Biogeography and Phylogeny of Sloanea

Biogeography and phylogeny of Sloanea

Matti A. Niissalo

August 2011

Thesis submitted in partial fulfilment for the MSc in the Biodiversity and Taxonomy of Plants

III

Abstract ______

Sloanea (Elaeocarpaceae) is a large genus of rainforest trees with a wide but disjunct distribution in the Old World and New World tropics. The genus has not been previously studied using molecular methods, and its biogeographic history has never been assessed in detail.

These factors make this genus a fascinating subject of study. Using two genes from 54 samples, The current study uses Bayesian and parsimony analysis methods to asses the relationship between the Old World and New World taxa as well as the formally described sections within the New World. The results suggest that both Old World and New World species form monophyletic clades. In the Old World, the S. section Paniculi forms a basal grade in which other sections are embedded.

Biogeographic analysis is carried out in order to identify dispersal patterns in the genus. Paleocene and Oligocene fossils are used to date molecular phylogenies, and the patterns of phylogenies were used to asses whether the family has dispersed through Boreotropics or through the Antarctica. According to the results, the family is of Southern Hemisphere, possibly Australian origin. The phylogenetic pattern suggests a dispersal to Old World and New World through the Antarctica.

Front cover

Flower of Sloanea terniflora Fruits of Sloanea (© Smithsonian woollsii Tropical Research (© Black Diamond Institute, Images, http://www. http://www. discoverlife.org) flickr.com)

Trunk of a Sloanea sp., Costa Rica (© Gary Braasch / Corbish, http://www.corbisimages.com)

Acknowledgements

This project is a joint effort from people from several organisations. It would not have been possible without the material provided by Dr. Terence Pennington from RBG Kew. This material formed the basis for the current study. He was also invaluable in providing information regarding morphological support for clades within Sloanea and acted as a co-supervisor during this study. It is unfortunate that there was no time during this study to visit him at Kew.

Professor Darren Crayn and Yumiko Baba from Australian Tropical Herbarium, James Cook University gave much support to the lab work for the phylogenetic analysis, and sequenced material from the genus especially for this study, in addition to the material they have already made available to GenBank.

Alain Rousteau from Laboratoire de biologie Végétale in Université des Antilles et de la Guyane provided many particularly valuable sequences from the Caribbean and French Guiana, and my thanks go to him and his team.

Dan Janzen from Costa Rica provided the project with DNA from a species that was particularly important for this study. Thanks also go to Dr. Alan Forrest for ultimately handing me this material. Euridice Honorio forwarded me material from Peru.

Drs. Toby Pennington and James Richardson from RBG Edinburgh were my main supervisors during this study, and their contribution to this project has been invaluable. I am grateful for their endless source of knowledge, always a source of answers when I’ve been in doubt.

Dr. Michelle Hollingsworth and Ruth McGregor provided the training in the molecular lab. Later they were always VI there to answer questions regarding laboratory problems. Dr. Michael Möller provided the phylogenetic training required for this study.

Tiina Särkinen, Natural History Museum, London, gave advice on extracting DNA from herbarium specimens.

Finally, I would like to thank my parents and the Natural Environment Resource Council for their financial support during my studies, and my fellow students for sharing it all.

Definitions

CI Consistency Index Ma megaannuum, million years (before present). MRCA most recent common ancestor. New World is here used to refer to the present-day Americas. Old World is here used to refer to present-day areas outside the Americas. pp posterior probability RI Retention Index TBR tree-bisection-reconnection

VII

Table of contents ______

Abstract ...... III Front cover ...... IV Acknowledgements ...... V Definitions ...... VI 1 Introduction ...... 1 1.1 Taxonomy ...... 1 1.1.1 Position of Elaeocarpaceae and Sloanea ...... 1 1.1.2 Infrageneric classification of Sloanea ...... 6 1.2 Biogeography ...... 10 1.2.1 Distribution patterns in Elaeocarpaceae ....10 1.2.2 Fossils ...... 11 1.2.3 Modern distribution of Sloanea ...... 15 1.2.4 Previous Biogeographic work ...... 16 1.2.5 Theories of tropical biogeography ...... 17 1.3 Hypotheses of infrageneric classification ...... 21 1.4 Biogeographic hypotheses for Sloanea ...... 22 2 Materials and methods ...... 29 2.1 Plant material ...... 29 2.2 Fossil material ...... 30 2.3 Laboratory materials and methods ...... 30 2.3.1 Health and safety ...... 34 2.4 Sequence editing and aligning ...... 34 2.5 Analyses ...... 35 2.5.1 PAUP ...... 35 2.5.2 Beast ...... 36 2.5.3 Morphological analysis ...... 37 3 Results ...... 39 3.1 Troubleshooting ...... 39 3.2 Polymorphism ...... 40 3.3 Parsimony ...... 41 3.3.1 trnL-F ...... 41 3.3.2 ITS1-2 ...... 42 3.3.3 Combined data set ...... 43 3.4 Bayesian analysis ...... 49 3.4.1 Tree statistics ...... 49 VIII

3.4.2 Tree shape ...... 49 3.4.3 Dating ...... 49 3.5 Phylogeography ...... 50 4 Discussion ...... 54 4.1 Species delimitation and identification ...... 54 4.2 Old World and New World clades of Sloanea ...... 54 4.3 Character evolution ...... 56 4.4 Evolution of the sections within New World Sloanea ...... 57 4.4.1 Sloanea subgenus Quadrisepala ...... 58 4.4.2 Sloanea subgenus Sloanea ...... 59 4.4.3 Sloanea porphyrocarpa ...... 60 4.4.4 Poorly placed species ...... 61 4.5 Biogeography ...... 61 4.5.1 Reliability of the Bayesian phylogeographic analysis ...... 61 4.5.2 Interpretation of phylogeography tree ...... 62 4.5.3 Northern origin scenario ...... 63 4.5.4 Southern origin scenario ...... 68 5 Conclusions ...... 70 5.1 Phylogeny ...... 70 5.2 Phylogeography ...... 71 6 Future work ...... 74 References ...... 76

Appendix 1 – Samples used in the current study ...... 87

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Appendix 2 – Aligned ITS1-2 matrix ...... 92

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Appendix 3 – Aligned partial trnL-F matrix ...... 107

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Appendix 4 – Additional trees ...... 126

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List of figures

Figure 1 . Elaeocarpaceae tree based on ITS1-2 and trnL-F (Crayn et al., 2006). Abbreviations: Aceratium (Ac), Aristotelia (Ar), Crinodendron (C), Dubouzetia (D), Elaeocarpus (E), Peripentadenia (Pe), Platytheca (Pl), Sericolea (Se), Sloanea (Sl), Tetratheca (Te), Tremandra (Tr), Vallea (V) (for authors see Appendix 1)...... 4 Figure 2 . Phylogenetic tree of some Fabids (Fabidae W.S. Judd, D.E. Soltis & P.S. Soltis in PhyloCode), including Oxalidales, based on 17 genes (Soltis et al., 2011)...... 5 Figure 3 . Two possible phylogenetic patterns for widespread tropical plants resulting from vicariance. The first one would suggest a boreotropical dispersal of the taxa, reaching South America latest. The second pattern suggests a southern dispersal of taxa to South America. In this case the species only reach North America after diversifying in South America (reproduced from Lavin & Luckow, 1993)...... 27 Figure 4a above and 4b below. Possible, simplified phylogenetic patterns for a genus originating from North America. Map outline from http://mapas.owjo.com...... 28 Figure 5 . A strict consensus tree of 40,000 most parsimonious trees using the trnL-F data set. Jackknife analysis values over 50% are shown...... 46 Figure 6 . A strict consensus tree of 10,000 most parsimonious trees using the ITS1-2 data set. Jackknife analysis values over 50% are shown above branches. Posterior probability values from the MCC tree are shown below branches...... 47 Figure 7 . A strict consensus tree of 1404 most parsimonious trees using the combined data set. Jackknife analysis values over 50% are shown above branches. Posterior probability values from the MCC X

tree are shown below branches. Clades referred to in the text are highlighted and labelled...... 48 Figure 8 . A dated phylogeny of Elaeocarpaceae using the combined data set. The tree is a Maximum Clade Credibility (mcc) tree formed of 45,001 trees from Bayesian analysis. All dates are mean dates. Fossil calibration nodes are displayed...... 51 Figure 9 . A dated phylogeny of Elaeocarpaceae using the ITS data set. The tree is a Maximum Clade Credibility (mcc) tree formed of 45,001 trees from Bayesian analysis. All dates are mean dates. Fossil calibration nodes are shown...... 52 Figure 10 .Results of the phylogeographic analysis of data. Aus = Australia (blue), NA = North America (incl. Central America and Caribbean), red, SA = South America (green), NC = New Caledonia (orange)...... 53

List of tables

Table 1 . Infrageneric classification for New World Sloanea with some key characters (CEJ Smith, 1954)...9 Table 2 . Infrageneric classification for Old World Sloanea with some key characters (Coode, 1983)...... 10

1 Introduction ______

1.1 Taxonomy

1.1.1 Position of Elaeocarpaceae and Sloanea

Sloanea L. is a widely accepted genus that has no homonyms. Linnaeus named the first species in the genus, S. dentata L., in Species Plantarum (Linnaeus, 1753). CEJ Smith (1954) gives a full account of the history of the taxonomy of this genus. It has usually been considered a “good” genus since AC Smith (1944) argued for recent segregates ( Echinocarpus Bl., Anoniodes Schlechter, Antholoma Labill. and Phoenicosperma Miq.) to be congeneric with Sloanea . The genus can be identified by its simple leaves (rarely pinnate on young plants), tree- like habit (rarely shrubby), flowers with a secondary increase in stamen numbers, anthers opening by pores or slits, connective extending into an awn or knob and valvate capsules, unarmed or with various spines, containing one to several anatropous seeds with axile placentation. The seeds are covered with an aril to various degrees. In the field the genus can often be identified by the large buttresses (CEJ Smith, 1954, Coode, 1983). The monophyly of Sloanea has found support from molecular analyses (Crayn et al., 2006). However, the Crayn et al. (2006) study included mostly Australian and New Guinean species ( Sloanea sogerensis Baker f., S. woollsii F. Muell., S. langii F. Muell. and an unnamed species), with only a single representative of New World taxa ( S. berteroana Choisy ex DC.).

The genus belongs to the family Elaeocarpaceae Jussieu. The family has been long considered close to Tiliaceae Jussieu in Malvales Berchtold & J. Presl (Malvaceae Jussieu sensu APG III, 2009), see for example Cronquist (1981) pp. 348-350 and Dahlgren (1980). Both of the most 2 recent reviews of Sloanea (CEJ Smith, 1954, Coode, 1983) maintain the family’s connection to Tiliaceae, without discussing it in detail - Coode (1983) acknowledges that there are differing views. Takhtajan (1997 pp. 226-227) suggested an affinity of Elaeocarpales Takht. (using his nomenclature – note that the order has been named earlier, as Elaeocarpales Juss. ex Berchtold & J. Presl.) to Flacourtiaceae Richard (which he considered to be a member of Violales Perleb) in Dilleniidae Takht. ex Reveal & Takht. APG (1998) moved the family into a re- erected order, Oxalidales Heintze. This led to Matthews and Endress (2002) to carry out a study in floral evolution in Oxalidales. This study was was inconclusive in its support to the Oxalidales as a clade, though it found support for the close relationship of Elaeocarpaceae and Tremandraceae Candolle. Stevens (2011) confirms that there is little morphological support for the order. Matthews and Endress (2002) also give an account on the history of the taxonomic position of Elaeocarpaceae. Further molecular analyses have supported a monophyletic Oxalidales (APG II, 2003, APG III, 2009, Soltis et al., 2011).

The only molecular analysis of the family Elaeocarpaceae so far was that carried out by Crayn et al. (2006). Their results suggest that a clade consisting of Sloanea , Aristotelia L’Hér. and Vallea Mutis ex L. f. is sister to the rest of Elaeocarpaceae. Crinodendron Molina and Peripentadenia L. S. Sm. are supported as an outgroup (or outgroups) to the remaining taxa, all of which are Australasian apart from the more widespread Elaeocarpus L. That study, and studies at order or higher level, suggests that the families closest to Elaeocarpaceae are from the Brunelliaceae Engl./Cephalotaceae Dumort. clade, followed by Cunoniaceae R. Br. (Crayn et al., 2006, Zhang & Simmons, 2006, Soltis et al., 2007, Soltis et al., 2011). See Figure 1 and Figure 2.

Sloanea is morphologically distinct from the genera Vallea and Aristotelia. These two genera share two obvious characters with Elaeocarpaceae at large: fleshy fruits and fringed petals, clearly different from sepals. 4

Figure 2. Phylogenetic tree of some Fabids (Fabidae W.S. Judd, D.E. Soltis & P.S. Soltis in PhyloCode), including Oxalidales, based on 17 genes (Soltis et al., 2011).

1.1.2 Infrageneric classification of Sloanea

Sloanea is a large genus with 62 species according to CEJ Smith (1954) in the New World and 52 species in the Old World according to Coode (1983). These numbers are outdated, as many new species have been described since these publications.

