Molecular Phylogenetics and Classification of Santalaceae

MOLECULAR PHYLOGENETICS AND CLASSIFICATION OF SANTALACEAE

by

Joshua P. Der

B. S. Biology and Botany, Humboldt State University, 2003

A Thesis

Submitted in Partial Fulfillment of the Requirements for the

Master of Science Degree

Department of Plant Biology

in the Graduate School

Southern Illinois University Carbondale

July 2005

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AN ABSTRACT OF THE THESIS OF

JOSHUA P. DER, for the Master of Science degree in Plant Biology, presented on 2 July

2005 at Southern Illinois University Carbondale.

TITLE: Molecular Phylogenetics and Classification of Santalaceae

MAJOR PROFESSOR: Dr. Daniel L. Nickrent

Santalaceae are a diverse group of root and stem hemiparasitic plants in the

sandalwood order (Santalales), which occur worldwide in both tropical and temperate

climates. As traditionally classified, 35 genera in four tribes (Amphorogyneae,

Santaleae, Anthoboleae and Thesieae) are included in Santalaceae. This family is

paraphyletic with respect to Viscaceae (seven genera) and Eremolepidaceae (three

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

parsimony, maximum likelihood and Bayesian inference. Phylogenies inferred from

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

earlier classifications. Generic circumscription within tribe Amphorogyneae also remains

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

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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.

Three distinct clades (Nanodeaceae, Pyrulariaceae, and Comandraceae) are segregated

from the polyphyletic tribe Santaleae and Buckleya and Kunkeliella are members of

Thesiaceae. Arjona and Quinchamalium (Thesieae) form the eighth well-supported clade

and are recognized as Arjonaceae.

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ACKNOWLEDGEMENTS

This research was made possible with financial support from the National Science

Foundation (DEB 0108229 to Daniel Nickrent).

I wish to express my sincere gratitude to the many people who helped to make

this work possible.

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

discussion and through the clearinghouse of information he has assembled on the

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

me in planning the analyses in this work.

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

contributions of these people.

I am also grateful to several people who granted me access to University

computing facilities to run my phylogenetic analyses. Sandy Hostetler enthusiastically

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

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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

resources, the analyses I ran would be finishing next year.

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

she has done and the times we have shared.

I want to thank Guille Amico, Roberta Torunsky, Justin Sipiorski, Alonso

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 encouragement throughout my stay in Southern Illinois.

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,

my companion in life and my best friend.

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TABLE OF CONTENTS

ABSTRACT.....................................................................................................................i

ACKNOWLEDGEMENTS............................................................................................iii

LIST OF TABLES ........................................................................................................vii

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

MATERIALS AND METHODS

Taxon Sampling ........................................................................................................13

Laboratory Methods and Sequence Alignment...........................................................13

Phylogenetic Analysis ...............................................................................................15

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

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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

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LIST OF TABLES

Table Page

Table 1: List of genera, number of species, geographical distribution and documented

parasitism in Santalaceae and related taxa. ...................................................41

Table 2: Published classification of Santalaceae and related families. ........................44

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

Bayesian analyses. .......................................................................................48

Table 5: Revised phylogenetic classification of Santalaceae s. lat. .............................49

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LIST OF FIGURES

Figure Page

Figure 1: Phylogenetic relationships among Santalalean families. ...............................50

Figure 2: Photographs showing floral diversity in Santalaceae. ...................................51

Figure 3: Nuclear SSU rDNA maximum parsimony strict consensus tree with bootstrap

support values and Bayesian posterior probabilities......................................52

Figure 4: Nuclear SSU rDNA maximum likelihood phylogram. ..................................53

Figure 5: rbcL maximum parsimony strict consensus tree with bootstrap support values

and Bayesian posterior probabilities. ............................................................54

Figure 6: rbcL maximum likelihood phylogram. .........................................................55

Figure 7: matK maximum parsimony strict consensus tree with bootstrap support values

and Bayesian posterior probabilities. ............................................................56

Figure 8: matK maximum likelihood phylogram. ........................................................57

Figure 9: Three-gene maximum parsimony strict consensus tree with bootstrap support

values and Bayesian posterior probabilities. .................................................58

Figure 10: Three-gene maximum likelihood phylogram...............................................59

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INTRODUCTION

Biology of Santalaceae

Santalaceae R. Br. is a very diverse family of hemiparasitic plants in the

sandalwood order (Santalales Dumort.) that occur worldwide in both temperate and

tropical climates (Kuijt, 1969). Defined primarily by plesiomorphic and absent

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

species and the advanced parasites in Amphorogyneae) and sometimes spines

(Acanthosyris) (Kuijt, 1969). Tropical and subtropical species often have thick leathery

leaves, but thin deciduous leaves are common in temperate representatives (e.g.

Pyrularia, Myoschilos, Buckleya and Geocaulon). Some species have dimorphic

branches, which may consist of short and long shoots (Acanthosyris) or an alternating

series of squammate and foliate branches (Dendromyza and Exocarpos). A number of

genera have determinate branches, resulting in dichasial (Buckleya) or sympodial (e.g.

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).

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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 in Quinchamalium are the exception). The 4-5-merous (rarely 3- or 6-merous)

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

this is reduced to a calyculus in Loranthaceae and lost completely in Opiliaceae. This

reversal, where a calyx is reinvented, isn’t parsimonious and doesn’t seem likely. In his

Integrated System of Classification, Cronquist (1981) stated that the undiferentiated

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

(esp. in Amphorogyneae) (Hieronymus, 1889; Pilger, 1935; Danser, 1940, 1955). In

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

ovary is found in Anthoboleae (Anthobolus, Exocarpos, Omphacomeria), while the

remainder of the family has an inferior, or partially inferior ovary (Pilger, 1935; Smith

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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

tissue. The embryo eventually becomes enclosed by a sclerenchymatous endocarp in

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).

Detailed information on parasitism in Santalaceae is limited, but a great deal of

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

documentation of their parasitism. Most members of Santalaceae are root parasites,

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

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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

parasitic only on other mistletoes in Santalaceae or Loranthaceae (Danser, 1939; Kuijt,

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

World, Australia and South America. The eremolepidaceous mistletoes (Antidaphne,

Eubrachion and Lepidoceras) are restricted to the New World (Kuijt, 1988) and the aerial

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parasites in Amphorogyneae are found in Southeast Asia and New Guinea (Danser, 1940,

1955; Macklin, 2000; Macklin and Parnell, 2002).

Historical Classification of Santalaceae

Santalaceae was first described by Robert Brown in his Prodromus Florae Novae

Hollandiae (1810), in which he summarizes the specimens he collected in Australia from

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

organization of santalaceous genera in a natural system of classification following de

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

Exocarpos Labill., Anthobolus R.Br. and Olax L. with the family.

The next treatment of Santalaceae was that of Heironymus (1889) in Engler and

Prantl’s Die Natürlichen Pflanzenfamilien. Heironymous recognized 26 genera

circumscribed within three tribes (Anthoboleae Bartl., Osyrideae Rchb., and Thesieae

Rchb.) as well as Calyptosepalum S. Moore (a taxon with uncertain affinity, but

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.)

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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

species as Dendromyza, Cladomyza and Hylomyza, synonymized the remainder with

Dendrotrophe, and described 19 new species (Danser, 1940, 1955). Stauffer revised

Anthoboleae to include Omphacomeria (Endl.) A. DC (1959) and erected a fourth tribe,

Amphorogyneae Stauffer (1969). Amphorogyneae, containing three genera segregated

from Santaleae (Choretrum, Leptomeria, and Phacellaria), can be recognized by their

peculiar anther structure. Amphorogyneae also included Spirogardnera Stauffer,

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

Kunkeliella. Recently, Macklin has done detailed taxonomic work on Santalaceae in

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

based on Pilger and subsequent workers is given in Table 2.

