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

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 i

supported as sister to some members of a polyphyletic tribe Santaleae + Eremolepidaceae. This clade, which contains the type species of Santalaceae (Santalum album), is recognized here as Santalaceae sensu stricto and includes all three genera of Eremolepidaceae, Exocarpos and Omphacomeria, and six genera from tribe Santaleae. 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 iii

Kolb, who authorized after-hours access to the College of Mass Communication/ Information Technology’s New Media Center. A special thanks also to Eric Rowan, whose cooperation and flexibility made it possible to simultaneously use twelve of the 2.0 GHz dual processor PowerMac G5 computers in New Media Center. Without these 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 v

APPENDIX I Primer sequences, PCR reagents, and Thermal Cycle Parameters .............................. 73 APPENDIX II Example PAUP* and MrBayes Command Blocks..................................................... 75 APPENDIX III Log Likelihood Plots from Bayesian Analyses .......................................................... 78 APPENDIX IV Supplemental Phylogenetic Trees.............................................................................. 79 Supplemental tree IV-1: Nuclear SSU rDNA BI majority rule consensus............. 80 Supplemental tree IV-2: rbcL BI majority rule consensus .................................... 81 Supplemental tree IV-3: matK MP strict consensus with Phacellaria ................... 82 Supplemental tree IV-4: matK ML phylogram with Phacellaria .......................... 83 Supplemental tree IV-5: matK BI majority rule consensus with Phacellaria ........ 84 Supplemental tree IV-6: matK BI majority rule consensus without Phacellaria ... 85 Supplemental tree IV-7: Three-gene MP strict consensus with Phacellaria.......... 86 Supplemental tree IV-8: Three-gene ML phylogram with Phacellaria .................. 87 Supplemental tree IV-9: Three-gene BI majority rule consensus without Phacellaria, partitioned by gene ....................................................................... 88 Supplemental tree IV-10: Three-gene BI majority rule consensus with Phacellaria, partitioned by gene and codon .......................................................................... 89 Supplemental tree IV-11: Three-gene BI majority rule consensus without Phacellaria, partitioned by gene and codon ...................................................... 90 VITAE .......................................................................................................................... 91

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LIST OF TABLES Table Table 1:

Page 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

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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, halfinferior 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,

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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

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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

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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

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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.

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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

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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

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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 threegene 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

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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

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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 Metropoliscoupled 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 burnin, 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).

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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%) parsimonyinformative 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 codonpartitioned analyses) are mapped on this tree. The ML analysis implemented the

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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 %) parsimonyinformative 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.

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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 %) parsimonyinformative 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

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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.

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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

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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.

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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.

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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, allinclusive 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 subfamilial/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,

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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

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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

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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

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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

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tropical and southern Africa and Thesium reaches its peak diversity in southern Africa, but extends throughout the Old World, Australia and South America. Buckleya has a distribution unlike all the other members of this clade, and is found disjunct between North America and China and Japan. There are four recognized species (Carvell and Eshbaugh, 1982; Li, Boufford, and Donoghue, 2001) which have an interesting biogeographic relationship. Two species pairs are formed in which the North American taxon is sister to one of the Chinese species, while the other Chinese species is sister to the Japanese species (Li, Boufford, and Donoghue, 2001). The affinity with and basal position of Buckleya in Clade 7 has interesting implications for the biogeography of this group. This clade is recognized here at the family level and the following clade definition is given: Thesiaceae are the least inclusive clade that contains Buckleya distichophylla and Thesium alpinum. The type species is Thesium alpinum L. (Species Plantarum 1: 207. 1753). ARJONACEAE Arjona and Quinchamalium form a well-supported clade (100% BS and PP, Clade 6, Figure 9). These two genera are quite speciose and morphologically similar. The 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.

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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

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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

32

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). This clade is recognized here at the family level and the following clade definition is given: Nanodeaceae are the least inclusive clade that contains Nanodea muscosa and Mida salicifolia. The type species is Nanodea muscosa Banks ex C. F. Gaertn. (De 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.

33

This whole group is characterized by having a woody habit and a large drupaceous fruit with a stony pit. Bhatnagar and Sabharwal (1969) noted the mesocarpic origin of the stony layer in the fruit of Jodina and suggested the term “pseudodrupe” as the fruit type (in contrast to endocarpic origin in a true drupe). Stauffer clearly recognized the affinities of the genera in each of these sub-clades in his Santalales 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). This clade is recognized here at the family level and the following clade definition is given: Pyrulariaceae are the least inclusive clade that contains Pyrularia pubera and Cervantesia tomentosa. The type species is Pyrularia pubera Michx. (Flora BorealiAmericana 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