The genus can be considered taxonomically difficult, though it has been a subject of taxonomic revision. It has been revised in two parts: New World species by CEJ Smith (1954) and Old World species by Coode (1983). A two-part review is practical in this genus, and not only because of the disjunct distribution in the genus, but also because there are morphological differences between the Old World and the New World taxa. Most notably, all but two New World species ( S. jamaicensis Hook. from Jamaica and S. petalata D. Samp. & V.C. Souza from Brazil) are monochlamydeous or apetalous (they only have a calyx), whereas all but two Old World species ( S. woollsii F. Muell. and S. macbrydei F. Muell. from Australia) are dichlamydeous or petalous (with two whorls in the perianth); also, the New World species have an aril that almost completely covers the seed – in the Old world aril characters are variable (CEJ Smith, 1954, Coode, 1983, Sampaio & Souza, 2010). No taxon occurs in both the New World and the Old World. There has been little speculation has been put to which of these two distinct types in the genus may be the ancestral state (Coode, 1983). The Crayn et al. (2006) study does bring into attention that all the closest relatives to Sloanea have petals of the “petals present and distinct” type of Coode (1983).

CEJ Smith (1954) gave some suggestions about evolution of fruit types in Sloanea . He believed that spineless fruit type is the ancestral state, with rigidly spined fruit, derived from it, giving rise to two other fruit types – 7 capsules with detachable spines (irritant type) and a small, flexible-spined fruit. However, this only makes sense in a phylogenetic sense if the various fruit types have emerged repeatedly, as reported from Old World by Coode (1983). It is not strictly consistent with another view of CEJ Smith (1954), according to which Sloanea section Paniculi is the most basal in the genus, as both the Sloanea sections Paniculi and Corymbo-racemi can have unarmed fruits.

Chromosome counts for Sloanea are almost completely lacking. Considering that this is a large and complex genus, cytological studies may reveal important information about speciation in Sloanea . Raven (1975) gave the chromosome number as 13 (from a single sample). The same publication gives the chromosome number of Aristotelia , also from a single sample, as 14.

Several subgenera and sections have been described for the genus. However, no cladistic analyses of these have so far been realised. The genus has been (validly and tentatively for New World and Old World species, respectively) divided into groups as shown in Table 1 and Table 2.

The sections of CEJ Smith (1954) are not very clear-cut, with highly variable characters (inflorescence type, fruit size, spines) used to separate them. The value of inflorescence type and fruit spines have both been questioned (Coode, 1983), and therefore I will only evaluate the sections when discussing the results.

Some validly described sections for Old World taxa are available from AC Smith (1944). This classification is not currently much used, but it provides a basis for nomenclature of future infrageneric classification. Another early classification is that by Bentham (1861).

Of the species included in the current study not known to CEJ Smith (1954), S. petalata and S. uniflora D. Samp. & V.C. Souza have been included in the S. subgenus Quadrisepala ; their suggested closest relatives belong in the S. section Corymbo-racemi (Sampaio & Souza, 2010, Sampaio & Souza, 2011).

Several taxa in the current study have not yet been formally described, nor placed within any taxonomic rank. These include the taxa here called “ S. myrmecophyta ” (of unknown relation) “S. cruciata “ (tentatively part of the S. subgenus Quadrisepala ) and “S. dazae ” (tentatively part of the S. section Paniculi in the S. subgenus Quadrisepala ). Unnamed samples with collection numbers “Pennington 18249” and “Pennington 18252” are tentatively from the S. section Paniculi , and may represent S. durissima (Terry Pennington, RBG Kew, personal communication)

The current study focuses on New World taxa. Coode (1983) was useful a useful reference for the study as it allowed me to ensure that the Old World taxa are covered relatively comprehensively. However, CEJ Smith (1954) is the most relevant classification for the current study, and it remains the definitive work on New World Sloanea . 9

Sloanea Plants with a calyx that does not cover subgenus other organs in the bud. Sepals are u nequal, Sloanea numbers ranging from four to eleven. Flowers in lateral racemes (Coode, 1983 questions the inflorescence types used by CEJ Smith). Stipules are present as the leaves develop. Sloanea E.g. semipersistent stipules, in current section study: S. faginea Standl., S. guianensis Brevispicae (Aubl.) Benth., S. massonii Sw., S. parviflora Planch. ex Benth., S. pubescens Radlk., S. robusta Uittien, S. spathulata Earle Sm., S. tuerckheimii Donn. Sm. Sloanea E.g. large stipules and reduced section inflorescences, in current study: S. dentata Sloanea L., S. grandiflora Sm. Sloanea Calyx covering the floral organs in bud, subgenus sepals usually four (up to five), lateral Quadrisepala (rarely) terminal inflorescences of various types, stipules falling from bud before leaf development. Sloanea E.g. fleshy dark sepals, in current study: section S. caribaea Krug & Urb., S. durissima Spruce Paniculi ex Benth., S. floribunda Spruce ex Benth., S. latifolia (Rich.) K. Schum, and S. synandra Spruce ex Benth.) Sloanea E.g. with membranous light sepal, in current section study: S. berteroana Choisy ex DC., S. Corymbo- brachytepala Ducke, S. dussii Urb., S. racemi garckeana K. Schum, S. ?kuhlmannii Ducke, S. monosperma Vell., S. porphyrocarpa Du cke, and S. terniflora (Sessé & Moc. ex DC.) Standl. Table 1. Infrageneric classification for New World Sloanea with some key characters (CEJ Smith, 1954). 10

Subgenus I Petals present, similar to sepals. Group A In current study: S. langii F. Muell.) Group B In current study: S. sogerensis Baker f. Group C No representatives in the current study Subgenus II Petals present, differentiated from sepals.

Group D Petal margin rounded, untoothed, in curr ent study: S. australis (Benth.) F. Muell. Group E Petal margin toothed, in current study: S. lepida Tirel and S. montana AC Sm. Subgenus III Petals absent. Group F In current study: S. woollsii F. Muell and S. macbrydei F. Muell. Table 2. Infrageneric classification for Old World Sloanea with some key characters (Coode, 1983).

1.2 Biogeography

1.2.1 Distribution patterns in Elaeocarpaceae

Given that the distribution patterns of plant families are often difficult to interpret due to dispersal (e.g. Cox & Moore, 2010 p. 338), and the closest relatives of Sloanea within Elaeocarpaceae have disjunct distributions (Vallea in New World, Aristotelia in New World, Australia and New Zealand), it has so far not been possible to infer clearly the geographical origin of Sloanea from the phylogenetic pattern. The situation is similar in the two basal genera of the “main” Elaeocarpaceae clade (which includes Elaeocarpus ), one ( Crinodendron ) of which is endemic to New World, and another ( Peripentadenia ) to Australia, and also in the sister clade to Elaeocarpaceae – Brunelliaceae is Endemic to New World, and Cephalotaceae to Australia (distributions from Mabberley, 2008). Baker et al. (1998) looked at the distribution of genera of Elaeocarpaceae in the Old World. Their work is best understood in the light of Crayn et al. (2006) 11 study. Further study in the basal clade of the Elaeocarpaceae, particularly in the genera Sloanea and Aristotelia may give further insights to the historical biogeography of Elaeocarpaceae. The Crayn et al. (2006) study gave some implication that the New World Aristotelia may be basal in the genus, a result contradicting morphological evidence (Coode, 1985).

Crayn et al. (2006) provided a dated phylogeny for the family using a single calibration point, 30 Ma for the Crown node of Elaeocarpus . The biogeographic part of their study, concentrated on former Tremandraceae, which were shown to be originating in Australia. For the clades of interest to the current study, the following minimum ages should be noted: most recent common ancestor (MRCA) of Sloanea /Aristotelia 78-79 Ma (megaannuum, million years [before present]); crown node of the current Sloanea clade 30 Ma. For dating Sloanea , Crayn et al. (2006) only sampled Old World species.

1.2.2 Fossils

Fossils are of key importance to the current study: they are needed to infer past distribution of modern-day taxa, and they are here used for dating phylogenies in absence or reliable geological dates (e.g. presence in oceanic islands) for this genus. Therefore, they need to be correctly identified beyond reasonable doubt to a currently living clade so that positioning them to a phylogenetic tree is possible, and they must be from formations that have been reliably dated.

1.2.2.1 Elaeocarpaceae fossils

For the current study, the only relevant fossil material for Elaeocarpaceae (excluding Sloanea ) was that of Elaeocarpus utilised by Crayn et al. (2006). As their study states, Elaeocarpus is readily identified by fruit characters, and their fossils are widespread in Australia (Crayn et al., 2006). As Cunoniaceae is used as an outgroup in the current study, fossils were also looked for this family. A reliable fossil is available for this family at rughly 83 Ma (Schönenberger et al., 2001).

1.2.2.2 Sloanea fossils

According to fossil evidence, the range of this genus has changed dramatically over time. Fossils from the Eocene, Oligocene and Miocene has been found in Austria, Italy, Hungary, Czech, Slovenia and Germany, and in parts of this range the genus has been the dominant forest tree (Raven & Axelrod, 1974, Kva•ek et al., 2001, Kva•ek, 2002, Sachse, 2005, Tamás & Hably, 2005, Hably & Kva•ek, 2006, Hably, 2007, Erdei & Rákosi, 2009, Collinson et al., 2010). Earlier, in the Paleocene, the genus has been present in Greenland. In continental North America it has been documented from Paleocene to Eocene formations, possibly even in the Cretaceous (Manchester, 1999, Manchester & Kva•ek, 2009). There are also possible Sloanea fossils from Miocene New Zealand (Pole, 1993). The Eocene Sloanea /Elaeocarpus fossils have been reported several places in Australia (Christophel & Greenwood, 1987, Christophel et al., 1992). However, none of the Southern Hemisphere fossils have been studied in detail, and therefore they cannot be used to confirm the presence of Sloanea there at this time.

The reliability of the fossil record is variable. For example, the only English fossils – from he London Clay – 13 assigned to Echinocarpus Bl. (synonym of Sloanea ) (Reid & Chandler, 1933 pp. 390-392), lack an important fruit character for the genus, a keel inside the locule, which is replaced by a groove, as noted by Kva•ek et al. (2001) – however, Kva•ek later co-authored an article which does not question the identification of these specimens (Manchester & Kva•ek, 2009). Two articles are particularly interesting for the current study: Kva•ek et al. (2001) and Manchester and Kva•ek (2009).

Kva•ek et al. (2001) give a good account on characters that can be used to identify Sloanea . Gross morphology of leaves cannot reliably be used to identify the genus, but trichome bases and structure of cyclocytic stomata help in identifying Sloanea . Leaf epidermis and cuticle are also of key importance, as well as fruit characters (loculised woody capsule that splits into 4-5(-7) valves with a median keen/septum on inner surface; often spiny). Samples from Budapest’s Nagybátony-újlak brickyard have been reliably dated to the Lower Oligocene (Rupelian) ca. 31 ± 3 Ma. Unfortunately I have not seen the plates published with the Kva•ek et al. (2001) study, and therefore I need to rely on their descriptions for the identification of material. I have no hesitation in doing so, as their arguments for identification do appear to be very well founded.

Based on foliage evidence, Kva•ek et al. (2001) and Rásky (1962) found the closest recent relatives of the fossil taxa they observed to be Asian species in “Group E” of Coode (1983) ( Sloanea sinensis (Hance) Hemsley, S. sigun (Bl. K. Schum, S. hemsleyana (Ito) Rehder and S. forrestii W.W. Smith).

Manchester and Kva•ek (2009) found that fruit fossils reliably identifiable as Sloanea are present in North America (including Greenland) from the Puercan (early Paleocene) to the Middle Eocene, and a possible Sloanea fruit has been found from the late Cretaceous (this 14 specimen with a rather arbitrary identification was not used in the current study). They highlight the fact that these are the oldest Elaeocarpaceae fossils available, a fact that may challenge the perceived southern origin of the family, however acknowledging that the northern historic range of Sloanea may be due to a secondary dispersal.

To the list of typical Sloanea characters listed before, Manchester and Kva•ek (2009) add a staminal disk persistent in fruit, short stout style and a pedicel that may be longer than the capsule. They also mention that the fruits can be separated from similar Aesculus L. fruits by their narrower, more crowded spines, and by the number of valves (more than three). Other species possibly causing confusion are also discussed, and the identification of the material as Sloanea is highly convincing.

Their suggestion to include these fossils in the crown node of the extant genus seems less strongly founded. The fruit type preserved in fossils is rather typical to the genus, but it could be ancestral to the genus.

1.2.2.3 Fossil ages

Young age of the Sloanea clade as suggested by Crayn et al. (2006), in contrast to older fossil evidence may be the result of small sampling in the genus, extinction of the fossil clades or unsuitability of the molecular dating method to the genus (however, the rate of substitutions didn’t vary significantly from the rest of Elaeocarpaceae). The possibility must be considered that Sloanea fossils have been misidentified, though there is strong evidence against this (Kva•ek et al., 2001). However, all Sloanea fossils are from the Tertiary, and thus more recent than the implied minimum age of the 15

Sloanea /Aristotelia MRCA, and therefore do not obviously contradict the results of Crayn et al. (2006).

1.2.2.4 Fossil implications to biogeography

The fossil evidence suggests that the genus originated from North America in the late Cretaceous or Palaeocene. It has then spread to Europe in the Eocene. The European fossils are related to Asian taxa, but the affinity of the North American taxa has not been confirmed. The dispersal to North America needs to have been long distance, possibly from South America, which was the tropical land mass closest (though still distant) to North America in the late Cretaceous and early Paleocene (Scotese, 2003). However, the early Paleocene fossils from Wyoming (and Montana further North) would suggest that Sloanea was not at this time strictly tropical (Wilf, 2000), some ten million years before large areas in Northern Hemisphere became tropical in the Paleocene- Eocene Thermal Maximum (Gingerich, 2006), and therefore dispersal from other subtropical/temperate areas remains a possibility.