Eremolepidaceae Tiegh. ex Kuijt is a group of 12 mistletoe species in three genera

(Antidaphne Poepp. & Endl., Eubrachion Hook., and Lepidoceras Hook.) restricted to the

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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

species (Table 1).

The related family of mistletoes, Viscaceae Miers, has recently been included in

Santalaceae by the Angiosperm Phylogeny Group (APG, 2003). This family was

previously classified as a subfamily of Loranthaceae Juss., but was shown to be distinct

based on morphological, karyological, embryological and molecular characters (Johri and

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

supporting the separation of these two mistletoe families.

Phylogeny of Santalales

While the familial composition of Santalales, as well as ordinal boundaries, have

varied substantially over time (Johri and Bhatnagar, 1960; Kuijt, 1968), current

classification includes six families: “Olacaceae”, Loranthaceae, Misodendraceae,

8

Opiliaceae, “Santalaceae” (including Eremolepidaceae), and Viscaceae (families in

quotations indicate paraphyletic assemblages; Figure 1) (Nickrent and Duff, 1996;

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

has traditionally been classified as a monogeneric subfamily (Schoepfioideae) of

Olacaceae. Opiliaceae represents the next family to diverge within the order, and is quite

similar (using both morphological and molecular characters) to santalaceous members

(Nickrent and Malécot, 2001). Santalaceae are currently considered paraphyletic with

respect to Viscaceae (Nickrent and Duff, 1996; Nickrent et al., 1998; Nickrent and

Malécot, 2000, 2001). Consequently, Viscaceae were subsumed within Santalaceae in

the APGII classification (2003). However, because Viscaceae is a well-established

monophyletic group of economic importance (Calder, 1983), and the major relationships

within the Santalaceae were not resolved, some authors have retained Viscaceae and

“Santalaceae” as distinct families in working classifications (Nickrent and Malécot, 2001;

Judd et al., 2002; Malécot et al., 2004).

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Modern Molecular Phylogenetics, Previous Work, and Gene Selection

Current taxonomic classification relies on a strong understanding of phylogenetic

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;

Steussy, 1990; de Queiroz and Gauthier, 1992).

Molecular methods involving DNA sequencing and computer-based phylogenetic

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

Nickrent lab at Southern Illinois University Carbondale for phylogenetic work in

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

was incomplete. Santalaceae was shown to be paraphyletic with respect to Viscaceae,

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

10

Santalaceae as well). These data increased resolution and showed promise for resolving

the primary relationships within Santalaceae (Malécot, 2002).

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

present and relatively conserved (due to functional constraints) in all photosynthetic

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;

Nickrent and Malécot, 2000).

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

11

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

The primary objectives of this research are to:

1. infer the generic-level phylogeny of Santalaceae and examine the monophyly

of this group using Opiliaceae as the outgroup.

2. assess the monophyly of major groups within Santalaceae, including

Viscaceae, Eremolepidaceae, each of the four previously described tribes and

the six largest genera in Santalaceae.

3. propose a revised classification of the family reflecting well-supported

phylogenetic relationships.

The uncertain affinity of Eremolepidaceae outlined above is grounds for increased

investigation of the phylogenetic placement of this group. Molecular work using nuclear

SSU rDNA and rbcL placed Eremolepidaceae within tribe Santaleae (Nickrent and

Malécot, 2001). However, the relationships and monophyly of the three

eremolepidaceaous genera (Antidaphne, Lepidoceras and Eubrachion) has varied

between studies with different taxon and gene sampling (Nickrent and Soltis, 1995;

Nickrent and Duff, 1996; Nickrent et al., 1998).

Tribe Amphorogyneae is an enigmatic group (which includes root and stem

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

12

resolution of relationships within Amphorogyneae, but show strong support for the

monophyly of the three sampled genera: Choretrum, Dendrotrophe, and Dufrenoya

(Nickrent and Malécot, 2001). Increased taxon sampling and sequence data provide the

necessary resolution to address these issues.

Aerial parasitism (i.e. mistletoes and dendroparasites) is a highly specialized habit

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

Dendromyza, Dendrotrophe, Dufrenoya and Phacellaria). A clearer understanding of

their phylogeny will allow us to examine the evolution of aerial parasitism in

Santalaceae.

A better understanding of the phylogenetic relationships in Santalaceae will

provide an evolutionary framework, within which future studies on their biology,

morphology and anatomy may operate, and which also serves as the basis for the

proposed classification presented in this work.

13

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

Biology at Southern Illinois University, Carbondale. Plants were field-collected by DLN

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

of the 35 currently recognized genera in Santalaceae were sampled, including two

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

study, Spirogardnera, is a monotypic endangered shrub endemic to western Australia for

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

for all taxa used in this study is given in Table 3.

Laboratory Methods and Sequence Alignment

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

Applied Biosystems (ABI) GeneAmp® PCR System 9700 thermocycler in 25 µL

reactions (see Appendix I for primer sequences, PCR reagents and thermal cycle

14

parameters). To check and quantify PCR reactions, 2-4 µL each was electrophoresed for

30 minutes at 110 volts in 1% agarose gels, stained in EtBr, and photographed on an

ultraviolet light transilluminator. Successful PCR amplifications were column purified

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

sodium acetate/ethanol precipitation and were dried in a Savant SpeedVac. Reactions

were resuspended in 2 µL of formamide/loading dye then denatured, and were

electrophoresed in 5% LongRanger XL polyacrylamide gels using an ABI Prism® 377

automated DNA sequencer.

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

15

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

codons into amino acids.

Phylogenetic Analysis

Datasets for all three genes were analyzed separately using maximum parsimony

(MP) and maximum likelihood (ML) as implemented in PAUP* v4.0b10 (Swofford,

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,

a member of tribe Amphorogyneae. Unfortunately, inclusion of this taxon obscured the

phylogenetic relationships within this tribe; subsequently, the matK and combined three-

gene datasets were also analyzed without Phacellaria to examine relationships among the

remaining taxa in Amphorogyneae.

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

step, and tree-bisection-reconnection (TBR) branch swapping. All of the most

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

16

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

phylogenetic signal and a preliminary bootstrap analysis failed to exceed 56 replicates

after 50 hours on a dedicated 2.0 GHz Dual Processor PowerMac G5.

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

17

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

lengths were saved.

Bayesian phylogenetic analyses were performed using the parallel Metropolis-

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

PowerPC computer processors using the message-passing interface (MPI) on small

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

of DNA sequence evolution (Rannala and Yang, 1996).

18

RESULTS

Nuclear SSU rDNA

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

tree. ML analysis of this dataset implemented the GTR+I+ model of molecular

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

trees after discarding the 50 000 generation burn-in was 8797.93022.

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

Eremolepidaceae). BS values and Bayesian PP (combined from separate runs of codon-

partitioned analyses) are mapped on this tree. The ML analysis implemented the

19

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

existence of rate heterogeneity in two mistletoe clades (Viscaceae and Eremolepidaceae).

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-

informative sites. Phacellaria contributed only one parsimony-uninformative variable

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

Santalaceae. ML analyses implemented the TVM+ model of molecular evolution with

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

which includes Phacellaria is shown in Appendix IV-4). Codon partitioned matK

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

Phacellaria was excluded.