34

confusion and many authors have reorganized the taxa in this traditional tribe in many different ways (Van Tieghem, 1896; Pilger, 1935; Rao, 1942a; Smith and Smith, 1943; Johri and Bhatnagar, 1960). The analyses presented in this study further corroborate the polyphyletic nature of these taxa. Several generic groups have been segregated out of the 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. This clade is recognized here at the family level and the following clade definition is given: Santalaceae s. str. are the least inclusive clade that contains Santalum album, 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

35

sampled) was outlined and described by Stauffer (1969). He recognized the distinctiveness of these taxa relative to other members of tribe Santaleae, and could distinguish them by the presence of peculiar characteristics of the anthers and placenta. Specifically, anthers are born on short-stout or nearly absent filaments where each thecum transversely dehisces independently (in contrast to many other Santalaceae where dehiscence occurs along a single longitudinal slit common to the two locules of a theca). In addition, the placenta is short-stout to nearly absent and more strongly associated with the ovary tissue. The ovules are borne in pockets at the base of the ovarian locule (in contrast to the stalked placental column with or without basal ovarian pockets in other Santalaceae). This group includes genera with a diversity of growth forms and levels of parasitism. Genera included in this clade are woody root parasitic shrubs, woody dendroparasites (sensu Stauffer, i.e. clambering, liana-like shrubs which are obligate root and opportunistic stem parasites, in contrast to the use of this term by Macklin, 2002, who uses it as synonymous with mistletoe) and both woody and herbaceous advanced mistletoes (i.e. exclusively aerial parasitic). The mistletoe habit is considered a highly derived trait. There is a distinct trend toward increased levels of parasitism and the associated loss of foliar leaves in this clade (Stauffer and Hürlimann, 1957). This is best 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

36

genera found in dry climates also tend to show a reduction of the leaves (e.g. Choretrum, Leptomeria and Spirogardnera), which parallels this reduction of leaves in the advanced 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

37

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. This clade is recognized here at the family level and the following clade definition is given: Amphorogynaceae are the least inclusive clade that contains Amphorogyne spicata, Leptomeria acida and Dendrotrophe varians. The type species is Amphorogyne spicata Stauffer & Hürl. (Vierteljahrsschr. Naturf. Ges. Zürich 102: 349. 1957). VISCACEAE 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

38

family. As such, Viscaceae remains intact and unchanged and classification should follow that of previous workers. A new clade definition is not given here. 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

39

assemblage relative to Viscaceae and Eremolepidaceae. Phylogenetic analyses of DNA sequences from three genes and nearly complete taxon sampling within Santalaceae and related families reveal eight well-supported clades, which represent more discreet and potentially better diagnosable units. These eight clades are recognized at the family level and are Comandraceae, Thesiaceae, Arjonaceae, Nanodeaceae, Pyrulariaceae, Santalaceae sensu stricto, Amphorogyneae and Viscaceae.

40

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

Genus SANTALACEAE R.Br. (1810) Acanthosyris (Eichl.) Griseb. (1957) Amphorogyne Stauffer & Hürl. (1957) Anthobolus R. Br. (1810) Arjona Cav. (1798) Buckleya Torr. (1843) Cervantesia Ruiz & Pav. (1794) Choretrum R. Br. (1810) Colpoon P. J. Bergius (1767) including Fusanus L. (1774)

Number Geographic distribution of species Total: 446 5 Temperate South America 3 New Caledonia 3 Australia 10 Tropical & temperate South America 4 Eastern United States and East Asia 4 Andean South America 6 Australia 1

Comandra Nutt. (1818)

1

Daenikera Hürl. & Stauffer (1957) Dendromyza Danser (1940) including Cladomyza Danser (1940) Dendrotrophe Miq. (1856) including Henslowia Blume Dufrenoya Chatin (1860) including Hylomyza Danser (1940) Exocarpos Labill. (1800) including Elaphanthera N. Hallé (1988) Geocaulon Fernald (1928) Jodina Hook. & Arn. ex Meissn. (1837) Kunkeliella Stearn (1972)

1 21 4 11 26 1 1 4

South Africa North America, Europe and the Mediterranean New Caledonia Southeast Asia, Indomalaysia, New Guinea Himalayas to the Philippines and Malaysia Indo-Malaysia

Documented parasitism Barroso, 1968 Stauffer and Hürlimann, 1957 Stauffer, 1959 Kuijt, 1969; Pilger 1935 Kusano, 1902; Piehl, 1965a — Hewson and George, 1984 Bean, 1990 Hedgecock, 1915; Piehl, 1965b Hürlimann and Stauffer, 1957 Danser, 1940 Macklin and Parnell, 2000, 2002 Macklin and Parnell, 2000, 2002

Benson, 1910; Stauffer, 1959; Fineran, 1962, 1963a-e, 1965a-b, 1979; Fineran and Bulluck, 1979; Philipson, 1959 Alaska and Canada Moss, 1926 Southern Brazil, Uruguay, Argentina Bhatnagar and Sabharwal, 1969 Canary Islands Anonymous, 2001 Southeast Asia, Malaysia to Hawaii