The North American fossils may or may not be related to current lineages of Sloanea . However, in the absence of Southern Hemisphere fossils, it seems reasonable to argue that the genus has first diversified in the Northern Hemisphere.

1.2.3 Modern distribution of Sloanea

The genus Sloanea is widespread in the tropics. In the New World, it is found in the central and northern parts of South America, in Central America and the Caribbean. In the Old World the distribution covers eastern 16

Australia, New Caledonia, Malesia, eastern Asia and Madagascar (CEJ Smith, 1954, Coode, 1983, Mabberley, 2008, GBIF, 2011,). Notably, the genus is absent from African continent and New Zealand. Its distribution doesn’t extend to temperate regions.

1.2.3.1 Implications to biogeography

The current distribution of Sloanea does not reflect the presence of the early Paleocene fossils in Northern United states (Wilf, 2000, Manchester & Kva•ek, 2009). The current distribution of the genus, extending only just to subtropics (Coode, 1983), would suggest that the MRCA of currently living Sloanea was tropical. Given this total absence of temperate species, it is possible that they became extinct during the climatic warming in the late Paleocene, though there is no direct evidence for this.

Given the northern origin suggested by fossil evidence, the current distribution of the genus could have been achieved though dispersal in the boreotropic after the late Paleocene, either through Antarctic, a North Atlantic land bridge or Bering Land Bridge (Lavin & Luckow, 1993, Pennington & Dick, 2004).

1.2.4 Previous Biogeographic work

Detailed biogeographical studies on Sloanea have not been made. In Crayn et al. (2006), which looked at biogeography, the internal structure of the Sloanea clade was unresolved using trnL-F (including only one New World species), and the ITS study by the same authors consisted only of three Old World species.

Raven and Axelrod (1974) suggested that Sloanea originated in from South America, and has dispersed from there to the Old World tropics. They based this on Sloanea only being present in the northern part of Australia. However, this is an over-simplification of the matter. In particular, it does not explain the wide distribution of the genus in the old world, including the very widespread fossils acknowledged in their study. The oldest fossils of the genus are from North America, from where no other Elaeocarpaceae are known, either in the fossil record or naturally extant.

1.2.5 Theories of tropical biogeography

Since the theory of plate tectonics has been accepted in the mainstream of science, there have been attempts to explain current plant distribution patterns with geological history. For a long time, vicariance biogeography resulting from plate tectonics was considered an overpowering contributor to distribution of taxa at various levels (e.g. Croizat et al., 1974, Raven & Axelrod, 1974, Rosen, 1978).

In recent years, the direct effects of plate tectonics have been challenged in respect to plant distribution. The extent of importance of long-distance dispersal and different aspects of climatic changes in earth history (for example warm periods in the Antarctic and in boreal regions) on distribution patterns is still argued, but it is widely accepted that their impact has been significant (e.g. Lavin & Luckow, 1993, Pennington & Dick, 2004, Richardson et al., 2004, Sanmartín & Ronquist, 2004). One way of assessing the influence of tectonic or climatic history on modern distributions of taxa is by using dated molecular phylogenies. A geographically distinct clade that has emerged at a time when dispersal to the area by land corridors was not possible is obvious evidence for 18 long distance dispersal. Meanwhile, if a certain time period shows an increased amount of dispersal between two geographical area, this is strong evidence that there has been an active dispersal route at this time. Evidence for such routes can initiate search for support from geological evidence. Therefore, dates of divergence give powerful information on geographic history.

Dated phylogenies have already been used in a large number of studies in this field, and these will be used in the interpretation of the results of the current study.

Geographic proximity of continents, evidence for presence other dispersal routes (such as island chains) and climate at geological periods should all be considered when interpreting dated molecular phylogenies.

1.2.5.1 Methods of dating molecular phylogenies

Whereas other methods of dating phylogenies do exist, it is most often done with the help of fossils. Each fossil will only give a minimum age for the node at which it is placed, but a comparison of the base of the tree to similar studies at higher taxonomic level can provide more confidence in the dates – for example, the earliest verifiable date for eudicots at ca. 124 Ma can be used as a reasonable oldest date for even old eudicot families (as new fossils are found, this date may need to be reviewed, by findings such as that described in Sun et al., 2011). These dates are then used as bases for calibrating case-specific molecular clocks.

There has been discussion about the validity of using phylogeny- and fossil-derived minimum ages as approximate absolute ages. The main opponent of this technique has been Heads (e.g. Heads, 2005a, 2005b, 2010). His arguments are not necessarily justified. It is not 19 within the scope of this project to discuss this rather philosophical question in detail, but counter-argument used by Goswami and Upchurch (2010) would find parallels in botany (in places even more convincingly, as plants are better than animals at dispersal, see Sanmartín & Ronquist, 2004). Also, the phylogenetic inferences by Heads himself at least regarding Asteraceae (Heads, 2009) are hardly logical, and rely on very poor fossil data (with fossils being constantly found only tens of millions of years after the clades arose). This appears not as an attempt to find a biogeographic theory which best explains the observed phylogeny and fossil data, but an attempt to force the observed data to fit a biogeographic theory.

1.2.5.2 Amphi-Pacific distribution in other families

The only other mostly tropical families with distribution pattern of mostly tropical taxa limited to Asia, Australasia, Tropical America (with or without Madagascar) are Actinidiaceae Van Tiegh., Bonnetiaceae Beauvis, Chloranthaceae R. Br., Clethraceae Klotzsch., Heliconiaceae (Endl.) Nak., Mitrastemonaceae Mak., Sabiaceae Bl. (incl. Meliosmaceae Endl.) Tapisciaceae (F. Pax) Takhtajan, Tetrameristaceae (H. Hallier) Hutch. and Trigoniaceae Endl. (Heywood et al., 2007, APG II, 2003). Of these, Bonnetiaceae, Mitrastemonaceae, Tapisciaceae, Tetrameristaceae and Heliconiaceae have such limited distribution that drawing parallels to Sloanea would be difficult. The remaining genera are remarkably similar in distribution to Sloanea /Elaeocarpaceae. Chloranthaceae however is much more ancient than Elaeocarpaceae (see for example Kuprianova, 1967), and may well have been distributed into the current locations much earlier. Clethraceae is limited further northeast in the Old World and further northwest in the New World (it is absent in much of Amazonia and Madagascar, for example), and does 20 not seem analogous to Elaeocarpaceae. Support has been found for it as an example of tropical North American and East Asian floristic disjunctions (Fior et al., 2003), a pattern discussed in detail by Zhengyi (1983). Sabiaceae and Trigoniaceae have not been extensively studied but could prove interesting in this context. Like Sloanea , they are evergreen trees, apart from Sabia Colebr. (Heywood et al., 2007). Interestingly (though admittedly later than Sloanea fossils), Middle Eocene North American (including Central America and the Caribbean) fossils are suggested for Clethraceae and Tetrameristaceae (Liu & Basinger, 2000, Martínez-Millán, 2010).

At the generic level more examples of such disjunct distributions are more common. Many have been discussed by Welzen et al. (2005), but the studies they refer to (main one being Steenis, 1962, not seen) are not with dated phylogenies. Thorne (1972) gives a list of genera with this “amphi-Pacific tropical” distribution. Interestingly, Scharaschkin and Doyle (2005) created dated phylogenies of the genus Anaxagorea St. Hil. (Annonaceae Juss.) that has a similar distribution to Sloanea (if a little more restricted in the Old World), but their dating relied on fossils outside the genus, and their strict consensus trees were completely unresolved, making their interpretation of biogeography doubtful. Richardson et al. (2004) placed the split of Anaxagorea within Annonaceae roughly at the late Cretaceous, with a crown node at around 44 million years ago, only some 10 Ma earlier than the age of the crown node of Sloanea found by Crayn et al. (2006). Genera Symplocos Jacq. (Symplocaceae Desf.) and Saurauia Willd. (Actinidiaceae Van Tiegh.) share many similarities with Sloanea (the Paleocene origin in North America, possible later dispersal to South America and the Old World (Wang et al., 1991, Keller et al., 1996), and further research may show that there is a significant pattern here. Genus Musa L. (Musaceae Juss.), an example of a genus now 21 restricted to Asia and Australasia, will be discussed later.

1.3 Hypotheses of infrageneric classification

Phylogenetic patterns can be used to identify ancestral character states in the genus. If the “petals distinct” type of flower is ancestral in Sloanea , and other petal types are derived from this then the phylogenetic pattern observed should show the “petals distinct” species in a derived position. The other petal types may or may not form a monophyletic clade within this grade, e.g. the monochlamydeous Australian taxa may or may not be related to the similar New World species.

Alternatively, if the “petals absent” or “petals different from sepals” types are ancestral, the phylogenetic pattern should be reversed.

For the New World sections, I hypothesise the following pattern of evolution: ( Paniculi ( Brevispicae ( Corymbo- racemi ( Sloanea )))). This is based on the insights of CEJ Smith (1954). He considered Paniculi to be the most primitive group in the New World. The S. ubgenus Sloanea has been kept monophyletic, as there appears to be no reason to contradict this – the species are very similar in fruit and stipule characters. He also thought that unarmed fruits are primitive in the genus. Both the S. sect. Corymbo-racemi and S. sect. Brevispicae have some species with unarmed fruits, whereas the S. sect. Sloanea always has armed fruits. Admittedly, support to the suggested phylogeny is very slim. The hypothesised phylogeny would mean that only the S. section Sloanea (and the S. subgenus Sloanea ) is monophyletic, but it is possible that clades not formally described will be identified during the current study.

1.4 Biogeographic hypotheses for Sloanea

The present-day distribution of Sloanea is limited to the tropics and subtropics, subtropics, but there is clear evidence, in the form of fossils, that the genus became extinct in temperate areas in the northern hemisphere (Europe and North America). The absence of this genus, and the family Elaeocarpaceae, from the wet tropics of Africa is peculiar from a biogeographic point of view, and requires an explanation. It does however suggest that the family originated after Africa was separated from the rest of Gondwana, i.e. ca. 100 Ma (McLoughlin, 2001).

Given the morphological differences between the New World and Old World Sloanea , it seems likely that there has been little movement of plants between the areas. Acquiring the current distribution by long distance dispersal is impossible to rule out; however, an attempt will be made to explain the distribution by vicariance where suitable dispersal routes have been present. The presence of fossil in both Old and New World does suggest that the current wide distribution is of ancient origin. Also, if the phylogenies will show a pattern in which taxa from the two regions are phylogenetically mixed, this would suggest that the transfer of material between the two areas has been more significant than now considered, and the likelihood of long-distance dispersal in creating the current distribution pattern would increase.

In creating a hypothesis of the phylogeny of Sloanea the age found by Crayn et al. (2006) for the MRCA of Sloanea and Aristotelia, approximately 80 Ma, can be used. This is an implied minimum age, but reasonably argued to be a fair estimate of absolute age, as a very soft maximum age of 124 Ma for the family was used (given that only a 23 single calibration point was used for the family, it is possible that the node in question may be younger than suggested).

Dispersal from North America to South America via the closing Caribbean Sea has been possible from the Middle Eocene onwards through island hopping via the Proto- Greater Antilles and GAARlandia, peaking in the closing of Panama Isthmus ca. 3 Ma (Pennington & Dick, 2004). Another important event for Sloanea biogeography is the crossing of Wallace’s Line. Unfortunately, samples are not available from West of Wallace’s line. Lineages would have been able to cross Wallace’s line from the Middle Miocene onwards (Morley, 1998), as the Australasian and Asian plates converged.

1.4.1.1 Northern vs. Southern Hemisphere origin

The area of origin of the genus is of importance in interpreting the phylogenetic patterns of Sloanea . A northern (North American/Eurasian) origin can find support in a dated phylogeny that has southern taxa embedded in clades of northern taxa, separating at times when dispersal between the areas has been possible. In the case of a southern origin (South American, Australasian or Antarctic), the pattern should be reversed.

Therefore, the “North American” taxa (i.e. species that now grow in the area north of South American continent, including Central America and the Caribbean)) are in a key role (Lavin & Luckow, 1993, also see Figure 3). This causes some issues in the current study. Sloanea is now completely absent from North America proper. Most of the South American taxa are widespread. No formally recognised species groups are limited to Central America. Only a few Central American species were successfully 24 included in this study; S. faginea is the only Central American endemic included ( S. tuerckheimii and S. terniflora are not exclusively Central American). Caribbean species are much more thoroughly sampled.

From the Old World, no species from Madagascar or West of Wallace’s Line are included in the current study, which limits the geographical scope of this study.

As it may turn out that separating the two areas of origin reliably won’t be possible with the material at hand, two different scenarios are given for the hypothesis, one with a Northern Hemisphere origin and another where the genus has originated in the Southern Hemisphere.

As stated above Sloanea was certainly present in North America from the Paleocene to the Eocene and in Europe from the Oligocene to the Miocene. The latter parts of these dates largely fit with tropical climates in the northern hemisphere (see Fossils and a review by Morley, 2003), and give reason to form a hypothesis where North America is where the MRCA of the genus lived. If the genus spread to Old World via Europe, the absence of the genus from the wet tropics of Africa would require an explanation. One such explanation would be a barrier of dispersal from Europe to Africa (climatic barriers are thought to have been present due to drying from the Oligocene onwards only Couvrer et al., 2008) or later extinction from Africa for example due to restriction of rain forest area in Africa in changing climate. Evidence from palms show that by the late Eocene plant diversity in Africa had started diminishing (Pan et al., 2006), and it could be argued that Sloanea reached Europe too late for it to be able to spread to the wet tropics of Africa.