20

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

ML topologies only in the placement of Phacellaria within Amphorogyneae (Appendix

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

Santalaceae as they have traditionally been circumscribed (Table 2) are

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.

members of Santalaceae, Viscaceae and Eremolepidaceae) are monophyletic (100% MP

bootstrap and Bayesian PP) with respect to Opiliaceae (outgroup). Tribe

21

Amphorogyneae is monophyletic and tribe Santaleae is polyphyletic. Arjona and

Quinchamalium are not included with other members of tribe Thesieae, while Buckleya

and Kunkeliella (Santaleae) are associated with this tribe.

Results from all three genes are largely congruent, but differ in the level of

resolution. In nuclear SSU analyses, Exocarpos and Omphacomeria (both Anthoboleae)

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

position of Korthalsella calls attention. MP places Korthalsella with other Viscaceae

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

not included in Viscaceae and is placed in a more intermediate position within

Santalaceae. ML places this taxon sister to a clade containing Acanthosyris, Jodina,

Cervantesia, Okoubaka, Scleropyrum and Pyrularia, while BI places it in a polytomy

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

other taxa varies (with low support) in various analyses.

22

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

SSU, rbcL and matK data partitions, respectively.

Despite substantial care during laboratory work and sequence alignment, some of

these anomalous results may have been caused by error introduced during manual

sequencing (for older sequences generated prior to the beginning of my work),

contamination during PCR amplification, replication errors introduced by Taq

polymerase or during cloning, accidentally swapping samples, or incorrect homology

assessment during manual sequence alignment. Additionally, disproportionately long

branches in Arjona (rbcL, Figure 6) and Korthalsella (matK, Figure 8) and missing rbcL

sequences for Quinchamalium (a close relative of Arjona) might also contribute to

artifactual results.

Despite these incongruencies, santalaceous genera occur in eight well-supported

clades recovered in analyses of all three genes in combination (indicated by circled

numbers in Figure 9). Viscaceae, as traditionally circumscribed, is monophyletic (Clade

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

Santalaceae (Santalum). Exocarpos and Omphacomeria (Anthoboleae) are sister taxa

and are basal in this clade. Eremolepidaceae is also included in Clade 3 as are several

additional members of tribe Santaleae (Colpoon, Rhoiacarpos, Nestronia, Osyris and

Myoschilos). Clade 4 (100% BS and PP) includes six genera segregated from tribe

23

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,

with exception of the position of Phacellaria within Clade 2.

24

DISCUSSION

Molecular Phylogenetics of Santalaceae

Santalaceae have not been the subject of a detailed, worldwide, generic-level

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

pollen characteristics, he ultimately suggested a relationship with Osyris, Colpoon, and

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

taxonomic breadth and have been subject to local biases.

Earlier molecular work in the Nickrent lab demonstrated the paraphyletic nature

of the traditional Santalaceae but as mentioned previously, taxon sampling and lack of

resolution prohibited any systematic revision. With nearly comprehensive taxon

sampling for three genes, strong patterns of evolutionary relationships have emerged.

25

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

should represent easily diagnosable biological units.

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

hierarchal rank-based system of nomenclature (that of the International Code of Botanical

Nomenclature, or ICBN) and name important, well-supported clades without reference to

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

provides a valid solution to numerous problems associated with rank-based

nomenclatural systems, it represents a dramatic shift from the established system of

nomenclature and fails to solve other problems inherent to both codes, for example,

stability of clade membership associated with a given taxon name. Additionally,

26

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

current rank-based framework, but incorporates phylogenetic clade-based definitions.

The traditional Santalaceae are quite diverse and heterogeneous with respect to

morphological, anatomical and embryological characters. Accordingly they have been

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

easier to diagnose and differentiate than a larger morphologically heterogeneous

Santalaceae s. lat. While the rank of family holds no biological or evolutionary

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

of organization in herbaria and floras, as well as an indicator used to describe diversity in

many ecological studies.

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

27

for each new family in turn, as well as generic circumscription and relationships within

each family. Taxonomic history, geographic distribution and morphological and

anatomical support are also discussed, where information is available.

While a full taxonomic treatment with comprehensive family and generic

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

phylogenetic framework. As this is done, clear synapomorphies may emerge and be

recognized.

Phylogenetic Classification of Santalaceae s. lat.

OPILIACEAE

Opiliaceae was used as the outgroup in these analyses, so no statement of

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

Omphacomeria) orphaned. As discussed later, these genera are included in Santalaceae

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

definition is not given here.

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

28

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.

Geocaulon is restricted to North temperate and arctic North America and

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

stalks from a creeping rhizome. Geocaulon is distinguished from Comandra by having

monoecious inflorescences (versus bisexual flowers in Comandra) and a thin herbaceous

rhizome (versus a thick woody rhizome).

Comandra was described in 1818 by Nuttall, from which C. lividum was

subsequently segregated as Geocaulon lividum in 1928 by Fernald. Johri and Bhatnagar

(1960) suggested that Comandra was distinctive enough from other groups in

Santalaceae that it should be recognized at the tribal level (Comandreae) based on

embryology and details of the ovary and placenta. Johri and Bhatnagar’s work

corroborated a tribal designation for this group by Van Tieghem (1896).

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

of North American Plants 1: 157. 1818).

THESIACEAE

Kunkeliella, Thesidium, Thesium, Osyridocarpos and Buckleya form a well

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

29

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

having dioeceous flowers (Pilger 1935).

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

Thesium (e.g. Austroamericium, Chrysothesium, Linosyris, Steinteitera, Linophyllum and

others). Miguel García is currently undertaking a systematic study of this genus in

collaboration with Daniel Nickrent.

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

subshrubs, herbaceous perennials and annuals.

As mentioned previously, Stearn (1972) noted the possible affinities of

Kunkeliella with members of this clade. Stearn noted that the habit of Kunkeliella

resembles Osyridocarpos, Austroamericium (included in Thesium), and some additional

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)

were not considered to lie with members of this group.

This clade is primarily African in distribution with Thesidium and Kunkeliella

endemic to South Africa and the Canary Islands, respectively. Osyridocarpos is found in

30

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.


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

monophyly of Quinchamalium is brought into question in this study. Quinchamalium

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

that are dry nuts or achenes.

31

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)

based on the presence of bracts surrounding an inferior ovary attached to an

undifferentiated perianth. This relationship was not recognized by Hieronymus (1889) or

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

32

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).

Molecular results corroborate this separation, and this clade (excluding

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

Quinchamalium chilense. The type species is Arjona tuberosa Cav. (Icones et

Descritiones Plantarum 4: 57. 1798).

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

33

recognized as a section of Fusanus in this treatment, but was later elevated to genus. The

remaining members of Fusanus were included in Santalum. This relationship with

Santalum was recognized by Bhatnagar (1960), but an examination of Nanodea was not

made in the series of publications on the embryology of Santalaceae out of the

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

Agarwal, 1965; Bhatnagar and Sabharwal, 1969; Bhatnagar, 1991).


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

Fructibus et Seminibus Plantarum 3: 251. 1807).

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

Acanthosyris, Cervantesia and Jodina sub-clade is strictly South American in

distribution, whereas Okoubaka, Scleropyrum and Pyrularia are all Old World taxa, with

the exception of one species of Pyrularia. Okoubaka is found in tropical Africa.

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.

34

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

Studien series. In Santalales Studien VII (1961), Stauffer discussed Acanthosyris,

Cervantesia and Jodina together and provided a table to compare and contrast these

genera. In Santalales Studien I (1957), Stauffer discussed the placement of Okoubaka

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

with large drupes (or pseudodrupes).


is given: Pyrulariaceae are the least inclusive clade that contains Pyrularia pubera and

Cervantesia tomentosa. The type species is Pyrularia pubera Michx. (Flora Boreali-

Americana 2: 231-233. 1803).