Table 1 (continued) Genus SANTALACEAE (continued) Leptomeria R. Br. (1810)

Number of species 17

Mida A. Cunn. ex Endl. (1837)

1

Myoschilos Ruiz & Pav. (1794)

1

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

1

Geographic distribution Australia Disjunct from New Zealand to Juan Fernandez Islands Chile Temperate South America (Patagonia, Tierra del Fuego, Falkland Islands)

Documented parasitism Herbert, 1920; Lepschi, 1999 Philipson, 1959 — Skottsberg, 1913

Nestronia Raf. (1836) including Darbya A. Gray (1846) Okoubaka Pellegr. & Normand. (1946) Omphacomeria (Endl.) A. DC. (1857) Osyridocarpus A. DC. (1894)

1

Eastern United States

Melvin, 1956

2 1 1

Osyris L. (1753)

2

Phacellaria Benth. (1880)

5

Stauffer, 1957; Veenendaal et al., 1996 Stauffer, 1959 — Ferrarini, 1950; Planchon, 1858; Pizzoni, 1906 Danser, 1939

Pyrularia Michx. (1803)

2

Quinchamalium Molina (1781) Rhoiacarpos A. DC. (1857) Santalum L. (1753) including Eucarya T. L. Mitch (1927) and Fusanus R. Br. (1774) Scleropyrum Arn. (1838) including Scleromelum K. Schum. & Lauterb. (1900)

25 1

Tropical Africa Southeast Australia Africa Europe, Mediterranean, Africa to India East India to Southern China Southeastern United States, China to Himalayas Andean South America South Africa

20

Indo-Malaysia to Australia, Hawaii

Barber, 1907a, 1907b, 1908

6

Malaysia, New Guinea, Southeast Asia, India

—

Leopold and Muller, 1983 Ruiz and Roig, 1958 —

Table 1 (continued) Genus SANTALACEAE (continued) Spirogardnera Stauffer (1968) Thesidium Sonder (1857) Thesium L. (1753) including Austroamericium Hendrych

Number of species 1 8 245

Geographic distribution Southwestern Australia (endemic) South Africa Europe, Africa, Asia, Australia, South America. Two species introduced into North America

Documented parasitism Stauffer, 1968 Hill, 1915; Visser, 1981 Mitten, 1847; Visser, 1981; Weber, 1977

EREMOLEPIDACEAE Tiegh. ex Kuijt Total: 12 Antidaphne Poep & Endl. (1838)

8

Eubrachion Hook. (1846)

2

Lepidoceras Hook. (1846) VISCACEAE Miers 1802

2 Total: 501

Arceuthobium M. Bieb (1819)

26

Dendrophthora Eichl. (1868) Ginalloa Korth. (1839)

68 5

Korthalsella Tiegh. (1896)

10

Notothixos Oliv. (1864)

8

Phoradendron Nutt. (1848)

234

Viscum L. (1753)

150

Central and South America, Kuijt, 1965, 1988 Caribbean, Mexico Jamaica, Dominican Republic, Puerto Rico, Brazil, Argentina, Uruguay, Kuijt, 1988 Venezuela Chile Kuijt, 1988 Kuijt, 1960; Scharpf, 1963 ; Hull and Leonard, 1964; Hawksworth and North America, Europe, Asia, Africa Wiens, 1972; Alosi and Calvin, 1984; Rey et al., 1991 Caribbean and South America Kuijt, 1961 Indonesia and Malaysia Mistletoe, no direct reference Africa, Madagascar, Himalayas to Stevenson, 1934 ; Danser, 1937 Japan, Australia, New Zealand Sri Lanka, Southeast Asia, Australia Mistletoe, no direct reference Hawksworth, 1966 ; Kuijt, 1994; Fineran North and South America and Calvin, 2000; Kuijt, 2003 Temperate and tropical Old World Kuijt, 1986

Table 2: Traditional classification of Santalaceae and related families. This classification is based on Pilger (1935) with modifications and additions by Danser (1955), Hewson & George (1984), Macklin (2000), Macklin and Parnel (2002), Stauffer (1959; 1968; 1969), Stauffer and Hürlimann (1957), and Stearn (1972). Classification of Viscaceae after Barlow (1964), Eremolepidaceae follows Kuijt (1988) and Opiliaceae follows Hiepko (Hiepko, 1979, 1982, 1985, 1987). Santalaceae R. Br. Tribe Anthoboleae (Dumort.) Spach 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.

