AN ABSTRACT OF THE DISSERTATION OF

Brian A. Atkinson for the degree of Doctor of Philosophy in Botany and Plant Pathology presented on April 13, 2017. Title: Unearthing the Cretaceous Diversification of Cornales.

Abstract approved:

______Aaron I. Liston

The order Cornales (dogwoods) is the earliest diverging lineage within the most diverse group of flowering plants, the asterids (>80,000 species). Although molecular phylogenetics have significantly improved our understanding of cornalean systematics, early phylogenetic relationships remain uncertain due to an initial rapid radiation. The fossil record of Cornales is extensive and indicates that the Late Cretaceous was an important time for the initial diversification of the order. Moreover, molecular divergence time estimates suggest that most of the major cornalean lineages diverged between 100-

84 Ma. Many cornalean fossils are represented by fruits, which contain numerous systematically informative characters. Therefore, cornalean fossil fruits are crucial for reconstructing the early evolution of Cornales.

The over-arching goal of this dissertation is to reconstruct the early evolution of

Cornales by utilizing the fossil record. There are three main objectives: 1) increase the taxon sampling density of Late Cretaceous cornalean fruits, 2) develop new hypotheses on the early evolution of Cornales, and 3) elucidate the initial diversification of the order by conducting phylogenetic and quantitative morphological analyses.

Herein, seven taxa, six of which are new, are characterized based on permineralized fruits from several Upper Cretaceous deposits. All fossils described in this study are anatomically preserved within shallow-marine calcareous concretions.

Specimens were prepared using the cellulose acetate peel technique and studied using light microscopy.

In Chapter 2 two permineralized cornalean fruits from the Campanian of

Washington Stae are described. Fruits are ellipsoidal, tetra-locular, and consist of valvate endocarps. Each locule is crescent-shaped in cross section with one apically attached seed and have valves that are restricted to the apical third of the endocarp. Endocarp tissue consists of isodiametric to elongate sclereids and secretory cavities. Vascular bundles occur in rows in each septum, there are rows of vascular bundles. These fossil fruits display unique character mosaic indicative of several cornalean families, but not conforming to any family or fruit from known extinct taxa. Therefore, the fossil fruits are assigned to a new cornalean genus and species, Suciacarpa starrii gen. et sp. nov.

In Chapter 3, a permineralized fossil fruit assignable to Cornus subg. Cornus is described from the Campanian of Vancouver Island, British Columbia, Canada. The fossil fruit consists of a tri-locular woody endocarp with dorsal germination valves.

Endocarp contains elongate and isodiametric sclereids and secretory cavities. Central vasculature is lacking and the endocarp is interpreted to have been vascularized by bundles located along the outer periphery of the septa. There is one seed per locule. The interpreted vascular pattern and presence of secretory cavities strongly suggest that the fossil fruit is assignable to Cornus subg. Cornus, with a morphology and anatomy o is

most similar to Cornus piggae, which was originally described from the Paleocene of

North Dakota. This Cretaceous fossil fruit is the earliest evidence for crown-group

Cornaceae and its discovery in western Canada contradicts the hypothesis that Cornaceae originated in Europe.

In Chapter 4, two new taxa are described based permineralized fruits from the

Campanian Shelter Point locality of Vancouver Island. One fruit represents a new species, Suciacarpa xiangae sp. nov., that is represented by a large ovoid tetra-locular sclerenchymatous endocarp. Locules are crescent shaped in cross section and accompanied by short dorsal germination valves. Valves consist of small isodiametric sclereids and numerous large secretory cavities. The septa and central axis are composed of transverse fibers and elongate sclereids. Central vascular bundles are lacking, but there are several small bundles within the septa. There is one apically attached seed in each fertile locule. The second fruit type represents a new cornalean genus and species,

Sheltercarpa vancouverensis gen. et sp. nov., that is characterized by a single smooth tetra-locular woody endocarp. The endocarp is made up of isodiametric and, to a lesser extent, elongate sclereids. Germination valves are short. The fruit lacks a central vascular bundle. Fruits of Suciacarpa xiangae and Sheltercarpa vancouverensis display character mosaics that are reminiscent of at least two different major cornalean clades. The cornalean fruit diversity from the Late Cretaceous suggests that the Campanian was a critical time for the initial diversification of Cornales.