SANTALACEAE sensu stricto

Tribe Santaleae (=Osyrideae) is the most heterogeneous group within the

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

35

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

traditional Santaleae (i.e. Pyrulariaceae, Buckleya + Kunkeliella, Mida + Nanodea and

Comandra + Geocaulon). This is shown clearly in Figure 9, where the historical

classification of these genera is indicated to the left of the taxon names.

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

clade is supported with 100% BS and PP.


is given: Santalaceae s. str. are the least inclusive clade that contains Santalum album,

Exocarpos cupressiformis and Antidaphne viscoidea. The type species is Santalum

album L. (Species Plantarum 1: 349. 1753).

AMPHOROGYNACEAE

Clade 2 (Figure 9) comprises one of the best-defined groups in the traditional

Santalaceae and is sister to Viscaceae with 85% BS and 100% PP. This clade includes all

of the sampled genera of tribe Amphorogyneae, which, with Spirogardnera (not

36

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

characterized by the stick-like shrub, Daenikeria, from New Caledonia, which

approaches a holoparasitic habit (Hürlimann and Stauffer, 1957), and the ultimate

mistletoe, Phacellaria, which is herbaceous, only parasitic on the stems of other

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

37

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

parasites (Stauffer and Hürlimann, 1957; Stauffer, 1968; Lepschi, 1999).

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

parasites (Dendrotrophe, Dufrenoya and Dendromyza). Amphorogyne and Daenikera

come out basal to these two sub-clades, but their sister relationship decays when

incomplete sequences of Phacellaria are included. Amphorogyne is most often

considered a root parasitic shrub, but photographs in Stauffer’s tribal description of

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

(Dendromyza, Dufrenoya and Dendrotrophe). The position of Dendrotrophe as derived

from within the mistletoes is interesting, as this genus has been considered “primitive”

among the stem parasites (Danser, 1940, 1955; Stauffer and Hürlimann, 1957).

Spirogardnera is a monotypic endangered shrub endemic to Western Australia and was

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

38

Leptomeria, with which it shares several features including anther morphology,

inflorescence structure and habit.

Additionally, the generic identity of Hylomyza and Cladomyza should be

investigated more thoroughly. These mistletoe genera were synonymized and included

within Dufrenoya and Dendromyza respectively by Macklin (Macklin, 2000; Macklin

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.


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

Viscaceae has long been recognized as a distinct monophyletic group of

mistletoes based on morphological, embryological, cytological, anatomical and molecular

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

in Europe and Phoradendron serotinum in North America). Because of the strongly

supported widely recognized nature of this group, it is least disruptive to retain it as a

39

family. As such, Viscaceae remains intact and unchanged and classification should

follow that of previous workers. A new clade definition is not given here.

Evolution of Aerial Parasitism in Santalaceae

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

also evolved in Loranthaceae and Misodendraceae, also families in Santalales. Of the

taxa examined in this study, aerial parasitism has evolved independently in Viscaceae,

Amphorogynaceae and in the eremolepidaceous taxa (now in Santalaceae s. str.). Until a

better understanding of the phylogeny of genera in Amphorogynaceae emerges with

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

on the biology of Amphorogyne and Daenikera must be acquired in order to completely

document this phenomenon. Dendrotrophe may represent the retention of an

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

statement cannot be made at this time.

Conclusion

Santalaceae, as they have traditionally been classified, are comprised of diverse

hemiparasitic plants which are difficult to differentiate from other families in Santalales.

The family has a cosmopolitan distribution and has primarily been characterized by

plesiomorphic or generalized traits occurring throughout Santalales. This study supports

results from previous work which show that Santalaceae represent a paraphyletic

40

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

and are Comandraceae, Thesiaceae, Arjonaceae, Nanodeaceae, Pyrulariaceae,

Santalaceae sensu stricto, Amphorogyneae and Viscaceae.

Table 1: List of genera, species counts, geographical distribution and documented parasitism in Santalaceae and related families.

Genus

Number

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)

including Fusanus L. (1774)1 South Africa Bean, 1990

Comandra Nutt. (1818) 1North America, Europe and the

MediterraneanHedgecock, 1915; Piehl, 1965b

Daenikera Hürl. & Stauffer (1957) 1 New Caledonia Hürlimann and Stauffer, 1957

Dendromyza Danser (1940)

including Cladomyza Danser (1940)21

Southeast Asia, Indomalaysia, New

GuineaDanser, 1940

Dendrotrophe Miq. (1856)

including Henslowia Blume4

Himalayas to the Philippines and

MalaysiaMacklin and Parnell, 2000, 2002

Dufrenoya Chatin (1860)

including Hylomyza Danser (1940)11 Indo-Malaysia Macklin and Parnell, 2000, 2002

Exocarpos Labill. (1800)

including Elaphanthera N. Hallé

(1988)

26 Southeast Asia, Malaysia to Hawaii

Benson, 1910; Stauffer, 1959; Fineran,

1962, 1963a-e, 1965a-b, 1979; Fineran

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)

Genus

Number


SANTALACEAE (continued)

Leptomeria R. Br. (1810) 17 Australia Herbert, 1920; Lepschi, 1999

Mida A. Cunn. ex Endl. (1837) 1Disjunct from New Zealand to Juan

Fernandez IslandsPhilipson, 1959

Myoschilos Ruiz & Pav. (1794) 1 Chile —

Nanodea Banks ex C. F. Gaertn. (1807) 1

Temperate South America

(Patagonia, Tierra del Fuego,

Falkland Islands)

Skottsberg, 1913

Nestronia Raf. (1836)

including Darbya A. Gray (1846)1 Eastern United States Melvin, 1956

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 —

Osyris L. (1753) 2Europe, Mediterranean, Africa to

India

Ferrarini, 1950; Planchon, 1858; Pizzoni,

1906

Phacellaria Benth. (1880) 5 East India to Southern China Danser, 1939

Pyrularia Michx. (1803) 2Southeastern United States, China to

HimalayasLeopold and Muller, 1983

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)

and Fusanus R. Br. (1774)

20 Indo-Malaysia to Australia, Hawaii Barber, 1907a, 1907b, 1908

Scleropyrum Arn. (1838)

including Scleromelum

K. Schum. & Lauterb. (1900)

6Malaysia, New Guinea, Southeast

Asia, India—

Table 1 (continued)

Genus

Number


SANTALACEAE (continued)

Spirogardnera Stauffer (1968) 1 Southwestern Australia (endemic) Stauffer, 1968

Thesidium Sonder (1857) 8 South Africa Hill, 1915; Visser, 1981

Thesium L. (1753)

including Austroamericium Hendrych245

Europe, Africa, Asia, Australia,

South America. Two species

introduced into North America

Mitten, 1847; Visser, 1981; Weber, 1977

EREMOLEPIDACEAE Tiegh. ex Kuijt Total: 12

Antidaphne Poep & Endl. (1838) 8Central and South America,

Caribbean, MexicoKuijt, 1965, 1988

Eubrachion Hook. (1846) 2

Jamaica, Dominican Republic, Puerto

Rico, Brazil, Argentina, Uruguay,

Venezuela

Kuijt, 1988

Lepidoceras Hook. (1846) 2 Chile Kuijt, 1988

VISCACEAE Miers 1802 Total: 501

Arceuthobium M. Bieb (1819) 26 North America, Europe, Asia, Africa

Kuijt, 1960; Scharpf, 1963 ; Hull and

Leonard, 1964; Hawksworth and

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

Korthalsella Tiegh. (1896) 10Africa, Madagascar, Himalayas to

Japan, Australia, New ZealandStevenson, 1934 ; Danser, 1937

Notothixos Oliv. (1864) 8 Sri Lanka, Southeast Asia, Australia Mistletoe, no direct reference

Phoradendron Nutt. (1848) 234 North and South AmericaHawksworth, 1966 ; Kuijt, 1994; Fineran

and Calvin, 2000; Kuijt, 2003

Viscum L. (1753) 150 Temperate and tropical Old World Kuijt, 1986

44

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

Anthobolus R. Br.