44

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 SANTALACEAE Acanthosyris falcata Amphorogyne celastroides Anthobolus leptomerioides Arjona tuberosa Arjona tuberosa Buckleya distichophylla Cervantesia tomentosa Choretrum pauciflorum Colpoon compressum Comandra umbellata Daenikera corallina Dendromyza sp. Dendromyza sp. Dendrotrophe varians Dendrotrophe varians Dufrenoya sphaerocarpa Exocarpos aphyllus Exocarpos bidwillii Geocaulon lividum Jodina rhombifolia

DLN Coll. Number Voucher Specimen 4053 4564 4311 4131 4566 2735 4273 4222 4084 2739 4876 4466 4483 2827 4014 2754 3094 2745 3047 4052

Michael Nee 46690 McPherson 18051 Lepschi and Craven 4352 Coll. V. Melzheimer, N.V. Coll. J. Puntieri, N.V. Coll. L. J. Musselman, N.V. L. J. Dorr & L. C. Barnett 6941 Lepschi, Lally & Murray 4237 Nickrent, Steiner & Wolfe 4084 Coll. G. Tonkovitch, N.V. Munzinger 2054 Nickrent, Kierang, & Sape 4466 Nickrent, Pop & Kairo 4483 Nickrent 2827 Nickrent & Calvin 4014 Coll. G. G. Hambali, N.V. Coll. A. Markey, N.V. Coll. B. Molloy, N.V. Coll. J. Fetzner, N.V. Michael Nee 46673

nuclear SSU rDNA

chloroplast rbcL

chloroplast matK

JPD JPD JPD DLN — X16598 JPD DLN DLN L24772 JPD DLN N.A. L24087 — AF039071 JPD L24142 AF039072 DLN

DLN N.A. JPD — JPD DLN DLN DLN DLN DLN JPD DLN JPD DLN — DLN DLN JPD DLN DLN

JPD JPD JPD — JPD JPD JPD JPD JPD DLN JPD JPD N.A. — JPD JPD JPD DLN JPD DLN

Table 3 (continued) Species SANTALACEAE (cont.) Kunkeliella subsucculenta Leptomeria aphylla Leptomeria spinosa Mida salicifolia Myoschilos oblonga Nanodea muscosa Nestronia umbellula Okoubaka aubrevillei Omphacomeria acerba Osyridocarpos schimperianus Osyris quadripartida Phacellaria compressum Pyrularia pubera Quinchamalium dombeyi Quinchamalium chilensis Rhoiacarpos capensis Santalum album Santalum mcgregorii Scleropyrum pentandrum Thesidium fragile Thesium fruticosum Thesium impeditum

DLN Coll. Number Voucher Specimen 4374 4609 3081 4233 4504 4893 2736 4173 4221 4110 4062 4911 2737 4283 4503 4117 2734 4499 4347 4102 4115 2845

Coll. A. Santos Guerra, N.V. Lepschi & Whalen 4875 Coll. A. Markey, N.V. Ogle 3413 Coll. R. Vidal Russell, N.V. Coll. L. Collado, N. V. Coll. L. J. Musselman, N.V. Cheek 6007 Lepschi & Murray 4213 Nickrent 4110 Nickrent, Aparicio & Sanchez García 4062 J. F. Maxwell 91-242 Coll. L. J. Musselman, N.V. Landrum 8087, MO 04628132 R. Vidall Russell, N.V. Nickrent & Marx 4117 Coll. R. Narayana, N.V. Nickrent & Beko 4499 Suddee, Paton, Jonganurak, and Chamchurmroon 1007 Nickrent & Wolfe 4102 Nickrent & Brink 4115 Coll. K. Steiner, N.V.

nuclear SSU rDNA

chloroplast rbcL

chloroplast matK

DLN N.A. JPD DLN JPD JPD L24399 N.A. DLN DLN JPD N.A. L24415 DLN JPD DLN L24416 JPD JPD

DLN N.A. DLN DLN JPD JPD DLN DLN DLN DLN JPD N.A. DLN N.A. N.A. DLN L26077 JPD DLN

JPD JPD JPD JPD JPD JPD JPD DLN JPD JPD AY042623 JPD JPD JPD JPD JPD AY042650 JPD JPD

JPD JPD L24423

JPD JPD DLN

JPD JPD DLN

Table 3 (continued) Species EREMOLEPIDACEAE Antidaphne viscoidea Eubrachion ambiguum Lepidoceras chilense VISCACEAE Arceuthobium verticilliflorum Dendrophthora clavata Ginalloa arnottiana Korthalsella lindsayi Notothixos leiophyllus Phoradendron californicum Viscum articulatum Viscum articulatum OPILIACEAE Agonandra macrocarpa Cansjera leptostachya Champereia manillana Lepionurus sylvestris Opilia amentacea

DLN Coll. Number Voucher Specimen

nuclear SSU rDNA

chloroplast rbcL

chloroplast matK

2730 2699 4065

Coll. S. Sargent, N.V. Nickrent, D. Clark & P. Clark 2699 Marticorena & Rodríguez 10043

L24080 L24141 DLN

L26068 L26071 DLN

JPD JPD JPD

2065

Nickrent & A. Flores C. 2065

L26067

N.A.