Chapter 5, three new taxa from the early Coniacian (~89 Ma) of North America, are characterized: Eydeia vancouverensis sp. nov., Obamacarpa edenensis gen. et sp.

nov., and Edencarpa grandis gen. et sp. nov. The fruits of each species have thick-walled woody endocarps with dorsal germination valves, one apically attached seed per locule, several rows of vascular bundles in each septum, but no central vascular bundle.

However, each species differs from the others, and from previously described cornaleans in endocarp size, sculpturing, and anatomy. The taxa characterized in this chapter are the same age as the previously recognized oldest cornalean, Hironoia fusiformis, from the early Coniacian of Japan. The diversity, geographic distribution, and stratigraphic age of these early cornalean fruits strongly suggest that the initial diversification of Cornales and asterids was well underway by the Coniacian.

In Chapter 6, the Cretaceous diversification of Cornales is reconstructed using phylogenetic, morphospace, and disparity analyses. A cladistic matrix consisting of 77 fruit morphological characters and 58 taxa (35 extant, 23 extinct) was constructed. This matrix was used for two main phylogenetic parsimony analyses: 1) fossil inclusive and 2) fossil exclusive (living taxa only). The fossil inclusive and exclusive parsimony analyses resulted in trees with a basal grade consisting of Loasaceae, Hydrangeaceae,

Hydrostachyaceae, Grubbiaceae, and Curtisiaceae, respectively, leading to a core cornalean clade comprising Cornaceae, Alangiaceae, Nyssaceae, Mastixiaceae, and

Davidiaceae. In the analysis excluding extinct taxa, relationships among the core cornalean clades are largely unresolved. However, inclusion of fossils in the analysis results in more highly resolved relationships among Cornaceae, Alangiaceae and a clade consisting of Nyssaceae, Mastixiaceae, and Davidiaceae. These results clearly demonstrate that the novel character mosaics found within Cretaceous cornalean fruits

play a vital role in resolving deep-node relationships within Cornales. In addition, a modified fruit morphological matrix (drupaceous fruits only) was used for morphospace and disparity analyses. The results of these analyses indicate that the morphological diversity of cornalean fruits has significantly changed and increased since the Late

Cretaceous. A combination of ecological and developmental factors most likely explain the morphological diversification of Cornales that occurred during the Paleogene (66-56

Ma).

© Copyright by Brian A. Atkinson April 13, 2017 All Rights Reserved

Unearthing the Cretaceous Diversification of Cornales.

by Brian A. Atkinson

A DISSERTATION

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Presented April 13, 2017 Commencement June 2017

Doctor of Philosophy dissertation of Brian A. Atkinson presented on April 13, 2017.

APPROVED:

______Major Professor, representing Botany and Plant Pathology

______Head of the Department of Botany and Plant Pathology

______Dean of the Graduate School

I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request.

______Brian A. Atkinson, Author

ACKNOWLEDGEMENTS

It would not have been possible for me to make it this far without all the mentoring, training, support, and love that I was blessed with throughout my time here.

Firstly, I must thank my two primary advisors, Drs. Ruth Stockey and Gar

Rothwell. I cannot begin to describe how grateful I am for their outstanding training, mentoring, support, and boundless patience. With fondness, I will remember all the laughs, meals (especially the sushi), and beers we shared.

Sincere appreciation goes to my third advisor, Dr. Aaron Liston, who has provided me so much great advice and support. Thanks to the other two members of my committee, Drs. Barb Lachenbruch and Dee Denver. I am truly grateful for all the genuine conversations we had and the outstanding professional and life advice they have given me.

There are several other people in Botany and Plant Pathology that have made life easier for me during my tenure here. Appreciation is expressed to Dianne Simpson and

Carleen Nutt for helping me navigate the inner workings of the department and university. Special thanks to Blaine Baker for always looking out for me. I would also like to thank Drs. Stephen Meyers and Paul Severns for their encouragement and support.

I have met so many great friends whom are fellow graduate students in this department and I’m so grateful for all of them! Sincere thanks to my past and current roommates, Kevin Weitemier, Javier Tabima, and Tyler Schappe for their technical assistance and overwhelming support. I must also express gratitude to Alisha Quandt for her wisdom and priceless peer mentoring.