Exocarpos Labill.

Omphacomeria (Endl.) A. DC.

Tribe Amphorogyneae Stauffer ex Stearn

Amphorogyne Stauffer & Hürl.

Choretrum R. Br.

Daenikera Hürl. & Stauffer

Dendromyza Danser

Dendrotrophe Miq.

Dufrenoya Chatin

Leptomeria R. Br.

Phacellaria Benth.

Spirogardnera Stauffer

Tribe Santaleae A, DC.

(syn. Osyrideae Rchb.)

Acanthosyris (Eichl.) Grieseb.

Buckleya Torr.

Cervantesia Ruiz & Pav.

Colpoon P. J. Bergius

Comandra Nutt.

Geocaulon Fernald

Jodina Hook. & Arn. ex Meissn.

Kunkeliella Stearn

Mida A. Cunn. ex Endl.

Myoschilos Ruiz & Pav.

Nanodea Banks ex C. F. Gaertn.

Nestronia Raf.

Okoubaka Pellegr. & Normand

Osyris L.

Pyrularia Michx.

Rhoiacarpos A. DC.

Santalum L.

Scleropyrum Arn.

Tribe Thesieae Rchb.

Arjona Cav.

Osyridocarpus A. DC.

Quinchamalium Molina

Thesidium Sonder

Thesium L.

Eremolepidaceae Tiegh. ex Kuijt

Antidaphne Poepp. & Endl.

Eubrachion Hook.

Lepidoceras Hook.

Viscaceae Miers.

Arceuthobium M. Bieb

Dendrophthora Eichl.

Ginalloa Korth.

Korthalsella Tiegh.

Notothixos Oliv.

Phoradendron Nutt.

Viscum L.

Opiliaceae Valeton

Agonandra Miers ex Benth.

Cansjera Juss.

Champereia Griffith

Gjellerupia Lauterb.

Lepionurus Blume.

Meliantha Pierre

Opilia Roxb.

Pentarhopalopilia Hiepko.

Rhopalopilia Pierre

Urobotrya Stapf.

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.).

Species

DLN Coll.

Number Voucher Specimen

nuclear

SSU rDNA

chloroplast

rbcL

chloroplast

matK

SANTALACEAE

Acanthosyris falcata 4053 Michael Nee 46690 JPD DLN JPD

Amphorogyne celastroides 4564 McPherson 18051 JPD N.A. JPD

Anthobolus leptomerioides 4311 Lepschi and Craven 4352 JPD JPD JPD

Arjona tuberosa 4131 Coll. V. Melzheimer, N.V. DLN — —

Arjona tuberosa 4566 Coll. J. Puntieri, N.V. — JPD JPD

Buckleya distichophylla 2735 Coll. L. J. Musselman, N.V. X16598 DLN JPD

Cervantesia tomentosa 4273 L. J. Dorr & L. C. Barnett 6941 JPD DLN JPD

Choretrum pauciflorum 4222 Lepschi, Lally & Murray 4237 DLN DLN JPD

Colpoon compressum 4084 Nickrent, Steiner & Wolfe 4084 DLN DLN JPD

Comandra umbellata 2739 Coll. G. Tonkovitch, N.V. L24772 DLN DLN

Daenikera corallina 4876 Munzinger 2054 JPD JPD JPD

Dendromyza sp. 4466 Nickrent, Kierang, & Sape 4466 DLN DLN JPD

Dendromyza sp. 4483 Nickrent, Pop & Kairo 4483 N.A. JPD N.A.

Dendrotrophe varians 2827 Nickrent 2827 L24087 DLN —

Dendrotrophe varians 4014 Nickrent & Calvin 4014 — — JPD

Dufrenoya sphaerocarpa 2754 Coll. G. G. Hambali, N.V. AF039071 DLN JPD

Exocarpos aphyllus 3094 Coll. A. Markey, N.V. JPD DLN JPD

Exocarpos bidwillii 2745 Coll. B. Molloy, N.V. L24142 JPD DLN

Geocaulon lividum 3047 Coll. J. Fetzner, N.V. AF039072 DLN JPD

Jodina rhombifolia 4052 Michael Nee 46673 DLN DLN DLN

Table 3 (continued)

Species

DLN Coll.


nuclear

SSU rDNA

chloroplast

rbcL

chloroplast

matK

SANTALACEAE (cont.)

Kunkeliella subsucculenta 4374 Coll. A. Santos Guerra, N.V. DLN DLN JPD

Leptomeria aphylla 4609 Lepschi & Whalen 4875 N.A. N.A. JPD

Leptomeria spinosa 3081 Coll. A. Markey, N.V. JPD DLN JPD

Mida salicifolia 4233 Ogle 3413 DLN DLN JPD

Myoschilos oblonga 4504 Coll. R. Vidal Russell, N.V. JPD JPD JPD

Nanodea muscosa 4893 Coll. L. Collado, N. V. JPD JPD JPD

Nestronia umbellula 2736 Coll. L. J. Musselman, N.V. L24399 DLN JPD

Okoubaka aubrevillei 4173 Cheek 6007 N.A. DLN DLN

Omphacomeria acerba 4221 Lepschi & Murray 4213 DLN DLN JPD

Osyridocarpos schimperianus 4110 Nickrent 4110 DLN DLN JPD

Osyris quadripartida 4062 Nickrent, Aparicio & Sanchez García 4062 JPD JPD AY042623

Phacellaria compressum 4911 J. F. Maxwell 91-242 N.A. N.A. JPD

Pyrularia pubera 2737 Coll. L. J. Musselman, N.V. L24415 DLN JPD

Quinchamalium dombeyi 4283 Landrum 8087, MO 04628132 DLN N.A. JPD

Quinchamalium chilensis 4503 R. Vidall Russell, N.V. JPD N.A. JPD

Rhoiacarpos capensis 4117 Nickrent & Marx 4117 DLN DLN JPD

Santalum album 2734 Coll. R. Narayana, N.V. L24416 L26077 AY042650

Santalum mcgregorii 4499 Nickrent & Beko 4499 JPD JPD JPD

Scleropyrum pentandrum 4347 Suddee, Paton, Jonganurak, and

Chamchurmroon 1007

JPD DLN JPD

Thesidium fragile 4102 Nickrent & Wolfe 4102 JPD JPD JPD

Thesium fruticosum 4115 Nickrent & Brink 4115 JPD JPD JPD

Thesium impeditum 2845 Coll. K. Steiner, N.V. L24423 DLN DLN

Table 3 (continued)

Species

DLN Coll.


nuclear

SSU rDNA

chloroplast

rbcL

chloroplast

matK

EREMOLEPIDACEAE

Antidaphne viscoidea 2730 Coll. S. Sargent, N.V. L24080 L26068 JPD

Eubrachion ambiguum 2699 Nickrent, D. Clark & P. Clark 2699 L24141 L26071 JPD

Lepidoceras chilense 4065 Marticorena & Rodríguez 10043 DLN DLN JPD

VISCACEAE

Arceuthobium verticilliflorum 2065 Nickrent & A. Flores C. 2065 L25700 or

L24042

L26067 N.A.