2182 2982 2740 2785 2689 2812 2782

Coll. M Melampy, N.V. Beaman 9074 Coll. B. Molloy, N.V. Nickrent 2785 Coll. J. Paxton, N.V. Nickrent 2812 Nickrent 2782

L25700 or L24042 L24086 L24144 L24150 L24402 AF039070 L24427 —

L26069 L26070 L26073 DLN DLN DLN —

N.A. JPD JPD N.A. N.A. — JPD

2764 2815 3014 2879 2816

Nickrent & Olson 2764 Nickrent 2815 Coll. W. Forstreuter, N.V. Coll. G. Hambali, N.V. Nickrent 2816

DLN DLN DLN DLN L26076

DLN DLN DLN DLN DLN

L24079 L24084 L24746 DLN L24407 or U42790

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.

Total characters 4516 1825

Variable characters 1691 414

hLRT GTR+I+
GTR+I+


matK

1258

830

GTR+
/TVM+


matK Pos1 matK Pos2 matK Pos3 rbcL rbcL Pos1 rbcL Pos2 rbcL Pos3

420 419 419 1428 476 476 476

274 248 308 447 90 50 307

GTR+
GTR+
GTR+
GTR+I+
GTR+I+
JC+
/JC+I GTR+


Data Partition Three-gene nuclear SSU

AICc (all characters) GTR+I+
GTR+I+
GTR+I+
/ TVM+I+
GTR+
GTR+I+
GTR+I+
GTR+I+
GTR+I+
F81+
GTR+


AICc (variable characters) GTR+I+
GTR+I+
GTR+I+
/ TVM+I+
GTR+
GTR+I+
GTR+I+
GTR+I+
GTR+I+
F81+
GTR+


Chosen Model GTR+I+
GTR+I+
GTR+
(BI)/ TVM+
(ML) GTR+
GTR+
GTR+
GTR+I+
GTR+I+
JC+
GTR+


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

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 49

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

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Figure 2: Floral diversity in Santalaceae. This plate illustrates some of the range in floral morphology in Santalaceae s. lat. Species (and photographer) names for each image are given below. A: Arceuthobium pusillum (D. L. Nickrent); B: Amphorogyne spicata (H. U. Stauffer); C: Choretrum spicatum (H. U. Stauffer); D: Lepidoceras chilense (G. Glatzel); E: Osyris alba (T. Zumbrunn, Botanical Images Database); F: Santalum freycinetianum (G. D. Carr, Hawaiian Native Plant Genera); G: Exocarpos gaudichaudii (G. D. Carr, Hawaiian Native Plant Genera); H: Cervantesia tomentosa (P. M. Jørgensen, TROPICOS Image Library, MO); I: Nanodea muscosa (J. Puntieri); J: Quinchamalium chilense (N. Tercero-Bucardo); K: Thesium bergeri (L. J. Musselman); L: Comandra umbellata (A. H. Bazell, CalPhotos). All images are used with permission and their respective owners retain copyright.

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Figure 3: Nuclear SSU rDNA MP strict consensus of 3211 trees (length=1135). BS support values greater than 50% are shown above branches (1000 replicates; MaxTrees limit 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) are shown below branches for those clades also sampled in the combined Bayesian analyses. See Appendix IV-1 for the full Bayesian topology and all posterior probabilities greater than 50%. Consistency index (CI) = 0.4714, homoplasy index (HI) = 0.5286, CI excluding uninformative characters = 0.3664, HI excluding uninformative characters = 0.6336, retention index (RI) = 0.5979 and rescaled consistency index (RC) = 0.2818.

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Figure 4: Maximum likelihood (ML) phylogram from the nuclear SSU rDNA data partition analyzed under the GTR+I+
model of molecular evolution (nucleotide frequencies of A=0.25210, C=0.19810, G=0.27050 and T=0.27930; substitution rate matrix of AC: 1.664200, AG: 4.398100, AT: 3.106500, CA: 1.664200, CG: 1.025000, CT: 12.216500, GA: 4.398100, GC: 1.025000, GT: 1.000000, TA: 3.106500, TC: 12.216500, TG: 1.000000; proportion of invariable sites = 0.6182; gamma distribution shape parameter (alpha) = 0.5786). The “*” indicates clades also found in three-gene analyses. The -lnL score of this single most optimal tree was 8722.54101.