1.4.1.2 Phylogenetic patterns with a Northern Hemisphere origin

If North America is considered to be the origin of the genus, as the oldest fossils would suggest, then the migration patterns that could have been taken to achieve modern distributions could have been either of those illustrated in figure 4. Dispersal through Antarctica (Figure 4a) should show a phylogenetic pattern where there has been diversification in South America before the dispersal into the Old World. Dispersal to South America via the Old World may not have been likely, as the dispersal to East of Wallace’s Line has only been happening since the cooling of Antarctic (Morley, 1998}, Francis, 2008). The other possibility is that migration occurred from North America to the old world tropics via a northern boreotropical route (Figure 4b).

1.4.1.3 Phylogenetic patterns with a Southern Hemisphere origin

Information on the climatic history of Antarctica is still poor and the fossil record is especially so (see also Morley, 2003 and Pennington & Dick, 2004). However, according to evidence reviewed by Francis et al. (2008), at the time of between stem and crown ages for Sloanea , 79-30 Ma (Crayn et al., 2006) Antarctica still existed as a land bridge between Australia and America and it may have supported tropical vegetation for parts of this period.

In the late Paleocene, possible fossils of Elaeocarpaceae have also been found in Antarctica, though there is no reason to connect these findings to Sloanea (Francis et al., 2008). However, the Antarctic started to cool some 20 million years later, before the crown node age of Sloanea suggested by the Crayn et al. (2006) study. It would not be surprising if fossil evidence of Sloanea 26 would have gone undetected. In this theoretical scenario, the genus must have dispersed to the Northern Hemisphere through long distance dispersal, and then become extinct there.

An origin in the southern hemisphere only allows dispersal through the southern route. The phylogenetic pattern should show dispersal in the Southern Hemisphere with northern taxa embedded in the phylogeny both in Old World and the New World (Figure 3 scenario 2).

These tree scenarios, scenario 2 of Figure 3 and both scenarios of Figure 4 are the hypotheses in the current study, and dated molecular phylogenies will be used to separate them.

Figure 3. Two possible phylogenetic patterns for widespread tropical plants resulting from vicariance. The first one would suggest a boreotropical dispersal of the taxa, reaching South America latest. The second pattern suggests a southern dispersal of taxa to South America. In this case the species only reach North America after diversifying in South America (reproduced from Lavin & Luckow, 1993). 28

Figure 4a above and 4b below . Possible, simplified phylogenetic patterns for a genus originating from North America. Map outline from http://mapas.owjo.com. 29

2 Materials and methods ______

2.1 Plant material

DNA from 33 samples of Sloanea (see Appendix 1) were extracted and sequenced at Royal Botanic Garden Edinburgh (RBGE). All material was provided by Terence Pennington from Royal Botanic Gardens, Kew (RBG Kew), apart from a single specimen sent by Euridice Honorio (EDNA11-0022672) and two further samples from Dan Janzen, Costa Rica, via the RBGE DNA bank (EDNA11-01679 and EDNA11-01680). Some was herbarium material, some collected in silica gel.

Additional Sloanea sequences were available from GenBank, from Université des Antilles et de la Guyane (UAG) and from James Cook University’s Australian Tropical Herbarium (JCU).

For all analyses, the sequences used in the combined analysis of Crayn et al. (2006) were included in the data matrix. These were available from GenBank.

Additionally, two samples from relatively closely related family Cunoniaceae were used as an outgroup, as ITS sequences from the sister families to Elaeocarpaceae (Brunelliaceae and Cephalotaceae) were not available.

2.2 Fossil material

Internal fossil calibration was possible due to the studies by Kva•ek et al. (2001). This gave an age of ca. 31 Ma for the stem lineage of “Group E” of Sloanea . In the ITS and combined data sets (which were used for dating analysis in the current study), this group was represented by S. lepida and S. montana .

Late Paleocene fossils from North America (Manchester & Kva•ek, 2009) give an age of ca. 63 Ma for the stem lineage of Sloanea . The original publication suggests that the fossils should be part of the crown group of the genus, but this has not been well argued, and in the current study I consider it to be a stem group representative.

Elsewhere in Elaeocarpaceae, calibration was done using the same fossil dates as in the Crayn et al. (2006) study (30 Ma for crown group of Elaeocarpus , which in current study was made to include Aceratium DC. as Elaeocarpus was otherwise not monophyletic).

A stem age for the outgroup, Cunoniaceae, was given as 83 Ma, as the oldest well studied fossil from the family is roughly that age (Schönenberger et al., 2001).

2.3 Laboratory materials and methods

DNA was mostly extracted using the following, standard RBG Edinburgh extraction method:

1. A small piece of leaf material (up to 0.5 cm 2) was placed in a vial, broken with sterilised tweezers and homogenised in Mixer Mill (Retsch) at 20- 31

times/second frequency. As the leaf material was hard, a relatively long treatment was necessary. 2. To the homogenised material, 1 ml of preheated 2% CTAB (hexadecyltrimethylammonium bromide) + 2 µl β- mercaptoethanol and a pinch of PVPP was added. Material was the incubated for 30 minutes at 65°C. 3. After allowing to cool to room temperature, 500 µl of chloroform + IAA (isoamyl alcohol, 24:1) was added to the tube. The samples were placed in orbital shaker for 30 min – 1 hour. 4. Samples were centrifuged for 10 minutes at 13,000 rpm to separate the chloroform. 5. The top (supernatant) layer was moved to a clean tube, and steps 3-4 were repeated. 6. The DNA was precipitated b adding 600 µl of ice cold isopropanol. The samples was gently rocked by hand. The DNA was left in the freezer overnight. 7. DNA was pelleted by centrifuging the precipitate for 10 minutes at 13,000 rpm. 8. The supernatant was removed, and 500 µl of wash buffer was added to the pellet, it was ensured that the pellet became loose by agitating the tube on vortex. 9. The tube was pelleted again by centrifuging for 5 minutes at 13,000. The supernatant was removed, and the pellet was dried in vacuum centrifuge for 5 minutes (until dry). 10. The pellet was dissolved in 100 µl of TE DNA dissolving buffer with mixing, and the samples was placed in freezer (-20°C).

For a single lot of samples (EDNA11-0021901 - EDNA11- 0021924), QIAxtractor (Qiagen) was used, following manufacturer’s protocol.

The success of DNA extractions was tested on a gel electrophoresis after extraction and again after the initial PCR. 32

Following advice from Tiina Särkinen (Natural History Museum, personal communication), some samples which failed to show bands in the gel were extracted with a method that combined the steps 1-4 of the standard Edinburgh extraction method as written above, and the steps of DNeasy Plant Mini Kit from step 11 of the manufacturer’s protocol (Qiagen). This gives the large amount of DNA that is the benefit of using CTAB extraction, and the purity of DNA provided by DNeasy.

Two regions were investigated for the study. The regions were those used by Crayn et al. (2006). Partial sequence of trnL-F was used to study cpDNA. Primers c and f (c 5’ primer, 5’-3’ CGA AAT CGG TAG ACG CTA CG; f 3’ primer, 5’-3’ ATT TGA ACT GGT GAG ACG AG) from Taberlet et al. (1991) were used. In troubleshooting, internal primers e and d were employed (e 5’ primer 5’-3’ GGT TCA AGT CCC TCT ATC CC, d 3’ primer, 5’-3’ GGG GAT AGA GGG ACT TGA AC). To provide an independent phylogenetic estimate from another genome, nuclear ITS1-2 (nrDNA) was studied using primers GN1 (5’ primer, 5’-3’ CGC GAG AAG TTC ATT GAA CC, Scott & Playford, 1996) and ITS4 (3’ primer, 5’-3’ TCC TCC GCT TAT TGA TAT GC, White et al., 1990). In troubleshooting, internal primers ITS2g* and ITS2g (ITS2g* 5’ primer 5’-3’ ACG TCT GCC TGG GTG TCA C, ITS2g 3’ primer 5’-3’ GTG ACA CCC AGG CAG ACG T) were utilised.

For PCR reactions, the following formula was used per sample:

2 µl dNTPs 2 µl 10 x Buffer

0.6 µl MgCl 2 0.2 µl Taq 4 µl CES 1.5 µl Primer 1 1.5 µl Primer 2 1 µl DNA template 33

7.2 µl H 2O

I trouble shooting, increased volume of DNA template was used (up to 3 µl) and the volume of H 2O was reduced to 5.2 µl, or 2 µl of stock (10 mg/ml) BSA was added without changing the volume of H 2O.

The following cycle was used for PCR of trnL-F:

94˚C for 3 min, then subjected to 25 cycles of the following profile 30s at 94˚C (denaturation) 30s at 55˚C (annealing) 30s at 72˚C (extension) After cycling, a final incubation for 5 minutes at 72˚C was carried out.

The PCR of ITS1-2 used the following cycle:

94˚C for 3 min, then subjected to 25 cycles of the following profile 30s at 94˚C (denaturation) 30s at 55˚C (annealing) 1min at 72˚C (extension) After cycling, a final incubation for 5 minutes at 72˚C was carried out.

In troubleshooting, increased cycles (up to 45 cycles) were sometimes used.

Purification of PCR was carried out using ExoSAP IT (GE Healthcare, 2 µml ExoSap-IT and 5 µl PCR product). This was subjected to 15 minute incubation at 37°C and heating for 15 minutes at 80°C.

Sequencing PCR was carried out using 0.5 µl Bigdye 2 µl 5 x Buffer 34

0.32 µl primer

6.18 µl H 2O 1 µl PCR template

The sequencing reaction required the following temperatures for 25 cycle: 30s at 95°C 20s at 50C° 4 min at 60°C

The samples were then kept at 4°C until ready for analysis. Sequencing PCR was not analysed at RBGE.

Samples which showed faint PCR bands in electrophoresis, increase amount (up to 2 µl) of PCR template was used.

2.3.1 Health and safety

β-mercaptoethanol, chloroform and SYBR Safe (used in gel electrophoresis) are irritant, harmful or toxic and were used and disposed of following RBGE COSHH procedures. Gloves were used during all laboratory work.

2.4 Sequence editing and aligning

The sequences were edited using Sequencher v4.7 (Gene Codes Corporation). Ambiguous ends of sequences were cut, and sequences were combined with each other either automatically or manually (if sequence quality was poor). The sequences were then visually checked for any ambiguities. In the case of ITS polymorphism, some areas could not be interpreted unambiguously.

The combined edited sequences were aligned manually using Mesquite v2.73 (Mesquite Project). For trnL-F, indels 35 were used as characters using a manually created two- state (presence/absence) character matrix. Very similar indels were treated as the same character. All gaps were treated as missing data in Bayesian analyses. Gaps were treated as missing data for ITS1-2 due to high variation in the genome. One region of 51 base pairs the ITS data could not be reliably aligned, and was therefore excluded from all analyses (see Appendix 2).

2.5 Analyses

2.5.1 PAUP

Heuristic parsimony analyses were done separately for ITS1-2 and trnL-F with PAUP v 4.0b10 (Sinauer Associates, Inc. Publishers).

10,000 addition sequence replicates with TBR (tree- bisection-reconnection) were used. No more than 5 trees of length greater than 5 steps were saved for each replicate.

The acquired trees were used as starting trees for another heuristic search, saving no more than 10,000 trees of length greater than 5 for each replicate. Due to a high number of starting trees with the trnL-F data (the initial heuristic search resulted in over 30,000 most parsimonious trees), the limit of the second heuristic search was set to 40,000 trees for this gene.

Congruence was tested between data sets using the partition-homogeneity test at default settings using PAUP at the freely accessible Oslo Bioportal (https://www.bioportal.uio.no). The two data sets were not significantly incongruent with either trnL-F gap data excluded (p = 0.95) or included (p = 0.17). Therefore, the data was combined and a parsimony analysis was run 36 with the combined data set using the same settings as for heuristic searches on individual genes.

MrModeltest was used at Oslo Bioportal to get substitution models for the two data sets. ITS1-2 data was analysed in its entirety and trnL-F data was analysed separately, excluding gap characters.

Support for clades was searched within each of the three datasets using jackknife analysis. 37% of data was used for each replicate, and 10,000 analysis replicates were performed. Quick analysis was performed.

2.5.2 Beast

Beast was used to acquire divergence dates and posterior probability values for nodes in the phylogeny through Bayesian analysis. One analysis was run with only the ITS1-2 data, and a separate analysis with a combined data matrix (with ITS of Ceratopetalum added to give a two- species outgroup, the minimum required for placing fossils). Because trnL-F data was not very variable, it was not used for Bayesian analysis. One of the most parsimonious trees, closely resembling the strict consensus tree from parsimony analyses of the combined data was rooted and selected as a starting tree for the analysis. Nodes were dated as discussed earlier.

Data was prepared for Beast v1.6.1 in the same version of BEAUti. Default settings were used apart from substitution models (used as separately defined for tnrL- F and ITS1-2), clock model (relaxed clock: uncorrelated lognormal) and tree prior (speciation: birth-dead process). A fully dichotomous tree was selected from a parsimony analysis as a starting tree. Only the Elaeocarpaceae clade was forced monophyletic. Priors were 37 set at lognormal where default settings were not accepted.

The resulting .xml file was run in Beast for 50,000,000 generations, saving a tree every 1,000 generations. The log files were combined using LogCombiner. Data was then viewed in Tracer v1.5. An initial burn-in of 10% (5,000 trees) was removed from further analyses. The remaining 45,001 tree files were prepared for viewing using TreeAnnotator, setting the shape of the tree to conform to a maximum clade credibility (mcc) tree. The resulting tree files were viewed in Mesquite v2.71 or FigTree v1.3.1.