Dendrophthora clavata 2182 Coll. M Melampy, N.V. L24086 L26069 N.A.

Ginalloa arnottiana 2982 Beaman 9074 L24144 L26070 JPD

Korthalsella lindsayi 2740 Coll. B. Molloy, N.V. L24150 L26073 JPD

Notothixos leiophyllus 2785 Nickrent 2785 L24402 DLN N.A.

Phoradendron californicum 2689 Coll. J. Paxton, N.V. AF039070 DLN N.A.

Viscum articulatum 2812 Nickrent 2812 L24427 DLN —

Viscum articulatum 2782 Nickrent 2782 — — JPD

OPILIACEAE

Agonandra macrocarpa 2764 Nickrent & Olson 2764 L24079 DLN DLN

Cansjera leptostachya 2815 Nickrent 2815 L24084 DLN DLN

Champereia manillana 3014 Coll. W. Forstreuter, N.V. L24746 DLN DLN

Lepionurus sylvestris 2879 Coll. G. Hambali, N.V. DLN DLN DLN

Opilia amentacea 2816 Nickrent 2816 L24407 or

U42790

L26076 DLN

Table 4: Optimal models of molecular evolution chosen using hierarchical Likelihood Ratio Tests (hLRT) and the second order

Akaike Information Criterion (AICc) implemented in Modeltest and MrModeltest. When alternative models were chosen with

different model hierarchies in MrModeltest, both are reported. Models chosen in both programs were identical, except for matK, in

which TVM+ was chosen with both hLRT and AICc in Modeltest when all 56 possible models implemented in PAUP* were

examined. This model was used in the ML analysis for this matK. The models chosen for the three-gene and matK partitions remained

the same when Phacellaria was excluded.

Data Partition Total characters

Variable

characters hLRT

AICc (all

characters)

AICc (variable

characters) Chosen Model

Three-gene 4516 1691 GTR+I+ GTR+I+ GTR+I+ GTR+I+

nuclear SSU 1825 414 GTR+I+ GTR+I+ GTR+I+ GTR+I+

matK 1258 830 GTR+ /TVM+GTR+I+ /

TVM+I+

GTR+I+ /

TVM+I+

GTR+ (BI)/

TVM+ (ML)

matK Pos1 420 274 GTR+ GTR+ GTR+ GTR+

matK Pos2 419 248 GTR+ GTR+I+ GTR+I+ GTR+

matK Pos3 419 308 GTR+ GTR+I+ GTR+I+ GTR+

rbcL 1428 447 GTR+I+ GTR+I+ GTR+I+ GTR+I+

rbcL Pos1 476 90 GTR+I+ GTR+I+ GTR+I+ GTR+I+

rbcL Pos2 476 50 JC+ /JC+I F81+ F81+ JC+

rbcL Pos3 476 307 GTR+ GTR+ GTR+ GTR+

49

Table 5: Revised classification of santalaceous genera based on phylogenetic results inthis study.

Viscaceae Miers

Arceuthobium M. Bieb

Dendrophthora Eichl.

Ginalloa Korth.

Korthalsella Tiegh.

Notothixos Oliv.

Phoradendron Nutt.

Viscum L.

Amphorogynaceae

Amphorogyne Stauffer & Hürl.

Choretrum R. Br.

Daenikera Hürl. & Stauffer

Dendromyza Danser

Dendrotrophe Miq.

Dufrenoya Chatin

Leptomeria R. Br.

Phacellaria Benth.

Spirogardnera Stauffer

Santalaceae R.Br.

Antidaphne Poepp. & Endl.

Colpoon P. J. Bergius

Eubrachion Hook.

Exocarpos Labill.

Lepidoceras Hook.

Myoschilos Ruiz & Pav.

Nestronia Raf.

Omphacomeria (Endl.) A.DC.

Osyris L.

Rhoiacarpos A.DC.

Santalum L.

Pyrulariaceae

Acanthosyris (Eichl.) Grieseb.

Cervantesia Ruiz & Pav.

Jodina Hook. & Arn. ex Meissn.

Okoubaka Pellegr. & Normand

Pyrularia Michx.

Scleropyrum Arn.

Nanodeaceae

Mida A. Cunn. ex Endl.

Nanodea Banks ex C. F. Gaertn.

Arjonaceae

Arjona Cav.

Quinchamalium Molina

Thesiaceae

Buckleya Torr.

Kunkeliella Stearn

Osyridocarpus A. DC.

Thesidium Sonder

Thesium L.

Comandraceae

Comandra Nutt.

Geocaulon Fernald

Opiliaceae Valeton

Agonandra Miers ex Benth.

Anthobolus R. Br.

Cansjera Juss.

Champereia Griffith

Gjellerupia Lauterb.

Lepionurus Blume.

Meliantha Pierre

Opilia Roxb.

Pentarhopalopilia Hiepko.

Rhopalopilia Pierre

Urobotrya Stapf.

Figure 1: Summary of phylogenetic relationships among the families of Santalales [afterNickrent et al. (1998), Nickrent and Malecot (2000; 2001) and Malecot et al. (2004)].

Figure 2: Floral diversity in Santalaceae. This plate illustrates some of the range in floralmorphology in Santalaceae s. lat. Species (and photographer) names for each image aregiven 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 permissionand their respective owners retain copyright.

Figure 3: Nuclear SSU rDNA MP strict consensus of 3211 trees (length=1135). BSsupport values greater than 50% are shown above branches (1000 replicates; MaxTreeslimit of 100 trees per BS replicate) and Bayesian posterior probabilities greater than 50%(5+5 million generations combined, 50 000 generation burn-in discarded for each run) areshown 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 greaterthan 50%. Consistency index (CI) = 0.4714, homoplasy index (HI) = 0.5286, CIexcluding uninformative characters = 0.3664, HI excluding uninformative characters =0.6336, retention index (RI) = 0.5979 and rescaled consistency index (RC) = 0.2818.

Figure 4: Maximum likelihood (ML) phylogram from the nuclear SSU rDNA datapartition analyzed under the GTR+I+ model of molecular evolution (nucleotidefrequencies of A=0.25210, C=0.19810, G=0.27050 and T=0.27930; substitution ratematrix of A C: 1.664200, A G: 4.398100, A T: 3.106500, C A: 1.664200, C G:1.025000, C T: 12.216500, G A: 4.398100, G C: 1.025000, G T: 1.000000, T A:3.106500, T C: 12.216500, T G: 1.000000; proportion of invariable sites = 0.6182;gamma distribution shape parameter (alpha) = 0.5786). The “*” indicates clades alsofound in three-gene analyses. The -lnL score of this single most optimal tree was8722.54101.

Figure 5: rbcL MP strict consensus of 76 trees (length=1053). BS support valuesgreater than 50% are shown above branches (1000 replicates) and Bayesian posteriorprobabilities (5+5 million generations combined, 50 000 generation burn-in discarded foreach run) greater than 50% are shown below branches for those clades also sampled inthe combined Bayesian analyses. See Appendix IV-2 for the full Bayesian topology withall posterior probabilities greater than 50%. The arrows highlight Arjona, whose positionvaries in other analyses, and Thesium impeditum, who is not sister to its congener withthis 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.

