Set Partition=full;
unlink revmat=(all);
unlink shape=(all);
unlink statefreq=(all);
unlink pinvar=(all);
mcmc ngen=5000000 nchains=4 printfreq=1000 samplefreq=1000
savebrlens=yes startingtree=random filename=3GeneFull;
sump filename=3GeneFull.p burnin=50;
plot filename=3GeneFull.p burnin=50 match=all;
sumt filename=3GeneFull.t burnin=50 showtreeprobs=no;
Quit;
End;
77
APPENDIX III
-32000
-34000
-36000
Ln Likelihood
-38000
-40000
-42000
-44000
-46000
-48000
-50000
Number of Generations
Log likelihood plot for burn-in region of the fully partitioned three-
gene analysis without Phacellaria
0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000
-30000
-32000
-34000
-36000
Ln Likelihood
-38000
-40000
-42000
-44000
-46000
-48000
-50000
Number of Generations
78
APPENDIX IV
79
Supplemental Tree IV-1: Nuclear SSU Bayesian majority rule consensus from two
identical BI runs with 5000000 generations, each sampled every 1000 generations. Trees
from the first 50000 generations (50 trees) were discarded as burn-in. Parameters were
estimated under the GTR+I+ model of molecular evolution. Posterior probabilities
from each run separately are below the branches and from both runs combined are above
the branches. Values in boldface italic type represent clades not recovered in the MP
strict consensus tree for this data partition.
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Supplemental Tree IV-2: rbcL Bayesian majority rule consensus from two identical BI
runs with 5000000 generations each sampled every 1000 generations. Trees from the
first 50000 generations (50 trees) were discarded as burn-in. Data were partitioned by
codon position and model parameters for the first, second and third positions were
estimated independently (i.e. unlinked) under the GTR+I+, JC+, and GTR+ models
of molecular evolution, respectively. Posterior probabilities from each run separately are
shown below the branches and from both runs combined are above the branches. Values
in boldface italic type represent clades not recovered in the MP strict consensus tree for
this data partition.
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Supplemental Tree IV-3: matK MP strict consensus of 188 trees of length 2519 which
include Phacellaria. This is a 6-fold increase in the number of trees and a tree length
increase of only six steps from analyses that excluded Phacellaria. There is a dramatic
loss of resolution in Amphorogyneae/Amphorogynaceae, but the topology and support
values of other groups remain unchanged. BS support values greater than 50% are shown
above branches (1000 replicates). CI = 0.5304, HI = 0.4696, CI excluding uninformative
characters = 0.4642, HI excluding uninformative characters = 0.5358, RI = 0.6491, and
RC = 0.3442.
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Supplemental Tree IV-4: ML phylogram from the matK data partition analyzed with
Phacellaria under the TVM+ model of molecular evolution (-lnL = 14021.11728). This
tree recovers the three distinct clades in Amphorogyne, but places Phacellaria in a
polytomy with the root parasites (Choretrum + Leptomeria and Amphorogyne +
Daenikera), a relationship not seen when Phacellaria is excluded.
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Supplemental Tree IV-5: matK Bayesian majority rule consensus including
Phacellaria, from two identical BI runs with 5000000 generations each sampled every
1000 generations. Trees from the first 50000 generations (50 trees) were discarded as
burn-in. Data were partitioned by codon position and model parameters for all three
positions were estimated independently (i.e. unlinked) under the GTR+ model of
molecular evolution. Posterior probabilities from each run separately are shown below
the branches and from both runs combined are above the branches.
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Supplemental Tree IV-6: matK Bayesian majority rule consensus. This is a similar
analysis to Supplemental Tree IV-5, but without Phacellaria. Two identical BI runs with
5000000 generations each were sampled every 1000 generations. Trees from the first
50000 generations (50 trees) were discarded as burn-in. Data were partitioned by codon
position and model parameters for all three positions were estimated independently (i.e.
unlinked) under the GTR+ model of molecular evolution. Posterior probabilities from
each run separately are shown below the branches and from both runs combined are
above the branches. Values in boldface italic type represent clades not recovered in the
MP strict consensus tree for this data partition.
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Supplemental Tree IV-7: Three-gene strict consensus of 12 trees of length 4804 which
include Phacellaria. Phacellaria dramatically reduces the level of resolution in
Amphorogyneae/Amphorogynaceae, but the topology of other groups remains similar to
other analyses of the three-gene dataset. BS support values greater than 50% are shown
above branches (1000 replicates). CI = 0.5098, HI = 0.4902, CI excluding uninformative
characters = 0.4287, HI excluding uninformative characters = 0.5713, RI = 0.6268, and
RC = 0.3196.
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Supplemental Tree IV-8: Three-gene ML phylogram analyzed with Phacellaria under
the GTR+I+ model of molecular evolution. Two equally optimal trees with -lnL score
of 32165.54345 were found in the heuristic search. These trees differ only in the
placement of Phacellaria (plotted with a dashed line in both positions). Relative branch
lengths in both trees are the same.
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Supplemental Tree IV-9: Three-gene Bayesian majority rule consensus partitioned by
gene without Phacellaria, from two identical BI runs with 5000000 generations each
sampled every 1000 generations. Trees from the first 50000 generations (50 trees) were
discarded as burn-in. Model parameters were estimated independently (i.e. the partitions
were unlinked) for each gene under the GTR+I+ model of molecular evolution.
Posterior probabilities from each run separately are shown below the branches and from
both runs combined are above the branches.
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Supplemental Tree IV-10: Three-gene Bayesian majority rule consensus fully
partitioned with Phacellaria, from two identical BI runs with 5000000 generations each
sampled every 1000 generations. Trees from the first 50000 generations (50 trees) were
discarded as burn-in. Data were fully partitioned by genes, and for the protein coding
genes, by codon position. Model parameters were estimated independently for each
partition (i.e. the partitions were unlinked). See Table 4 for the models of molecular
evolution used for each partition. Posterior probabilities from each run separately are
shown below the branches and from both runs combined are above the branches.
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Supplemental Tree IV-11: Three-gene Bayesian majority rule consensus fully
partitioned without Phacellaria, from two identical BI runs with 5000000 generations
each sampled every 1000 generations. Trees from the first 50000 generations (50 trees)
were discarded as burn-in. Data were fully partitioned by genes, and for the protein
coding genes, by codon position. Model parameters were estimated independently for
each partition (i.e. the partitions were unlinked). See Table 4 for the models of molecular
evolution used for each partition. Posterior probabilities from each run separately are
shown below the branches and from both runs combined are above the branches. Values
in boldface italic type represent clades not recovered in the MP strict consensus tree for
this data partition.
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VITAE
Graduate School
Southern Illinois University
Thesis Title:
Molecular Phylogenetics and Classification of Santalaceae
Major Professor:
Dr. Daniel L. Nickrent
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