For a biogeographic analysis, each of the samples was given a geographic character state: North America (including Central America and the Caribbean), South America, Australasia (Australia and New Guinea) or New Caledonia. The data was incorporated into the .xml file using R (some manual adjustment was also needed), and scripts for the analysis were manually added to the .xml file.

2.5.3 Morphological analysis

The genus is poorly represented in the RBGE herbarium. Only a few species included in the current study were available at the time of the project, and the material did not allow a full morphological analysis of the various clades observed in molecular data. Therefore, the morphological analysis was largely dependent on literature. The major source of descriptions was CEJ Smith (1954). An attempt was made to find illustrations (drawing or scanned specimens) for enough species to aid in the analysis, but this goal was largely abandoned. Some illustrations were seen in Silveira (2009) and various floras, but the material wasn’t adequate enough 38 to make a full analysis. Fruit types were recorded for each New World species, as this appears to be a particularly interesting character for the genus. The characters states can be seen in Appendix 1.

3 Results ______

3.1 Troubleshooting

Sequencing silica-gel collected samples was always successful (see Appendix 1). However, in the case of S. uniflora , only somewhat poor quality ITS1-2 sequence could be retrieved. Its quality was adequate for the current study.

Acquiring sequences from air-dried or herbarium samples was very difficult, a character that appears common in the family (Yumiko Baba, James Cook University, personal communication). However, using more DNA, longer PCRs and 10 mg/ml BSA as discussed in the methodology, the success rate increased significantly. Extraction method did not have apparent effect on DNA quality, however, this was not rigorously tested. Reliable percentages of success in sequencing herbarium specimens cannot be given, because various troubleshooting methods were only applied on particularly interesting samples. Nine out of nineteen herbarium samples were successfully sequenced. Terence Pennington selected, where possible, relatively recent, air-dried herbarium specimens for this project (RBG Kew, personal communication).

Internal primers had to be used with species that failed to give good quality sequences of adequate length. In most cases this was due to ITS polymorphism, but in a few cases due to poor quality of trnL-F sequences, probably due to a very low amount of DNA.

From S. grandiflora , it was not yet possible to get good trnL-F sequences. The PCR bands for this sample were extremely faint. ITS1-2 sequences for a single accession of S. ? durissima were so polymorphic that they could not 40 be used. All other samples sequences especially for the current study provided both ITS and trnL-F sequences.

All aligned sequences, including indel characters, can be found in Appendices 2 and 3. Two successfully sequenced silica collections turned out to be unrelated to Elaeocarpaceae.

3.2 Polymorphism

The term (molecular) polymorphism in the current study is used to refer to coexistence of several alleles of the same genes in the genome of a single individual (or, more accurately, within the sample from which the DNA was acquired). This is only possible where several copies of the same gene exist, e.g. in the current study, only with ITS1-2. It is inherited from both parents, and is present in a large number of copies. Only one parent contributes towards the chloroplast genome of each individual, and only a single copy of this is normally present.

Such polymorphism turned out to be commonplace in Sloanea . Percentages of samples that display polymorphism cannot be given, as separating polymorphism from poor quality sequences is not always possible when the two sequences are very similar. In roughly a quarter of the samples it was an issue that required special attention. Where polymorphism was present, it was usually present in a few base changes, or as short indels. In a single case (S. pubescens , collection number Pennington 18233), the two genomes present were slightly more diverged. It was possible to report the two sequences separately in this case, and the two versions of its genome are included in the study as different samples. Only the “version 1” was used in combined analyses.

None of the polymorphism observed suggested hybridisation between any two species included in the current study. Therefore, the polymorphisms are best explained as infraspecific variation. Cloning methods, which are used to separate two coexisting genes in a sample may prove useful especially if population-level studies on Sloanea are conducted. They were not utilised in the current study, as the level of variation did not appear significant enough to affect the outcome of the study.

3.3 Parsimony

3.3.1 trnL-F

A strict consensus tree was constructed from 40,000 most parsimonious trees (Figure 5). Support values were only acquired through jackknife analysis, as the tree was of relatively little value within Sloanea . There were 1234 characters (24 of which were gap characters), of which 1001 were constant and 114 were parsimony-informative. The length of the most parsimonious trees was 300 steps. The strict consensus tree received the following consistency and retention index values: Consistency Index (CI) 0.715, which is a little high and Retention Index (RI) 0.907, a very good value.

Besides Sloanea , the tree is almost identical to tnrL-F consensus tree by Crayn et al. (2006) where the species selection agrees (it was slightly smaller in the current study, as only samples used in the combined analysis by Crayn et al., 2006 were used). The position of two genera, Crinodendron and Peripentaderia , was more resolved. Sloanea is monophyletic with strong (98%) jackknife support. Variation within Sloanea was small in the data matrix, and the parsimony analysis resulted in polytomy within Sloanea , with a few internal clades. S. lepida and S. montana came out as sister species (91% 42 support). The two samples of S. floribunda were also sisters (87% support). The two samples of S. dussii and another, unidentified specimen formed a clade (81% support). One sample of S. guianensis and S. massonii formed a clade with an unidentified specimen (52% support). Sloanea jamaicensis came out as a sister to a clade consisting S. garckana , S. terniflora (two samples forming a clade with 63% support – note that these samples had identical sequences) plus a clade consisting of S. ?kuhlmannii , S uniflora and S. porphyrocarpa . One clade was only seen in Jackknife analysis: a clade consisting S. macbrydei , S. woollsii , S. lepida and S. montana received 52% support.

3.3.2 ITS1-2

A strict consensus tree was constructed from 10,000 most parsimonious trees (Figure 6). Support values are available from Jackknife analysis and Bayesian analysis (posterior probability). There were 852 characters (after exclusion of 51 characters), of which 384 were constant and 278 were parsimony-informative. The length of the most parsimonious trees was 1255 steps. The strict consensus tree received the following consistency and retention index values: CI 0.547, a little better than for trnL-F, and RI 0.719, worse than for trnL-F. Where support values are given below, the jackknife percentage value is given before posterior probability (pp) value.

The overall tree closely resembles the tree from Crayn et al. (2006). A major difference is that the genus Crinodendron comes at the base of the tree. This is probably a result of that there was no outgroup in the Crayn et al. (2006) study.

Sloanea comes out as a strongly supported (83%, 100) monophyletic group. The lower clades within the genus do 43 not find jackknife support. However, the strict consensus tree shows Sloanea monosperma and S. “ myrmecophyta” as sisters to the rest of the genus. Clade consisting of S. floribunda , S. latifolia and S. durissima (all three from the S. section Paniculi ), and a clade consisting of Old World species and S. spathulata are then in polytomy with the rest of the genus.

In the remaining part of the tree, a clade with S. “dazae ”, S. berteroana , S. pubescens , S. “ cruciata ”, S. brachytepala (94%, 100) are a sister to the rest. Sloanea caribaea follows as a next clade on its own. Similarly to trnL-F results, S. garckeana , S. ?kuhlmannii , S. uniflora , S. jamaicensis , S. synandra and S. terniflora form a clade (94%, 100). This clade is sister to the remaining species, which form a clade with 99 pp support.

The clade sister to the remaining taxa ( S. dussii and an unidentified specimen, 100%, 100) is also present in the trnL-F consensus tree. A clade consisting of the S. sect. Sloanea species and S. faginea is then sister to the rest (79%, 100). The remaining clade (57%, 100) consists of unidentified specimens, S. massonii , S. guianensis (both samples), S. tuerckheimii , S. porphyrocapra , S. parviflora and S. robusta , with a few internal clades.

3.3.3 Combined data set

A strict consensus tree was constructed from 1404 most parsimonious trees (Figure 7). Jackknife and posterior probability values were acquired for the clades. There were 2121 characters, of which 1466 were constant and 357 were parsimony-informative. The length of the most parsimonious trees was 1460 steps. The strict consensus tree received the following consistency and retention index values: CI 0.596 and RI 0.78; both are intermediate 44 between trnL-F and ITS1-2 values, closer to the ITS1-2 values.

The tree was visually examined for obvious differences in rates of evolution (not shown), and apart from the taxa previously included in the family Tremadraceae, the substitution rates were not unusually varied.

Overall, the tree very closely resembles the tree by Crayn et al. (2006). Sloanea comes out monophyletic with very strong support (99%, 100). The internal structure of Sloanea does not find support from jackknife analysis, but the Bayesian analysis does give low support for a monophyletic Old World clade (63).

The Old World clade (52%, 97) are a sister to the rest of the genus. This is from here onwards referred to as the “Old World clade”, and the remaining part of the tree will be referred to as the “New World clade”. This is done for practical reasons, despite the fact that support values for the monophyly of the New World clade is small.

The clade that is sister to the remaining part of the New World clade is formed by S. floribunda , S. durissima and S. monosperma , S. “ myrmecophyta ” and S. spathulata (all from the S. subgenus Quadrisepala apart from S. spathulata , for which CEJ Smith, 1954 only had young material). This will be referred to as “core Paniculi clade”, rather artificially as the only species from this clade are S. durissima and S. floribunda . This is done on the basis that these two species always appear in all analysis (with sufficient resolution) in a position similar to the one seen here, whereas the other three species in the calde appear elsewhere in other analyses. This clade gets remarkably poor support values.

The rest of the genus is divided into two large clades. One of them, the “core Corymbo-racemi clade” (67 pp support), can be further divided to two clades.

One of these (“umbellate clade”, 93%, 100) includes specimens from umbellate or single-flowered members of the S. subgenus Paniculi section Corymbo-racemi as well as S. synandra . S. caribaea (S. section Paniculi ), is sister to this clade, with low support. The other clade (“unarmed clade”, 97%, 100) includes all of the S. sect. Corymbo-racemi species with unarmed fruits, as well as S. “dazae ”, S. “ cruciata ” (both presumably with unarmed fruits) and the quite different S. pubescens .

S. latifolia ( S. section Paniculi ) is sister to the remainder of the genus, mainly formed of species from the S. subg. Sloanea (62%, 100). The basal clade of this is formed of a S. section Corymbo-racemi species S. dussii and an unidentified specimen ()100%, 100).

S. faginea and S. dentata then form the lowest clade (71%, 100) of the “subg. Sloanea clade”.

The final clade (99 pp) includes unidentified specimens as well as all sampled species from the S. subg. Sloanea section Brevispicae (apart from S. spathulata , S. faginea and S. pubescens embedded elsewhere in the genus).

Therefore, this tree shows both New World and Old World Sloanea to be monophyletic, and the S. subgenus Sloanea to be embedded within the S. subgenus Paniculi in a single clade, with a few exceptions.

Figure 5. A strict consensus tree of 40,000 most parsimonious trees using the trnL-F data set. Jackknife analysis values over 50% are shown. 47

Figure 6. A strict consensus tree of 10,000 most parsimonious trees using the ITS1-2 data set. Jackknife analysis values over 50% are shown above branches. Posterior probability values from the MCC tree are shown below branches. 48

Figure 7. A strict consensus tree of 1404 most parsimonious trees using the combined data set. Jackknife analysis values over 50% are shown above branches. Posterior probability values from the MCC tree are shown below branches. Clades referred to in the text are highlighted and labelled. 49

3.4 Bayesian analysis

3.4.1 Tree statistics

The frequency of CG for the ITS1-2 was 63.42% (AT = 36.57%), and the model selected by MrModeTest was GTR+G.

The frequency of CG for the trnL-F was 32.57% (AT = 67.43%), and the model selected by MrModelTest was HKY+G.

3.4.2 Tree shape

The topology of the maximum clade credibility (mcc) trees can be seen in Figure 8 and Figure 9. Within Sloanea , the clades present here largely correspond to the trees acquired from heuristic parsimony search. However, the family-wide structure of the ITS1-2 mcc tree differs markedly from the parsimony search. Additionally, the relationships within Sloanea are in this tree are very similar to the strict consensus tree from the combined parsimony analysis. The only marked difference in the position of S. latifolia , now basal to the core Corymbo- racemi clade.

3.4.3 Dating

Initial short Beast runs were done with and without dating the Cunoniaceae outgroup. Without the outgroup date, the age of the lowest node of the tree appeared at > 100 Ma (not shown), whereas with the outgroup fossil added, this was reduced to ca. 83 Ma. As we were looking for minimum dates, and as the latter dates fit better with fossil evidence (oldest Cunoniaceae fossils are from the early Cretaceous, Schönenberger et al., 2001), full 50 analyses were only run on trees with this fossil calibration added. Also, all combinations of exclusion and inclusion of internal fossils for Elaeocarpaceae were attempted. Variation between these sets was small (not shown), but including all available data gives the most reliable dates. None of the fossils were much younger than the nodes where they were assigned in trees that resulted when they were excluded, which gives confidence that these fossil dates are probably in the proximity of absolute ages of the nodes.

The ITS tree closely resembles the Combined analysis tree. It can be seen in Figure 8. For clarity’s sake I am using the combined analysis values when discussing node ages. The dates from the combined data analysis can be seen in Figure 9.

3.5 Phylogeography

The results of the phylogeographic analysis can be seen in Figure 10 . According to these results, both the family and the genus Sloanea originate from Australasia, from where they have spread to South America and finally several time to North America (which, in this analysis, includes the Caribbean and Central America).