Figure 6: ML phylogram from the chloroplast rbcL data partition analyzed under theGTR+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 A C: 1.402200, A G: 2.389100,A T: 0.520800, C A: 1.402200, C G: 0.627400, C T: 3.150100, G A: 2.389100,G C: 0.627400, G T: 1.000000, T A: 0.520800, T C: 3.150100, T G: 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, andThesium impeditum, who is not sister to its congener in this data partition. Note the longbranches found in these two taxa. The “*” indicates clades also found in three geneanalyses. The –lnL score of the single most optimal tree was 8136.63960.

Figure 7: matK MP strict consensus of 31 trees (length=2513). BS support valuesgreater than 50% are shown above branches (1000 replicates) and Bayesian posteriorprobabilities (5+5 million generations, 50 000 generation burn-in discarded for each run)greater than 50% are shown below branches for those clades also sampled in thecombined Bayesian analyses. See Appendix IV-6 for the full Bayesian topology and allposterior probabilities greater than 50%. The arrow highlights the position ofKorthalsella, which is not sister to Ginalloa, and varies in other analyses of this datapartition. CI = 0.5312, HI = 0.4688, CI excluding uninformative characters = 0.4653, HIexcluding uninformative characters = 0.5347, RI = 0.6490, and RC = 0.3448.

Figure 8: ML phylogram from the matK data partition analyzed without Phacellaria

under the TVM+ model of molecular evolution (nucleotide frequencies of A=0.31010,C=0.15800, G=0.15790 and T=0.37400; substitution rate matrix of A C: 1.375300,A G: 1.740400, A T: 0.264300, C A: 1.375300, C G: 0.537500, C T: 1.740400,G A: 1.740400, G C: 0.537500, G T: 1.000000, T A: 0.264300, T C: 1.740400,T G: 1.000000; shape parameter of the gamma distribution (alpha) =1.1338). The –loglikelihood score of this single most optimal tree was 13988.94084. See Appendix IV-4for ML analysis with Phacellaria. The arrow highlights the position of Korthalsella,which varies in other analyses of this data partition. The “*” indicates clades also foundin three gene analyses.

Figure 9: Three-gene MP strict consensus of 2 trees (length=4798). BS support valuesgreater than 50% are shown above branches (1000 replicates) and Bayesian posteriorprobabilities greater than 50% (5+5 million generations combined, 50 000 generationburn-in discarded for each run) are shown below branches for those clades also sampledin 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 whenPhacellaria is included. Circled numbers indicate the eight well-supported clades, onwhich the revised classification of Santalaceae s. lat. is based. The organization of taxain 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 allposterior probabilities greater than 50%. CI = 0.5102, HI = 0.4898, CI excludinguninformative characters = 0.4292, HI excluding uninformative characters = 0.5708, RI =0.6267, and RC = 0.3198.

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 A C: 1.519300,A G: 2.190700, A T: 0.678000, C A: 1.519300, C G: 0.532900, C T: 3.201500,G A: 2.190700, G C: 0.532900, G T: 1.000000, T A: 0.678000, T C: 3.201500,T G: 1.000000; proportion of invariable sites = 0.4468; gamma distribution shapeparameter (alpha) = 0.7908). The –log likelihood score of this tree was 32132.07152.Brackets indicate familial circumscription according to the proposed classification in thisstudy; “‡” marks eremolepidaceous taxa. See Appendix IV-8 for ML results withPhacellaria.

60

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73

APPENDIX I

Primer Sequences

Nucleotide primers used in PCR amplification and cycle sequencing reactions.

Name Length Position Primer Sequence 5'-3'

nuclear SSU12F 20 12 - 21 TCC TGC CAG TAS TCA TAT GC1131R 21 1130-1150 CAA TTC CTT TAA GTT TCA GCC1769R 19 1769-1787 CAC CTA CGG AAA CCT TGT T

rbcL

1F 20 1-20 ATG TCA CCA CAA ACA GAR AC3'R 20 rbcL-accd spacer TAG TAA AAG ATT GGG CCG AG

matK

78F 20 73-92 CAG GAG TAT ATT TAT GCA CT834F 20 834-853 GCA TTA TGT TAG GTA TCA AG833R 21 833-853 CTT GAT ACC TAA CAT AAT GCA1420R 21 1291-1310 TCG AAG TAT ATA CTT TAT TCG

PCR Reaction Reagents

Typical amounts and concentrations of PCR reagents used to amplify the target DNA

sequences.

PCR reagent

Initial

concentration Volume (µL)Final

concentration

Buffer 10 X 2.5 1 XMgCl2 25 µM 1.5 1.5 µMdNTPs 2.5 mM each 0.5 0.05 mM

Forward primer 10 µm 1.0 0.4 µMReverse primer 10 µm 1.0 0.4 µM

Taq ~0.6 units/ L 1.0 ~ 2.5 units / 100 LGenomic DNA 5-10 ng/ L 1.0 0.2-0.4 ng/ L

H2O - 16.5 0.66 µMTotal 25.0

74

Appendix I (continued)

Thermal Cycle Parameters

Nuclear SSU rDNA

Temperature (°C) 94 94 48 72 94 52 72 72 4Time (min:sec) 5:00 1:00 1:00 2:00 0:30 0:30 1:30 10:00 continuousNumber of Cycles X 5 X 33

Chloroplast rbcL


Chloroplast matK


75

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;

[Maximum Parsimony heuristic search and bootstrap analysis.]BEGIN PAUP;

Log File=3gene.MP.log;Set Criterion=Parsimony Increase=Auto AutoInc=100

AutoClose=yes;HSearch AddSeq=random NReps=100 Hold=2;SaveTrees File=3gene.MPTrees.tre;DescribeTrees All;ConTree All / Strict;Bootstrap NReps=1000 TreeFile=3gene.BSTrees.tre /

AddSeq=Simple Hold=1;END;

76

Appendix II: PAUP* commands (continued)

[Maxiumum Likelihood heuristic search.]BEGIN PAUP;

Log File=3gene.ML.log;[Getting a Parsimony Starting Tree.]

Set Criterion=Parsimony Increase=Auto AutoInc=100AutoClose=yes;HSearch AddSeq=Simple;Set Criterion=Likelihood;

[Model of molecular evolution chosen in Modeltest.]Lset Base=(0.2770 0.1862 0.2235) Nst=6 Rmat=(1.5193

2.1907 0.6780 0.5329 3.2015) Rates=GammaShape=0.7908 Pinvar=0.4468;

HSearch Start=1 MulTrees=yes;DescribeTrees All / Plot=Both BrLens=yes;SaveTrees File=3gene.ML.tre BrLens=yes;

End;

77

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;

[Models of molecular evolution chosen in MrModeltest.]prset applyto=(1,2,4,5,6,7) statefreqpr=dirichlet(1,1,1,1);prset applyto=(3) statefreqpr=fixed(equal);lset applyto=(1,2) nucmodel=4by4 nst=6 rates=invgamma

ngammacat=4;lset applyto=(3) nucmodel=4by4 nst=1 rates=gamma

ngammacat=4;lset applyto=(4,5,6,7) nucmodel=4by4 nst=6 rates=gamma

ngammacat=4;

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;

78

APPENDIX III

Typical Log-Likelihood Plots from Bayesian Analyses

Full log likelihood plot of the fully partitioned three-gene analysis

without Phacellaria

-50000

-48000

-46000

-44000

-42000

-40000

-38000

-36000

-34000

-32000

-30000

0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 5000000

Number of Generations

Ln

Lik

elih

oo

d

Log likelihood plot for burn-in region of the fully partitioned three-

gene analysis without Phacellaria

-50000

-48000

-46000

-44000

-42000

-40000

-38000

-36000

-34000

-32000

-30000

0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000

Number of Generations

Ln

Lik

elih

oo

d

79

APPENDIX IV

Supplemental Phylogenetic Trees

Supplemental Tree IV-1: Nuclear SSU Bayesian majority rule consensus from twoidentical BI runs with 5000000 generations, each sampled every 1000 generations. Treesfrom the first 50000 generations (50 trees) were discarded as burn-in. Parameters wereestimated under the GTR+I+ model of molecular evolution. Posterior probabilitiesfrom each run separately are below the branches and from both runs combined are abovethe branches. Values in boldface italic type represent clades not recovered in the MPstrict consensus tree for this data partition.