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Figure 5: rbcL MP strict consensus of 76 trees (length=1053). BS support values greater than 50% are shown above branches (1000 replicates) and Bayesian posterior probabilities (5+5 million generations combined, 50 000 generation burn-in discarded for each run) greater than 50% are shown below branches for those clades also sampled in the combined Bayesian analyses. See Appendix IV-2 for the full Bayesian topology with all posterior probabilities greater than 50%. The arrows highlight Arjona, whose position varies in other analyses, and Thesium impeditum, who is not sister to its congener with this data partition. The “*” indicates BI support for clades which did not include Arjona in BI or ML analyses. CI = 0.5489, HI = 0.4511, CI excluding uninformative characters = 0.4540, HI excluding uninformative characters = 0.5460, RI = 0.6720, and RC = 0.3688.

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model of molecular evolution (nucleotide frequencies of A=0.2715, C=0.1917, G=0.2399 and T=0.29690; substitution rate matrix of AC: 1.402200, AG: 2.389100, AT: 0.520800, CA: 1.402200, CG: 0.627400, CT: 3.150100, GA: 2.389100, GC: 0.627400, GT: 1.000000, TA: 0.520800, TC: 3.150100, TG: 1.000000; proportion of invariable sites = 0.4536; gamma distribution shape parameter (alpha) = 0.7296). The arrows highlight Arjona, whose position varies in other analyses, and Thesium impeditum, who is not sister to its congener in this data partition. Note the long branches found in these two taxa. The “*” indicates clades also found in three gene analyses. The –lnL score of the single most optimal tree was 8136.63960.

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Figure 7: matK MP strict consensus of 31 trees (length=2513). BS support values greater than 50% are shown above branches (1000 replicates) and Bayesian posterior probabilities (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 the combined Bayesian analyses. See Appendix IV-6 for the full Bayesian topology and all posterior probabilities greater than 50%. The arrow highlights the position of Korthalsella, which is not sister to Ginalloa, and varies in other analyses of this data partition. CI = 0.5312, HI = 0.4688, CI excluding uninformative characters = 0.4653, HI excluding uninformative characters = 0.5347, RI = 0.6490, and RC = 0.3448.

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Figure 8: ML phylogram from the matK data partition analyzed without Phacellaria 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 AC: 1.375300, AG: 1.740400, AT: 0.264300, CA: 1.375300, CG: 0.537500, CT: 1.740400, GA: 1.740400, GC: 0.537500, GT: 1.000000, TA: 0.264300, TC: 1.740400, TG: 1.000000; shape parameter of the gamma distribution (alpha) =1.1338). The –log likelihood score of this single most optimal tree was 13988.94084. See Appendix IV-4 for ML analysis with Phacellaria. The arrow highlights the position of Korthalsella, which varies in other analyses of this data partition. The “*” indicates clades also found in three gene analyses.

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Figure 9: Three-gene MP strict consensus of 2 trees (length=4798). BS support values greater than 50% are shown above branches (1000 replicates) and Bayesian posterior probabilities greater than 50% (5+5 million generations combined, 50 000 generation burn-in discarded for each run) are shown below branches for those clades also sampled in the combined Bayesian analyses. The dashed line indicates the position of Phacellaria from BI and smaller numbers right of the slash indicate reduction in support when Phacellaria is included. Circled numbers indicate the eight well-supported clades, on which the revised classification of Santalaceae s. lat. is based. The organization of taxa in the historical classification is indicated at the branch tips (V=Viscaceae, Am=Amphorogyneae, E=Eremolepidaceae, S=Santaleae, An=Anthoboleae, T=Thesieae, O=Opiliaceae). See Appendix IV-9, 10 & 11 for the full Bayesian topology and all posterior probabilities greater than 50%. CI = 0.5102, HI = 0.4898, CI excluding uninformative characters = 0.4292, HI excluding uninformative characters = 0.5708, RI = 0.6267, and RC = 0.3198.

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model of molecular evolution (nucleotide frequencies of A=0.27700, C=0.18620, G=0.22350 and T=0.31330; substitution rate matrix of AC: 1.519300, AG: 2.190700, AT: 0.678000, CA: 1.519300, CG: 0.532900, CT: 3.201500, GA: 2.190700, GC: 0.532900, GT: 1.000000, TA: 0.678000, TC: 3.201500, TG: 1.000000; proportion of invariable sites = 0.4468; gamma distribution shape parameter (alpha) = 0.7908). The –log likelihood score of this tree was 32132.07152. Brackets indicate familial circumscription according to the proposed classification in this study; “‡” marks eremolepidaceous taxa. See Appendix IV-8 for ML results with Phacellaria.