Figure 8. A dated phylogeny of Elaeocarpaceae using the combined data set. The tree is a Maximum Clade Credibility (mcc) tree formed of 45,001 trees from Bayesian analysis. All dates are mean dates. Fossil calibration nodes are displayed. 52

Figure 9. A dated phylogeny of Elaeocarpaceae using the ITS data set. The tree is a Maximum Clade Credibility (mcc) tree formed of 45,001 trees from Bayesian analysis. All dates are mean dates. Fossil calibration nodes are shown. 53

Figure 10 .Results of the phylogeographic analysis of data. Aus = Australia (blue), NA = North America (incl. Central America and Caribbean), red, SA = South America (green), NC = New Caledonia (orange). 54

4 Discussion ______

4.1 Species delimitation and identification

The results illustrate problems with species delimitation and identification in this genus. Of the seven species for which more than one sequence was available for the combined parsimony analysis, three ( S. pubescens , S. “dazae ” and S. guianensis ) were not monophyletic. Of these, the second specimen of S .guianensis was not confidently identified. CEJ Smith (1954) notes that “Sloanea guianensis [] has been one of the most confused species-complexes in the genus” (p. 34), whereas S. pubescens is said to be morphologically distinct (CEJ Smith, 1954).

There were also five unidentified specimens in the current study. In some cases this may have resulted from collection of vegetative material, but this could not be always confirmed, as I have not seen the specimens.

4.2 Old World and New World clades of Sloanea

The Old World Sloanea species are strongly supported to form a monophyletic clade in the combined analyses. This makes sense in the light of the fossil evidence. However, S. spathulata , a poorly known and genetically distinct species, appears as a part of this clade in the ITS1-2 parsimony analysis. In the mcc tree it is grouped with the rest of the New World species in all analyses.

Based on the analyses, the Old World monochlamydeous species may or may no be basal to the clade, and more species are needed to find this out – there wasn’t strong support for the position of the , the only 55 monochlamydeous Old World species for which both ITS1-2 and trnL-F sequences were available. Whereas parsimony analyses found the “petals distinct” type of the Old World to be embedded with the rest of the petal types, this was contradicted in the mcc trees, as the “petals distinct” type was basal here. Being able to separate these two scenarios would be important for study of character evolution in the family, as discussed later.

Also, the umbellate clade, which includes the only two New World Sloanea with petals (as S. petalata is said to be closely related to S. garckeana ), is not closely related to Old World taxa. Therefore, the study could not identify morphological grounds for the most basal clades of both New World and Old World clades, and it is clear that presence or lack of petals are inconsistent character stats in the family at large.

The study could not identify if the New World clade, seen in the combined data analysis, is monophyletic, as support for it was low, and it was not present in its entirety in ITS1-2 parsimony tree. Considering this genus is obviously old, such contradicting results are not very surprising. This is particularly true if a North American origin is assumed, as the genus may have reached some level of diversity before spreading to Old World and South America at roughly the same time, as shown by dated phylogenies. This would suggest that a single dispersal was successful in colonising the Old World, whereas several lineages may have moved to South America. It is clear, however, that the Old World clade is not deeply embedded in the New World clade, a key observation for biogeography.

Though it does illustrate that the data set has some room for improvement, the presence of S. monosperma and S. “myrmecophyta ” in a node below the Old World clade in the ITS1-2 parsimony tree (but not mcc tree) is not very strong evidence against the monophyly of the New World 56 clade, as analysis with a larger data set disagrees with this. Visual inspection of these two sequences does confirm their distinctness within the genus. There appears to be no morphological grounds to placing S. monosperma to such a basal position ( S. “ myrmecophyta ” remains poorly understood, as discussed elsewhere).

4.3 Character evolution

As discussed above, the parsimony analaysis results contradict with Bayesian analysis for the origin of petals in the Old World clade of the genus – this may r may not be ancestral. Therefore, the study can’t be used to identify if the MRCA of genus Sloanea had “typical” fringed Elaeocarpaceae petals. As Sloanea is one of the basal genera in Elaeocarpaceae, this would affect finding out the character state of MRCA of the entire family. Other lineages in the family, with the exclusion of some deeply embedded taxa, have petals that are clearly distinct from sepals, and fringed or lacerated (Crayn et al., 2006, Heywood et al., 2007, Stevens, 2011). However, the sister group of Elaeocarpaceae (Cephalotaceae and Brunelliaceae) are apetalous.

In parsimony terms, it would seem more likely that the “petals distinct” group represents the state of the MRCA of crown group of Sloanea , as otherwise consequent losses and re-acquirement of this state are required. As the petals are quite uniform within the family, it is tempting to think that the dichlymadeous state is ancestral for the family, but increased genetic sampling, further sampling in the Old World Sloanea , and analyses of flower development genes may bring further light to this issue. As Sloanea has gained this state on its own, it is possible this would have also taken place for Vallea /Aristotelia and “core Elaeocarpaceae” clades independently. 57

The results increase confidence in inflorescence types (at least if they have been accurately identified), fruit spines and stipules as taxonomically significant characters, valuable in characterising major clades within Sloanea .

4.4 Evolution of the sections within New World Sloanea

Genus Sloanea is very strongly supported as a monophyletic taxon according to all analyses conducted. The general structure of parsimony analysis trees and Bayesian analysis trees is similar to the hypothesis that was formulated for the relationship of sections within the genus, though several species do hold a highly surprising position in the tree. In the New World, the basic phylogenetic structure can be presented as ( S. sect. Paniculi (core S. sect. Corymbo-racemi + ( S. dussii + S. subg. Sloanea ))), with little support for the core Corymbo-racemi clade, and the relationship of the S. sect. Paniculi to the rest of the genus as well as the relationship of sections within the S. subgenus Sloanea .

This evidence suggests that the New World S. subgenus Paniculi is not monophyletic, just as hypothesised. Also according to hypothesis, with the exception of a few species, the S. subg. Sloanea did turn out to be monophyletic. Only two species, the poorly known S. spathulata (not reliably placed in the current study), and surprisingly, S. pubescens (reliably placed within the unarmed clade) fall outside this clade.

4.4.1 Sloanea subgenus Quadrisepala

The only section for which this study gives poor resolution is S. sect. Paniculi . There is no support for the monophyly of this section, and indeed several species from this clade appear elsewhere in the genus in phylogenetic trees. This is probably because the section is paraphyletic, as is evidenced by a number of taxa that are poorly placed in the analyses. Some of these come up in very surprising positions, particularly S. latifolia and S. caribaea which have basal positions in a grade of species from the S. sect. Corymbo-racemi in most of the trees produced.

The mostly monophyletic S. section Corymbo-racemi is divided into two well-supported clades, which do also find morphological support. Apart from S. synandra , all the species in the umbellate clade have umbellate inflorescence (if more than one flower) with one to three flowers, a character which seems to be remarkably stable. The other clade, namely the unarmed clade, has only a few formally named species, but it appears to hold all the species of the section with unarmed fruits. It should be noted that S. “ cruciata ” and S. “ dazae ” are of suggested relation to S. synandra based on morphology (Terence Pennington, RBG Kew, personal communication), but in the current study they are unrelated to that species.

Apart from the poorly placed S. monosperma , the only S. sect. Corymbo-racemi species that falls outside the core Corymbo-Racemi clade is S. dussii (also see notes for S. porphyrocarpa below). Of S. dussii , no flowering material was available to CEJ Smith (1954), but its position close to the S. subg. Sloanea is supported by the subpersistent stipules and relatively long spines in the fruit. From a phylogenetic point of view, it may be necessary to move this species into the S. subgenus Sloanea , but better herbarium material is needed than was available to CEJ 59

Smith (1954). This would make the S. section Corymbo- Racemi monophyletic.

The study does suggest that all species from the core Corymbo-racemi clade have either unarmed fruits or fruits covered densely with very short spines, possibly always shorter than 2 mm. The only exception is S. pubescens . S. dussii has spines up to 6mm long. Including S. echinocarpa in phylogenetic studies would be interesting, as it has spines similar to S. dussii .

4.4.2 Sloanea subgenus Sloanea

The Sloanea subgenus Sloanea is monophyletic, and seems to share an ancestor with the S. section Corymbo-racemi . Within this subgenus, the S. section Sloanea is also monophyletic (though it may include S. faginea ), but the S. section Brevispicae most probably forms a grade. This is not surprising, as the S. section Brevispicae is rather more variable in its characters (CEJ Smith, 1954). The sampling of the S. section Sloanea was small.

As discussed above, the expansion of the S. subg. Sloanea to include S. dussii , a species with which is shares stipule characters in particular, would make the S. section Corymbo-racemi monophyletic. This would somewhat change the circumscription of this subgenus, as S. dussii is unusual in having fruits densely covered with small spines, and with flowers that have four valves. Another possible solution would be a creation of a new section for S. dussii alone.

The position of S. pubescens amidst the Corymbo-racemi - clade with unarmed fruits is surprising. On morphological bases it has been placed in the S. subgenus Sloanea , but the results of this study do not support this. There ______60 appears to be no documented morphological reasons to place the species in the S. sect. Corymbo-racemi. A slim possibility remain of misidentification of the material samples, but this is highly unlikely, considering the material was collected and verified by the expert of this genus (Terence Pennington, RBG Kew). This said, I have not seen the material.

4.4.3 Sloanea porphyrocarpa

With trnL-F data, this species was well supported as a part of clade with the S. section Corymbo-racemi species with umbellate inflorescences, but according to the ITS1- 2 analysis results, it should be placed within the S. section Brevispicae , also with strong support, a surprising result as the two clades are well-defined and distinct.

This would seem as evidence that the species has acquired ITS1-2 genes from the S. section Brevispicae , most likely through hybridisation. Following hybridisation, homogenation has been shown to take place with nrDNA (see for example Hillis & Dixon, 1991, Pillon et al., 2007). If this is commonplace in the genus, it may explain some of the other unusual patterns in the phylogeny of the genus. This is the only case where a conflicting position for a species between data sets is strongly supported.

As this issue was identified early on in the study, S. terniflora was added to the sampled taxa on the suggestion of Terence Pennington (RBG Kew, personal communication), as he considers the two taxa to be conspecific. No incongruence between genes could be detected on S. terniflora .

4.4.4 Poorly placed species

The positions of S. caribaea , S. latifolia , S. monosperma , S. “ myrmecophyta ” and S. spathulata are radically different when ITS results are compared to combined analysis results. Their position in any clade did not find strong support from jackknife or Bayesian analysis. Therefore, the position of these species could not be determined in the current study. Therefore, unless further data can help in determining the position of these taxa, discussing their position in the phylogenetic trees is of little value. Other species that found no support for a place in a clade in jackknife analyses were S. durissima and S. floribunda , but they were placed in a basal clade in both ITS and combined data analysis.

The position of the “unplaced” taxa in the combined analysis strict consensus tree doesn’t receive strong support from morphology. S. “ myrmecophyta ”, with its large stipules that support ant colonies, is certainly anomalous within the genus, but it is not known well enough to place it in any section based on morphology alone. There is no known fertile material from this species. Outside this species, large stipules are only found in the S. subg. Sloanea clade.

4.5 Biogeography

4.5.1 Reliability of the Bayesian phylogeographic analysis

Superficially, the results of the Biogeographic analysis appear to be biased by taxon selection, in particular outgroup selection (which was limited by available sequences) – both of the outgroup taxa are from Australia, but Cunoniaceae also grows in Australia (It also grows in the southernmost tip of Australia) (Heywood 62 et al., 2007). The closest outgroup to Elaeocarpaceae is formed by the families Brunelliaceae (South American) and Cephalotaceae (Australian), neither of which had full sequences available in the GenBank for the genes under study. This does however mean that even all outgroup are Southern Hemisphere.

As it was considered likely that including South American members in the outgroup would have affected at least the confidence values for some of the nodes, this was tested using a mock analysis where the country of origin of Ceratopetalum was changed into South America was carried out. The tree and associated confidence values can be found in Appendix 4. This was done for illustration purposes only, and the values suggested by the tree should not be used in further work. They values are relatively similar to values available from the original tree. This test suggests that the outgroup selection did not affect the outcome of the analysis to a notable degree.

4.5.2 Interpretation of phylogeography tree

The tree in Figure 10 is consistent with that depicted in Figure 3 scenario 2. This suggests that the family has a southern origin and is also consistent with an Australasian origin. Sloanea would have migrated to South America at the separation of the Old World and New World lineages –around the same time as Vallea and basal Aristotelia got to South America. Also, Crinodendron could also migrated to South America at this time.

It is clear from the phylogeographic analysis that an early ancestor of Sloanea must have originated from the Southern Hemisphere - whether this was South America or Australia could be debated. A South American origin finds support in two factors: the basal species of the 63

Vallea /Aristotelia clade are South American (Crayn et al., 2006) and the oldest Sloanea fossil known is from North America, very far from Australia ever since the breakup of Pangaea (Scotese, 2003). Even then the only current explanation of arrival to North America is long distance dispersal, as there wasn’t a close connection between North and South America at the time of divergence of Sloanea from Vallea /Aristotelia (Scotese, 2003). The presence of the MRCA of Sloanea in Australia 37-63 Ma as suggested by the phylogeographic analysis would be surprising, considering the genus grew in North America at this time, and the split to Old World and New World clades had not yet taken place – it should be noted that the Old World clade is strongly supported to be monophyletic, so if the genus originated from Australasia, it was probably not very diverse at the time of separation of the Old World and New World clades.

The coverage of morphological groups from Old World has been comprehensive, so it is unlikely that further sampling would reveal older lineages within the Old World taxa not monophyletic with the rest of the Old World taxa.

The phylogeographic analysis does confirm that dispersal through the Antarctica has actively taken place in the family at the time of the divergence of the genus, i.e. ca. 37 Ma (or a little earlier, considering this is a minimum age). In the context of the family, this dispersal via Antarctica was already discussed by Crayn et al. (2006).