My success with fieldwork and obtaining specimens for study would not have been possible without the help of Graham and Tina Beard, Dr. Dirk Meckert, Rick Ross,

Ludo Le Renard, Dr. Nathan Jud, Ashley Klymiuk, Steve Karafit, Stan Yankowski, and

Dr. Steve Manchester. Further appreciation is expressed to Drs. Max Weigend, Steven

Manchester, and Rudy Serbet for sending images for data collection.

I would also like to thank my paleobotany friends and colleagues, Kelly

Matsunaga, Dori Contreas, Alex Bippus, as well as Drs. Nathan Jud, Carla Harper,

Selena Smith, Mihai Tomescu, Ignacio “Nacho” Escapa, Randal Mindell, Mike Dunn,

Steven Manchester, Thomas Taylor, Edie Taylor, and Rudy Serbet for their encouragement throughout my doctoral pursuits.

Special thanks to Drs. Harufumi Nishida and Toshihiro Yamada for their aide in fieldwork in Hokkaido, Japan. I realize that they took a lot of time out of their personal schedules to help me obtain fossils in museums and in the field, and for that I am sincerely grateful.

I must also acknowledge my funding sources. Thanks to the National Science

Foundation Graduate Research Program for financially supporting my dissertation for the past three years. I have also received research funds from the Department of Botany and

Plant Pathology including the Moldenke Plant Systematics Endowment, the Leslie and

Vera Gottlieb Research Fund, and the Anita S. Summers Graduate Student Travel Fund.

Additional funds for travel were provided by the International Organisation of

Paleobotany and the Paleobotanical Section of the Botanical Society of America. Thanks to Pankaj Jaiswal for providing me funding during my first summer here. Further

appreciation goes to the herbarium for giving me financial support when I first moved to

Corvallis just before the official start of my Ph.D.

A very special shout out goes to LaTreese Denson, Gabe Sheoships, Maritza

Mendoza, and Sean Roone. Even though they have all graduated and moved on to better things elsewhere, I still feel their love and support, and they will always be considered crew.

I further wish to express appreciation to the OSU Black Graduate Student

Association and black community of the Corvallis and Albany area for their overwhelming support and love. Over these past five years there has been so much going on with racial injustice, which had been a source of conflict for me because I was constantly in the lab working, and it felt like I was doing nothing in response. However, a friend of mine, Daryl Adkins, once said, “When we [black students] obtain our graduate degrees in a system that was never meant for us, we are actually performing a beautiful act of defiance.” Those words immediately sparked a light in me that I will hold on to for the rest of my life.

Much love goes to my family for being my biggest supporters. I would like to thank my mom for her unconditional love and encouragement, my father for making sure that I never take myself too seriously, my sister, Jae, for her endearing support, and my dogs, Azia and Zoey, for their boundless love. My deepest expression of love goes to my grandmother who has always believed in me.

Last, but certainly not least, I must thank my best friend, Ana Barros. Through highs and lows she was always there for me.

CONTRIBUTION OF AUTHORS

Chapter 3: Drs. Ruth A. Stockey and Gar W. Rothwell assisted in editing the manuscript.

Chapter 4: Drs. Ruth A. Stockey and Gar W. Rothwell helped design the study and edit the manuscript.

Chapter 5: Drs. Ruth A. Stockey and Gar W. Rothwell aided in editing the manuscript.

TABLE OF CONTENTS

Page

Chapter 1: Introduction ...... 1

The Cretaceous record: Initial phases of angiosperm diversification ...... 3

Asterids ...... 4

Cornales: The dogwood order ...... 5

Content of dissertation: Pursued goals and objectives ...... 9

Chapter 2: Early diverging asterids of the Late Cretaceous: Suciacarpa starrii gen. et sp. nov. and the initial radiation of Cornales...... 20

Introduction ...... 21

Materials and methods ...... 22

Systematic paleobotany ...... 24

Discussion ...... 28

Cornaceae/Alangiaceae and the NMD group ...... 29

Evolutionary patterns within Cornales ...... 37

Evolutionary implications of Suciacarpa ...... 38

Literature cited ...... 41

Chapter 3: Cretaceous origin of dogwoods: an anatomically preserved Cornus (Cornaceae) fruit from the Campanian of Vancouver Island...... 60