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______. 1972. Kunkeliella, a new genus of Santalaceae in the Canary Islands. Cuaderno Botanica Canariensis 16: 11-26. STEUSSY, T. F. 1990. Plant Taxonomy: The Systematic Evaluation of Comparative Data. Columbia University Press, New York. STEVENSON, G. B. 1934. The life history of the New Zealand species of the parasitic genus Korthalsella. Transactions Proceedings Royal Society of New Zealand 64: 175-190. SWOFFORD, D. L. 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, Sunderland, MA. UEDA, K., AND K. YOSHINAGA. 1996. Can rbcL tell us the phylogeny of green plants? In M. Nei and N. Takahata [eds.], Current Topics on Molecular Evolution, 97-103. Institute of Molecular Evolutionary Genetics, The Pennsylvania State University, USA & Graduate School for Advanced Studies, Hayama, Japan. VAN TIEGHEM, P. 1896. Sur les Phanerogames a ovule sans nucelle, formant le group des Innucellees ou Santalinees. Bulletin de la Société Botanique de France 43: 543577. ______. 1910. Classification nouvelle du groupe des Inovulées. Compt. Rend. Acad. Sci. Paris 150: 1715-1720. VEENENDAAL, E. M., I. K. ABEBRESE, M. F. WALSH, AND M. D. SWAINE. 1996. Root hemiparasitism in a West African rainforest tree Okoubaka aubrevillei (Santalaceae). New Phytologist 134: 487-493. VISSER, J. 1981. South African Parasitic Flowering Plants. Juta and Co., Cape Town. WARRINGTON, P. D. 1970. The haustorium of Geocaulon lividum, a root parasite of the Santalaceae. Canadian Journal of Botany 48: 1669-1675. WEBER, H. C. 1977. Anatomische Studien an den Haustorien (Kontaktorganen) von Thesium-Arten (Santalaceae). Berichte der Deutsche Botanische Geselschaft 90: 439-458. WIENS, D., AND B. A. BARLOW. 1971. The cytogeography and relationships of the viscaceous and eremolepidaceous mistletoes. Taxon 20: 313-332. WOJCIECHOWSKI, M. F., M. LAVIN, AND M. J. SANDERSON. 2004. A phylogeny of legumes (Leguminosae) based on analysis of the plastid matK gene resolves many well-supported subclades within the family. American Journal of Botany 91: 1846-1862. XIA, N., AND M. G. GILBERT. 2003. Santalaceae, Flora of China, 208-219.

71

XIANG, Q., D. SOLTIS, AND P. SOLTIS. 1998. Phylogenetic relationships of Cornaceae and close relatives inferred from matK and rbcL sequences. American Journal of Botany 85: 285-279.

72

APPENDIX I Primer Sequences Nucleotide primers used in PCR amplification and cycle sequencing reactions. Name nuclear SSU 12F 1131R 1769R rbcL 1F 3'R matK 78F 834F 833R 1420R

Length

Position

Primer Sequence 5'-3'

20 21 19

12 - 21 1130-1150 1769-1787

TCC TGC CAG TAS TCA TAT GC CAA TTC CTT TAA GTT TCA GCC CAC CTA CGG AAA CCT TGT T

20 20

1-20 rbcL-accd spacer

ATG TCA CCA CAA ACA GAR AC TAG TAA AAG ATT GGG CCG AG

20 20 21 21

73-92 834-853 833-853 1291-1310

CAG GAG TAT ATT TAT GCA CT GCA TTA TGT TAG GTA TCA AG CTT GAT ACC TAA CAT AAT GCA 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 Buffer MgCl2 dNTPs Forward primer Reverse primer Taq Genomic DNA H2O Total

Initial concentration 10 X 25 µM 2.5 mM each 10 µm 10 µm ~0.6 units/
L 5-10 ng/
L -

Volume (µL) 2.5 1.5 0.5 1.0 1.0 1.0 1.0 16.5 25.0

73

Final concentration 1X 1.5 µM 0.05 mM 0.4 µM 0.4 µM ~ 2.5 units / 100
L 0.2-0.4 ng/
L 0.66 µM

Appendix I (continued) Thermal Cycle Parameters Nuclear SSU rDNA Temperature (°C) Time (min:sec) Number of Cycles

94 94 48 72 5:00 1:00 1:00 2:00 X5

94 0:30

52 0:30 X 33

72 72 4 1:30 10:00 continuous

94 94 52 72 5:00 0:30 0:30 0:30 X5

94 0:30

48 0:30 X 33

72 72 4 1:00 10:00 continuous

94 94 46 72 5:00 1:00 1:00 2:00 X5

94 0:30

50 0:30 X 35

72 72 4 1:30 10:00 continuous

Chloroplast rbcL Temperature (°C) Time (min:sec) Number of Cycles Chloroplast matK Temperature (°C) Time (min:sec) Number of Cycles