4.5.3 Northern origin scenario

If we assume a northern origin for the genus, as suggested by the fossil evidence, then the separation of Sloanea to Old World and New world clades at ca . 37 Ma 64 corresponds to a northern dispersal, as the genus does not show significant diversification prior to the separation into the two geographically distinct areas. This scenario requires that the genus would have got to Europe around this time, a scenario consistent with dispersal via the boreotropics during the Eocene thermal maxima (Lavin & Luckow, 1993). In the Northern Origin scenario, the genus must have therefore dispersed through a northern route. The split would have occurred after the Eocene thermal maxima and the age of the split between New and Old World taxa presented here are consistent with this.

In the Northern Origin scenario, the genus must have therefore dispersed through a northern route.

The diversification of Old World taxa appears to have started around the same time as for the New World taxa. If diversification was actively happening in North America before this, the pattern would suggest that some lineages present in North America for some 20 Ma prior to dispersal to Old World must have gone extinct. Unlike in the Australian origin scenario described above, this is not particularly unlikely as the climate of North America in the modern sense has changed so dramatically, and the genus is now absent from there. As the New World “clade” is not necessarily monophyletic, it is possible that several lineages have given birth to the New World species.

The arrival of the genus to North America, far from the rest of the family, at ca. 60 Ma or earlier requires an explanation. The sister clade ( Vallea /Aristotalis ) has never been found in Laurasia, nor has any other species of Elaeocarpaceae apart from Elaeocarpus , which has probably dispersed there much later according to fossil evidence (Crayn et al., 2006). Interestingly, the closely related Cunoniaceae, although also largely Gondwanan, 65 appears to have grown in Laurasia some 83 Ma (Schönenberger et al., 2001).

4.5.3.1 Evidence for survival in North America

The apparent lack of “North America” taxa at the base of the New World clade requires further attention. Two of the observed clades (umbellate clade, S. subg. Sloanea clade) have North American plants at somewhat basal positions. Also, several species implied by CEJ Smith, 1954 or DA Smith, 1996 to be closely related to S. massonii or S. guianensis (basal in the S. section Brevispicae according to the current study) are Central American or Caribbean: S. meianthera Donn. Sm., S. picapica Standl., S. geniculata Dam. A. Sm., S. trichosticha , S. brenesii Standl., S. rugosa Dam. A. Sm., S. ilicifolia Urb. and S. curatellifolia Griseb. S. guianensis , a largely South American species itself, also occurs in Caribbean. None of species which could currently be suggested to exist at the base of the S. section Brevispicae (i.e. suggested to be relatives of S. guianensis , S. massonii) is endemic to South America – however, not all species recently described from Columbia by Leonardo Palacios-Duque have been studied by me.

Other S. section Brevispicae species are mostly found in South America, apart from S. faginea and S. massonii ( S. massonii is basal to the section according to the current study – S. faginea is not part of a monophyletic S. section Brevispicae ).

There is, however, a Central American clade apparently embedded in the section’s South American species: the closely related (CEJ Smith, 1954) S. guapilensis Standl. and S. tuerckheimii (only latter was included in the current study, and only the former is endemic to Central America). Overall the only Central American specimens 66 included in the current study embedded in phylogeny in South American clade are S. terniflora and S. tuerckheimii , both also found in South America.

Therefore, the only Central American endemic taxon of Sloanea that according to current knowledge is deeply embedded in a largely South American clade is S. guapilensis , and only if it really is a close relative of S. tuerckheimii . No molecular material is yet available for this species.

The only Caribbean species deeply bedded in clearly North American clade is S. berteroana .

4.5.3.2 Evidence for extinction in North America

With the above, knowledge, it is still impossible to identify any North American clades older than from the Miocene, and the phylogeographic analysis clearly suggests that almost all of the New World clades have a South America origin. While this could be explained due to very effective dispersal and migration across Sloanea clades from North America to South America during the Eocene. However, as the pattern is very clear, it seems more likely that the genus would have gone extinct in North America in the late Eocene, following an early Eocene dispersal to South America.

In particular support for the latter scenario is the near complete absence of the S. section Paniculi from North America. This paraphyletic group only has a single Caribbean representative, S. caribaea , even that not endemic. Also, interestingly, other poorly placed, obviously early diverging species are South American narrow endemics: S. monosperma and probably S. “myrmecophyta”. S. spathulata, though apparently rare, is a little more widespread. 67

The clade consisting S. faginea and S. dentata is probably much larger in reality, as it may include all species of the S. section Sloanea . These species are mainly South American, de-validating some of the arguments in previous section. With the very small sample size it is impossible to determine if South or North America is the ancestral state for this clade. Also, at least S. froesii Earle Sm. endemic to Brazil and S. chocoana Pal.-Duque endemic to Columbia are close relatives of S. faginea (CEJ Smith, 1954, Palacios-Duque, 2007). Similarly confusing case is the “ S. dussii clade”, which includes an unidentified sample from South America, and possibly also the South American S. echicarpa Uittien, a suspected close relative of S. dussii (CEJ Smith, 1954).

The unarmed clade lacks any particularly early diverging species that would occur in the previous North American plate. The Caribbean S. berteroana is placed here, but it has close relatives in South America (CEJ Smith, 1954).

There may be more cases where a genus has become extinct in North America in the Eocene, surviving in South America and Asia into the Oligocene. Interestingly, this is one explanation for fossil history of now strictly Asian/Australasian genus Musa (Berry, 1925, Boyd, 1992 for a review).

In conclusion all the basal species of the New World clade are North American. Dispersal to North America can only be shown to have taken place from the Miocene onwards. This means that the current clades in South America are of South American origin. This is in contrast to the phylogenetic pattern expected with the northern origin, but it could be explained with extinction of the genus in North America following dispersal to South America. As the southern origin scenario also requires extinctions in the Northern Hemisphere, it can be 68 concluded that the genus became temporarily extinct in North America during the Eocene.

4.5.4 Southern origin scenario

If the phylogeographic analysis results are taken as true, then the genus has originated in the Southern Hemisphere (Australasia, but see discussion above). Additional support for this scenario is found from other dispersals in the family ( Vallea , Crinodendron ) across the Antarctic that took place at a similar time period to the divergence of Sloanea .

A Gondwanan MRCA for the genus implies a divergence due to dispersal across the Antarctica – the Eocene/Oligocene dispersal route across the closing Caribbean Sea, and the Miocene dispersal across Wallace’s line leave little time for a northern dispersal. Such a late divergence would show the phylogeny would show a pattern where one of the clade is clearly embedded in another, which is not the case. Of course, the presence of Sloanea in Northern Hemisphere in fossil evidence does mean that the genus must have got there through long distance dispersal Together with a Southern Hemisphere MRCA this would suggest first of all a very early (ca. 63 Ma) dispersal from South America to North America, followed by a dispersal into Asia and extinction in North America and Europe. The fact that the Old World and New World clades receive similar ages of diversification in the dated phylogenies is strong evidence against this scenario, as the suggested scenario would show Old World clade diversifying some 30 Ma later than the New World clade.

Similar phylogenetic patterns have been seen in Myrtaceae and related genera which have been implied to have moved between continents through Antarctic dispersal (Sytsma et al. 2004). 69

As the picture remains somewhat confusing due to An obvious way of testing this scenario is an inclusion of species West from Wallace’s Line in further molecular analyses. To get a final confirmation for a southern MRCA for the genus, the species West from Wallace’s line should form clades no older than Miocene.

The wide dispersal of fossils in the Northern Hemisphere would need to be explained by long distance dispersal followed by extinction. It is particularly difficult to explain the fossils apparently from “Group E.” of the Old World clade in the Northern Hemisphere in the Oligocene (Kva•ek et al., 2001), though again, long distance dispersal remains a possibility.

Meanwhile, this is the scenario which is most strongly supported by phylogenetic evidence.

5 Conclusions ______

5.1 Phylogeny

The genus Sloanea is a strongly supported monophyletic clade within the family Elaeocarpaceae, strongly supported as a sister to a clade consisting of Aristotelia and Vallea . There is no support for Sloanea forming a clade with the genus Vallea .

The most exhaustive available evidence provided by the combined analysis of the current study as well as all Bayesian analysis mcc trees suggest that the genus Sloanea is divided into two monophyletic clades: the Old World taxa and the New World taxa, of which former is strongly supported. Morphology strongly supports this division. Taxa in the two regions are most obviously separated by floral characters (absence of petals in the Old World), but interestingly the New World taxa with flowers resembling the Old World taxa are not related to them, but have acquired petals independently.

In the New World, the S. subgenus Quadrisepala is basal. Within it the S. section Paniculi forms a grade in which the rest of the taxa have emerged from. Two species from other sections, S. monosperma ( S. sect. Corymo-racemi), S. spathulata ( S. sect. Brevispicae ) as well as the unplaced S. “ myrmecophyta ” have a similar basal position, but the position of these taxa does not get support from jackknife or posterior probability values, and varies between data sets. It is obvious, however, that they do not belong to any of the well defined clades within the genus.

The S. section Corymbo-racemi is monophyletic if S. dussii is excluded from it. S. dussii is sister to the S. 71 subg. Sloanea clade, also with obvious stipules. The S. sect. Corymbo-racemi can be further divided into two clades, one with mostly species that have a one-to-three- flowered inflorescence, plus S. synandra (for which there appears to be no morphological bases). The other one is characterised by absence of spines in fruits, though it does also include S. pubescens (also no obvious morphological support). These clades are robustly supported.

S. subgenus Sloanea is clearly monophyletic, but unfortunately the S. section Sloanea was not densely sampled. It appears that the S. sect. Sloanea may be embedded close to the base of the subgenus.

As the study concentrates on New World taxa, the relationships between the Old World groups could not be reliably assessed. Further sampling is needed to find out how the “groups” of Coode (1983) correspond to natural clades. In the New World, the sampling was dense enough to give a good appreciation of the evolution of sections and subgenera within the genus.

5.2 Phylogeography

The Phylegeogrpahic analyses completed suggest a Southern Hemisphere origin for the genus Sloanea . It is suggested to have originated from Australasia, but an alternative possibility of a MRCA in South America also seems very likely. The analyses suggest that the divergence of the genus into New and Old World clades is due to dispersal across the Antarctica some 37 Ma.

This scenario is contradicted by fossils. According to this evidence, Sloanea grew in North America ca. 63-40 Ma, possibly even earlier (Manchester & Kva•ek, 2009), as the only Northern Hemisphere representative in the family 72

(Elaeocaprus may have dispersed to West of Wallace’s Line at a later time). North America at this time was far from the Gondwanan land mass formed of Australia, Antarctic and South America, where the rest of family Elaeocarpaceae are found both currently and in fossil record, and no obvious channels of dispersal existed between the two land masses at this time. Later fossils from Europe (morphologically similar to some modern Old World taxa) can be linked to the North American fossils due to Eocene thermal maxima, which allowed dispersal between the two continents. Therefore, it is obvious that the genus arrived to North America by long distance dispersal. This suggests that an origin for the genus in North America is also possible, but this theory finds no support in phylogenetic analyses.

The dated phylogeny shows that the genus has separated into Old and New World clades at least 37 Ma (this is conservative estimate, as the North American taxa in the analyses have been placed in the stem of the genus). This date fits well a theory of origin of the genus in North America and dispersal from the northern route, or for division within Southern Hemisphere. The fact that Central American and Caribbean taxa are embedded in the phylogeny of South American Clade suggests that the second scenario is true.

The theory of a Northern MRCA for the genus remains intriguing due to the fossil evidence. A possibility remains that the genus became extinct in North America after a dispersal into both South America and the Old World. This could be due to climatic changes. Final evidence to separate these two potential scenario can be searched for by adding species from the Old World West of the Wallace’s Line.

Within the New World Clade, it is clear that the S. section Brevispicae originates from North America, as only species deeply bedded in its phylogeny are 73 exclusively South American. This can be explained by a Miocene dispersal into North America, where the section diversified. It is possible that the entire S. subgenus Sloanea originated in North America due to a Oligocene dispersal, but the current evidence is not strong enough to find this out. 74

6 Future work ______

The taxon sampling in the current study was wide. However, more species from the S. section Sloanea would be necessary to consolidate the position of this section and its early divergence from the S. section Brevispicae .

The support to some clades was not as strong as would be ideal. In particular, the support to the monophyly of New World taxa remains small. It may be impossible to confirm its monophyly. It may be very closely related to the Old World clade, as both appear to have migrated from North America in the Eocene (if the North American origin theory holds true). However, sequencing more regions from the DNA already extracted could well increase the support for various clades.

Further sequences may help to better determine the position of taxa within the New World that could not be reliably placed in the current study, i.e. S. caribaea , S. durissima , S. latifolia , S. monosperma , S. “myrmecophyta ” and S. spathulata .

Biogeographical work would greatly benefit from Old World material from Malesia/Asia north of Wallace’s line and Madagascar. In particular, the time of crossing of Wallace’s line should be obvious in dated phylogenies. If the North American origin for the genus is true, the Australasian taxa should form clades within the phylogeny of the rest of Old World Sloanea , diversifying no older than the Miocene. The origin of Madagascan species remains a mystery. If they are basal to Old World Sloanea , this could be implication that the genus has once been present in mainland Africa. DNA of S. rhodantha (Baker) Capuron from Madagascar is available from Missouri Botanic Gardens. Some new air-dried herbarium material from several Chinese taxa is available from RBGE 75 from 2000-2005. Considering the difficulty of acquiring sequences from herbarium material, silica gel dried material from Asia would be most useful, however, trying these relatively resent specimens would be a good start.