Supplemental Tree IV-2: rbcL Bayesian majority rule consensus from two identical BIruns with 5000000 generations each sampled every 1000 generations. Trees from thefirst 50000 generations (50 trees) were discarded as burn-in. Data were partitioned bycodon position and model parameters for the first, second and third positions wereestimated independently (i.e. unlinked) under the GTR+I+ , JC+ , and GTR+ modelsof molecular evolution, respectively. Posterior probabilities from each run separately areshown below the branches and from both runs combined are above the branches. Valuesin boldface italic type represent clades not recovered in the MP strict consensus tree forthis data partition.

Supplemental Tree IV-3: matK MP strict consensus of 188 trees of length 2519 whichinclude Phacellaria. This is a 6-fold increase in the number of trees and a tree lengthincrease of only six steps from analyses that excluded Phacellaria. There is a dramaticloss of resolution in Amphorogyneae/Amphorogynaceae, but the topology and supportvalues of other groups remain unchanged. BS support values greater than 50% are shownabove branches (1000 replicates). CI = 0.5304, HI = 0.4696, CI excluding uninformativecharacters = 0.4642, HI excluding uninformative characters = 0.5358, RI = 0.6491, andRC = 0.3442.

Supplemental Tree IV-4: ML phylogram from the matK data partition analyzed withPhacellaria under the TVM+ model of molecular evolution (-lnL = 14021.11728). Thistree recovers the three distinct clades in Amphorogyne, but places Phacellaria in apolytomy with the root parasites (Choretrum + Leptomeria and Amphorogyne +Daenikera), a relationship not seen when Phacellaria is excluded.

Supplemental Tree IV-5: matK Bayesian majority rule consensus includingPhacellaria, from two identical BI runs with 5000000 generations each sampled every1000 generations. Trees from the first 50000 generations (50 trees) were discarded asburn-in. Data were partitioned by codon position and model parameters for all threepositions were estimated independently (i.e. unlinked) under the GTR+ model ofmolecular evolution. Posterior probabilities from each run separately are shown belowthe branches and from both runs combined are above the branches.

Supplemental Tree IV-6: matK Bayesian majority rule consensus. This is a similaranalysis to Supplemental Tree IV-5, but without Phacellaria. Two identical BI runs with5000000 generations each were sampled every 1000 generations. Trees from the first50000 generations (50 trees) were discarded as burn-in. Data were partitioned by codonposition and model parameters for all three positions were estimated independently (i.e.unlinked) under the GTR+ model of molecular evolution. Posterior probabilities fromeach run separately are shown below the branches and from both runs combined areabove the branches. Values in boldface italic type represent clades not recovered in theMP strict consensus tree for this data partition.

Supplemental Tree IV-7: Three-gene strict consensus of 12 trees of length 4804 whichinclude Phacellaria. Phacellaria dramatically reduces the level of resolution inAmphorogyneae/Amphorogynaceae, but the topology of other groups remains similar toother analyses of the three-gene dataset. BS support values greater than 50% are shownabove branches (1000 replicates). CI = 0.5098, HI = 0.4902, CI excluding uninformativecharacters = 0.4287, HI excluding uninformative characters = 0.5713, RI = 0.6268, andRC = 0.3196.

Supplemental Tree IV-8: Three-gene ML phylogram analyzed with Phacellaria underthe GTR+I+ model of molecular evolution. Two equally optimal trees with -lnL scoreof 32165.54345 were found in the heuristic search. These trees differ only in theplacement of Phacellaria (plotted with a dashed line in both positions). Relative branchlengths in both trees are the same.

Supplemental Tree IV-9: Three-gene Bayesian majority rule consensus partitioned bygene without Phacellaria, from two identical BI runs with 5000000 generations eachsampled every 1000 generations. Trees from the first 50000 generations (50 trees) werediscarded as burn-in. Model parameters were estimated independently (i.e. the partitionswere unlinked) for each gene under the GTR+I+ model of molecular evolution.Posterior probabilities from each run separately are shown below the branches and fromboth runs combined are above the branches.

Supplemental Tree IV-10: Three-gene Bayesian majority rule consensus fullypartitioned with Phacellaria, from two identical BI runs with 5000000 generations eachsampled every 1000 generations. Trees from the first 50000 generations (50 trees) werediscarded as burn-in. Data were fully partitioned by genes, and for the protein codinggenes, by codon position. Model parameters were estimated independently for eachpartition (i.e. the partitions were unlinked). See Table 4 for the models of molecularevolution used for each partition. Posterior probabilities from each run separately areshown below the branches and from both runs combined are above the branches.

Supplemental Tree IV-11: Three-gene Bayesian majority rule consensus fullypartitioned without Phacellaria, from two identical BI runs with 5000000 generationseach 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 proteincoding genes, by codon position. Model parameters were estimated independently foreach partition (i.e. the partitions were unlinked). See Table 4 for the models of molecularevolution used for each partition. Posterior probabilities from each run separately areshown below the branches and from both runs combined are above the branches. Valuesin boldface italic type represent clades not recovered in the MP strict consensus tree forthis data partition.

91

VITAE

Graduate School

Southern Illinois University

JOSHUA P. DER Date of Birth: 6 August 1979

1846 Pine Street # 1, Murphysboro, IL 62966

15071 Neece Street, Westminster, CA 92683

Humboldt State University

Bachelor of Science, Biology and Botany, May 2003

Special Honors and Awards:

Omicron Delta Kappa, member, May 2003 to Present

Outstanding Student of the Year, Humboldt State University, May 2003

Who’s Who Among Students in American Universities and Colleges, May 2003

Thesis Title:

Molecular Phylogenetics and Classification of Santalaceae

Major Professor:

Dr. Daniel L. Nickrent

Publications and Abstracts:Nickrent, D. L., J. P. Der and F. E. Anderson. 2005. Discovery of the photosynthetic

relatives of the “Maltese mushroom” Cynomorium. BMC Evolutionary Biology,

5:38. doi:10.1186/1471-2148-5-38.

Der, J. P. and D. L. Nickrent. 2005. Molecular Phylogeny and Classification of

Santalaceae. Midwestern Ecology and Evolution Conference, Southern Illinois

University, Carbondale, Illinois USA.

Nickrent, D. L. and J. P. Der. 2004. Santalaceae: phylogeny, taxonomy, and

biogeography. Botany 2004, Snowbird, Utah USA.

Der, J. P. and D. L Nickrent. 2004. Phylogeographic investigations of parasitic plants in

Santalaceae. Midwestern Ecology and Evolution Conference, University of Notre

Dame, Bend, Indiana USA.

Der, J. P., Jordan, S. and Vega, V. 2003. Investigation of Wind Pollination in the

Humbodt Bay Wallflower (Erysimum menziesii ssp. eurekense) at the Lanphere-

Christensen Dunes. Final report submitted to Humboldt Bay National Wildlife

Refuge, May 2003.

Documents

Molecular Phylogenetics and Classification of Santalaceae