74

APPENDIX II Some representative command blocks used in this study. Phacellaria was both included and excluded from analyses containing matK sequence data. Models of molecular evolution used in Bayesian and likelihood analyses varied between data partitions (see Table 4). PAUP* command block (used for MP and ML analysis of the three-gene dataset) BEGIN SETS; CHARSET CHARSET CHARSET CHARSET CHARSET CHARSET CHARSET CHARSET CHARSET TAXSET TAXSET TAXSET TAXSET END;

18S = 1-1827; rbcL = 1828-3255; rbcLpos1 = 1828-3253\3; rbcLpos2 = 1829-3254\3; rbcLpos3 = 1830-3255\3; matK = 3256-4516; matKpos1 = 3256-4516\3; matKpos2 = 3257-4514\3; matKpos3 = 3258-4515\3; Opiliaceae = 51-55; missing18S = 20 30 36 40; missingrbcL = 9 30 40 42-43; missingmatK = 1-2 5-6 20;

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;

75

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=100 AutoClose=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=Gamma Shape=0.7908 Pinvar=0.4468; HSearch Start=1 MulTrees=yes; DescribeTrees All / Plot=Both BrLens=yes; SaveTrees File=3gene.ML.tre BrLens=yes; End;

76

Appendix II (continued) MrBayes command block (used for the fully partitioned three-gene analyses) [Bayesain Inference.] Begin MrBayes; Log Start File=3GeneFull.mb.log; Set Autoclose=yes; Delete Phacellaria; Outgroup Opilia; CharSet 18s = 1-1827; CharSet rbcl = 1828-3255; CharSet rbcLpos1 = 1828-3253\3; CharSet rbcLpos2 = 1829-3254\3; CharSet rbcLpos3 = 1830-3255\3; CharSet matk = 3256-4516; CharSet matKpos1 = 3256-4516\3; CharSet matKpos2 = 3257-4514\3; CharSet matKpos3 = 3258-4515\3; TaxSet viscaceae = 1-7; TaxSet opiliaceae = 51-55; TaxSet missing18s = 20 30 36 40; TaxSet missingrbcl = 9 30 40 42-43; TaxSet missingmatK = 1-2 5-6 20; Partition full=7:18S,rbcLpos1,rbcLpos2,rbcLpos3, matKpos1,matKpos2,matKpos3; Partition gene=3:18S,rbcL,matK; Set Partition=full; [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;

77

APPENDIX III Typical Log-Likelihood Plots from Bayesian Analyses

Full log likelihood plot of the fully partitioned three-gene analysis without Phacellaria 0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

-30000 -32000

Ln Likelihood

-34000 -36000 -38000 -40000 -42000 -44000 -46000 -48000 -50000

Number of Generations Log likelihood plot for burn-in region of the fully partitioned threegene analysis without Phacellaria 0

5000

10000

15000

20000

25000

30000

-30000 -32000

Ln Likelihood

-34000 -36000 -38000 -40000 -42000 -44000 -46000 -48000 -50000

Number of Generations 78

35000

40000

45000

50000

APPENDIX IV Supplemental Phylogenetic Trees

79

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

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

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

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

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Supplemental Tree IV-5: matK Bayesian majority rule consensus including Phacellaria, from two identical BI runs with 5000000 generations each sampled every 1000 generations. Trees from the first 50000 generations (50 trees) were discarded as burn-in. Data were partitioned by codon position and model parameters for all three positions were estimated independently (i.e. unlinked) under the GTR+
model of molecular evolution. Posterior probabilities from each run separately are shown below the branches and from both runs combined are above the branches.

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Supplemental Tree IV-6: matK Bayesian majority rule consensus. This is a similar analysis to Supplemental Tree IV-5, but without Phacellaria. Two identical BI runs with 5000000 generations each were sampled every 1000 generations. Trees from the first 50000 generations (50 trees) were discarded as burn-in. Data were partitioned by codon position and model parameters for all three positions were estimated independently (i.e. unlinked) under the GTR+
model of molecular evolution. Posterior probabilities from each run separately are shown below the branches and from both runs combined are above the branches. Values in boldface italic type represent clades not recovered in the MP strict consensus tree for this data partition.

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

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

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

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

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Supplemental Tree IV-11: Three-gene Bayesian majority rule consensus fully partitioned without Phacellaria, from two identical BI runs with 5000000 generations each sampled every 1000 generations. Trees from the first 50000 generations (50 trees) were discarded as burn-in. Data were fully partitioned by genes, and for the protein coding genes, by codon position. Model parameters were estimated independently for each partition (i.e. the partitions were unlinked). See Table 4 for the models of molecular evolution used for each partition. Posterior probabilities from each run separately are shown below the branches and from both runs combined are above the branches. Values in boldface italic type represent clades not recovered in the MP strict consensus tree for this data partition.

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VITAE Graduate School Southern Illinois University 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 LanphereChristensen Dunes. Final report submitted to Humboldt Bay National Wildlife Refuge, May 2003. 91