The sampling from Central America remains small. Further sampling from species endemic to this region would be very useful. RBG Edinburgh has samples identified as S. meianthera , S. cf. schippii Standl. and S. cf. petenensis Standl. & Steyerm. (all endemic to Central America) from 2004-2007.

The genus is currently subject to revision by Dr. Terence Pennington, RBG Kew. Once this work is done, it will be easier to do morphological analyses in lack of herbarium specimens. A parsimony analysis with morphological characters would be interesting in seeing how compatible molecular data is with morphology. The current study will contribute towards morphological analysis, as it may reveal importance of some characters that are not currently considered important in classification of the genus. In particular, support for S. dussii as a part of a sister clade to the S. subgenus Sloanea sought be searched. Also the placement of S. synandra in the umbellate clade and S. pubescens in the unarmed clade are very surprising results that require further attention from morphological studies.

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Appendix 1 – Samples used in the current study

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This table shows all samples used during the current study. Unsuccessful extractions are coloured grey. The fruit types, given for New World Sloanea species, are as follows: 1 = absent, 2 = detachable, 3 = stout, 4 = densely covered with short spines, 5 = flexible, 6 = curved (this is from literature sources, and may not be completely accurate – I particular, the types 2 and 3 seem to be sometimes occurring as variations of other fruit types).

Accession no Species Fruit type Country Type of Source, Collector no material AF299191 and Sloanea australis (Benth.) Australia N/A GenBank AF299244 F.Muell. (trnL-F only) DQ444656 Sloanea berteroana 1 Dominican N/A GenBank Choisy ex DC. (trnL-F only) Republic GUAD 85 Sloanea berteroana Guadeloup N/A Université des Antilles et de la e Guyane EDNA11-0021812 Sloanea brachytepala 1 Peru Silica RBG Kew, Terence Pennington Ducke 18055 EDNA11-0021902 Sloanea brenesii Standl. Costa Rica Hebrarium RBG Kew, Barry Hammel 20056 GUAD 91 Sloanea caribaea Krug & 3 (few) Guadeloup N/A Université des Antilles et de la Urb. e Guyane EDNA11-0021813 Sloanea crassifolia Earle Guyana Hebrarium RBG Kew, Hugh Clarke et al. Sm. 9383 EDNA11-0021814 Sloanea “cruciata ” 1? Peru Silica RBG Kew, Terence Pennington 18199 EDNA11-0021815 Sloanea davidsei Steyerm. Venezuela Herbarium RBG Kew, J ohn Wurdack & Lincoln Adderley 43260 EDNA11-0021922 Sloanea “dazae ” 1? Peru Silica RBG Kew, Terence Pennington 18061 EDNA11-0021923 Sloanea “dazae ” Peru Silica RBG Kew, Terence Pennington 18186 EDNA11-0021816 Sloanea “dazae ” Peru Silica RBG Kew, Terence Pennington 18239 GUAD 82 Sloanea dentata L. 3 Guadeloup N/A Université des Antilles et de la e Guyane 88

EDNA11-02285 Sloanea durissima Spruce 3 Peru Silica RBG Kew, Terence Pennington ex Benth. 18197 EDNA11-0021829 Sloanea ?durissima Peru Silica RBG Kew, Terence Pennington 18249 EDNA11-0021831 Sloanea ?durissima (trnL- Peru Silica RBG Kew, Terence Pennington F only) 18252 MART DU TRN and Sloanea dussii Urb. 4? Martinique N/A Université des Antilles et de la MART DUGN1 Guyane GUAD 93 Sloanea dussii Urb. Guadeloup N/A Université des Antilles et de la e Guyane EDNA11-02286 Sloanea eichleri K. Schum. French Hebrarium RBG Kew, Scott Mori et al. Guiana 14964 EDNA11-0021905 Sloanea faginea Standl. 3 Costa Rica Hebrarium RBG Kew, William Haber & Eric Bello 4268 EDNA11-0021906 Sloanea floribunda Spruce 1 Peru Silica RBG Kew, Terence Pennington ex Benth. 18228 EDNA11-0021817 Sloanea floribunda Peru Silica RBG Kew, Terence Pennington 18253 EDNA11-02287 Sloanea fragrans Rusby Ecuador Herbarium RBG Kew, Terence Pennington et al. 12245 EDNA11-0021818 Sloanea garckeana K. 4 Brazil Herbarium RBG Kew, Geovane? Siqueira Schum. 480 EDNA11-0021819 Sloanea grandiflora Sm. 6 Guyana Herbarium RBG Kew, Tom Hollowell et al. (ITS only) 457 EDNA11-0021909 Sloanea guianensis (Aubl.) 3 Peru Silica RBG Kew, Terence Pennington Benth. 18184 GUY 157 Sloanea ?guianensis French N/A Université des Antilles et de la Guiana Guyane EDNA11-02288 Sloanea ilicifolia Urb. Dominican Herbarium RBG Kew, Pimental & Cabral Republic 1063 EDNA11-0021820 Sloanea jamaicensis 4 Jamaica Herbairum RBG Kew, Grady Webster & Hook. Kenneth Wilson 5132 EDNA11-0021821 Sloanea ?kuhlmannii 4 Brazil Silica RBG Kew, Daniela Zappi et al. Ducke 1425 DQ444655 Sloanea langii F. Muell. Australia N/A GenBank (trnL-F only) EDNA11-02289 Sloanea latifolia (Rich.) K. 1/3 Peru Silica RBG Kew, Terence Pennington Schum. 18176 EDNA11-0021912 Sloanea latifolia Peru Silica RBG Kew, Terence Pennington 18185 N/A Sloanea lepida Tirel New N/A James Cook University Caledonia N/A Sloanea macbrydei F. Australia N/A James Cook University Muell. EDNA11-0021822 Sloanea macrophylla Venezuela Hebrarium RBG Kew, Ron Liesner et al. Benth. ex Turcz. 20919 89

GUAD 84 Sloanea massonii Sw. 5 Guadeloup N/A Université des Antilles et de la e Guyane EDNA11-0021914 Sloanea medusula K. Costa Rica Hebrarium RBG Kew, Damon Smith et al. Schum. & Pittier 1300 N/A Sloanea montana A.C. Sm. New N/A James Cook University Caledonia EDNA11-0021823 Sloanea monosperma 2 Brazil Herbarium RBG Kew, Adelaide? Kegler 417 Vell. EDNA11-0021830 Sloanea “myrmecophyta ” ? Peru Silica RBG Kew, Terence Pennington 18250 EDNA11-0021916 Sloanea parviflora Planch. 5 Guyana Herbarium RBG Kew, Marion Jansen- ex Benth. Jacobs et al. 2308 EDNA11-0021827 Sloanea petalata D.Samp. 4 Brazil Herbarium RBG Kew, Hermogenes Leitao- & V.C.Souza Filho 12087 EDNA11-0021824 Sloanea picapica Standl. Costa Rica Herbarium RBG Kew, Damon Smith et al. 1266 EDNA11-02291 Sloanea porphyrocarpa 2 Peru Silica RBG Kew, Terence Pennington Ducke 18407 EDNA11-0021918 Sloanea pubescens Radlk. 5 Peru Silica RBG Kew, Terence Pennington 18173 EDNA11-0021825 Sloanea pubescens Peru Silica RBG Kew, Terence Pennington 18233 EDNA11-0021835 Sloanea rhodantha Madagasca Hebrarium RBG Kew, Gordon McPherson (Baker) Capuron r & Henk van der Werff 16440 EDNA11-0021826 Sloanea robusta Uittien 5 (2?) Peru Silica RBG Kew, Terence Pennington 18181 DQ444657 and Sloanea sogerensis Baker Papua New N/A GenBank DQ448658 f. Guinea EDNA11-02292 Sloanea spathulata Earle 3 Peru Silica RBG Kew, Terence Pennington Sm. 18169 EDNA11-02290 Sloanea synandra Spruce 1 French Herbarium RBG Kew, Scott Mori 25655 ex Benth. Guiana EDNA11-01679 Sloanea terniflora (Sessé 4 Costa Rica N/A Dan Janzen (355) & Moc. ex DC.) Standl. EDNA11-01680 Sloanea terniflora (trnL-F Costa Rica N/A Dan Janzen (684) only) EDNA11-0021834 Sloanea tuerckheimii 5/6 Costa rica Herbarium RBG Kew, Brian Jacobs 3123 Donn. Sm. EDNA11-0021833 Sloanea uniflora D.Samp. 4 Brazil Silica RBG Kew, Daniela Zappi et al. & V.C.Souza 1491 DQ444654 and Sloanea woollsii F. Muell. Australia N/A GenBank DQ448657 EDNA11-0022672 Sloanea sp. 3 Peru Silica RBGE, Aniceto Daza 5454 DQ444658 and Sloanea sp. Papua New N/A GenBank DQ448659 Guinea GUY 142 Sloanea sp. French N/A Université des Antilles et de la 90

Guiana Guyane GUY 155 Sloanea sp. French N/A Université des Antilles et de la Guiana Guyane GUY 156 Sloanea sp. French N/A Université des Antilles et de la Guiana Guyane GUY 158 Sloanea sp. French N/A Université des Antilles et de la Guiana Guyane EDNA11-0021832 sp. (Euphorbiaceae) Silica RBG Kew, Terence Pennington 18260 EDNA11-0021828 sp. (Euphorbiaceae) Silica RBG Kew, Terence Pennington 18403 DQ444678 and Aceratium concinnum (S. - N/A GenBank DQ448684 Moore) C.T. White DQ444681 and Aceratium ferrugineum - N/A GenBank DQ448685 C.T. White DQ444677 and Aceratium ledermannii - N/A GenBank DQ448683 Schltr. AF299162, Ackama rosifolia A. Cunn. - N/A GenBank AF299215 and DQ499074 DQ444662 and Aristotelia fruticosa Hook. - N/A GenBank DQ448662 f. DQ444663 and Aristotelia serrata Oliv. - N/A GenBank DQ448663 DQ444661 and Aristotelia australasica F. - N/A GenBank DQ448661 Muell. DQ444659 and Aristotelia peduncularis - N/A GenBank DQ448664 Hook. f. DQ444660 and Aristotelia chilensis Stuntz - N/A GenBank DQ448660 DQ499071 Ceratopetalum - N/A GenBank succirubrum C.T. White DQ444666 and Crinodendron - N/A GenBank DQ448674 hookerianum Gay DQ444665 and Crinodendron patagua - N/A GenBank DQ448673 Molina DQ444668 and Dubouzetia caudiculata - N/A GenBank DQ448675 Sprague DQ444670 and Dubouzetia kairoi Coode - N/A GenBank DQ448677 DQ444669 and Dubouzetia saxatilis - N/A GenBank DQ448676 A.R.Bean & Jessup DQ444689 and Elaeocarpus angustifolius - N/A GenBank DQ448689 Blume DQ444685 and Elaeocarpus bancroftii F. - N/A GenBank DQ448687 Muell. 91

DQ444690 and Elaeocarpus - N/A GenBank DQ448690 ferruginiflorus C.T. White DQ444686 and Elaeocarpus hookerianus - N/A GenBank DQ448688 Raoul DQ444684 and Elaeocarpus largiflorens - N/A GenBank DQ448686 C.T. White subsp. retinervis B. Hyland & Coode DQ444693 and Elaeocarpus williamsianus - N/A GenBank DQ448691 Guymer DQ444676 and Elaeocarpus sp. ‘Rocky - N/A GenBank DQ448682 Creek’ DQ444672 and Peripentadenia mearsii - N/A GenBank DQ448679 (C.T.White) L.S.Sm. DQ444671 and Peripentadenia phelpsii - N/A GenBank DQ448678 B.Hyland & Coode DQ444675 and Sericolea calophylla (Ridl.) - N/A GenBank DQ448681 Schltr. subsp. grossiserrata Coode DQ444674 and Sericolea gaultheria - N/A GenBank DQ448680 Schltr. DQ444698 and Tetratheca ciliata Lindl. - N/A GenBank DQ448669 DQ444695 and Tetratheca filiformis - GenBank DQ448666 Benth. DQ444696 and Tetratheca juncea Sm. - N/A GenBank DQ448667 DQ444697 and Tetratheca parvifolia Joy - N/A GenBank DQ448668 Thomps. DQ444699 and Tetratheca pubescens - N/A GenBank DQ448670 Turcz. DQ444700 and Tetratheca stenocarpa - N/A GenBank DQ448671 J.H. Willis DQ444701 and Tremandra diffusa R. Br. - N/A GenBank DQ448672 ex DC. DQ444664 and Vallea stipularis L. f. - N/A GenBank DQ448665

Appendix 2 – Aligned ITS1-2 matrix

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The ITS data matrix contains 903 bases, of which base numbers 254-304 were excluded from all analyses as they could not be reliably aligned. Gap characters were not coded. Missing data is indicated with ?. Gaps are indicated with -. 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107

Appendix 3 – Aligned partial trnL-F matrix ______

The ITS data matrix contains 1210 bases. Additionally, 24 gap characters were used in parsimony analyses. Missing data is indicated with ?. Gaps are indicated with -. 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125

126

Appendix 4 – Additional trees ______

Some trees are shown here in addition to the most informative trees included in the main part of the thesis.

127

Node bars showing 95% date confidence of the ITS1-2 mcc dated tree. 128

Node bars showing 95% date confidence of the mcc dated tree from combined data. 129

State sets of the phylogeographic analysis mcc tree using original data. 130

Confidence values for the previous tree. 131

State sets of a mock analysis mcc tree, where Ceratopetalum has been given a state SA instead of Aus. 132

Confidence values for the previous tree.