Introduction ...... 61

Materials and methods ...... 62

Systematic paleobotany ...... 63

TABLE OF CONTENTS (Continued)

Page

Discussion ...... 65

Early evolutionary patterns of dogwoods and other cornaleans...... 69

Conclusions ...... 72

Literature cited ...... 74

Chapter 4: The early diversification of Cornales: Permineralized cornalean fruits from the Campanian (Upper Cretaceous) of western North America...... 84

Introduction ...... 85

Material and methods ...... 87

Systematic paleobotany ...... 88

Discussion ...... 92

Affinities of Suciacarpa xiangae...... 93

Affinities of Sheltercarpa vancouverensis...... 94

Initial diversification of Cornales ...... 97

Conclusions ...... 100

Literature cited ...... 102

Chapter 5: Tracking the initial diversification of asterids: Anatomically preserved cornalean fruits from the early Coniacian (Late Cretaceous) of western North America...... 119

Introduction ...... 120

Materials and methods ...... 121

Systematic paleobotany ...... 122

TABLE OF CONTENTS (Continued)

Page

Discussion ...... 128

Comparison of Eden Main taxa to extant cornaleans ...... 129

Affinities of Eydeia vancouverensis ...... 131

Comparison of Obamacarpa and Edencarpa to extinct taxa ...... 132

Implications for the initial radiation of asterids ...... 133

Conclusions ...... 136

Literature cited ...... 138

Chapter 6: Exploring the initial phylogenetic and morphological diversification of Cornales...... 160

Introduction ...... 161

Materials and methods ...... 162

Phylogenetic analyses ...... 163

Morphospace and disparity analyses ...... 164

Results ...... 165

Phylogenetic analyses ...... 166

Time-scaled phylogenetic tree ...... 167

Morphospace and disparity analyses of cornalean drupaceous fruits ...... 167

Discussion ...... 168

Inclusion vs. exclusion of fossil taxa in phylogenetic analyses ...... 168

Phylogenetic relationships ...... 169

General trends in cornalean fruit evolution ...... 171

TABLE OF CONTENTS (Continued)

Page

Timing of the initial diversification of Cornales ...... 173

Morphological diversification of drupaceous fruits ...... 174

Conclusions ...... 176

Chapter 7: Conclusions ...... 192

Bibliography ...... 197

Appendices ...... 215

Appendix 1: Cornalean fruit characters ...... 216

Appendix 2: R code for time scaling, morphospace, and disparity analyses ...... 231

LIST OF FIGURES

Figure Page

Figure 2.1. Suciacarpa starrii gen. et sp. nov...... 46

Figure 2.2. Suciacarpa starrii gen. et sp. nov...... 48

Figure 2.3. Suciacarpa starrii gen. et sp. nov...... 50

Figure 2.4. Suciacarpa starrii gen. et sp. nov...... 52

Figure 2.5. Three-dimensional reconstruction of Suciacarpa starrii gen. et sp. nov...... 54

Figure 2.6. Structure of extant Cornus fruits representing the cornelian cherry clade...... 55

Figure 2.7. Structure of extant fruits representing Nyssaceae, Mastixiaceae, and Davidiaceae...... 56

Figure 3.1. Permineralized fruit of Cornus...... 78

Figure 3.2. Permineralized fruit anatomy of Cornus...... 79

Figure 3.3. Seed anatomy of Cornus...... 81

Figure 4.1. Suciacarpa xiangae sp. nov...... 107

Figure 4.2. Suciacarpa xiangae sp. nov...... 109

Figure 4.3. Suciacarpa xiangae sp. nov...... 111

Figure 4.4. Suciacarpa xiangae sp. nov...... 113

Figure 4.5. Sheltercarpa vancouverensis gen. et sp. nov...... 114

Figure 5.1. Eydeia vancouverensis sp. nov...... 145

Figure 5.2. Eydeia vancouverensis sp. nov...... 147

Figure 5.3. Obamacarpa edenensis gen. et sp. nov...... 149

Figure 5.4. Obamacarpa edenensis gen. et sp. nov...... 151

Figure 5.5 Edencarpa grandis gen. et sp. nov...... 153

Figure 5.6. Edencarpa grandis gen. et sp. nov...... 155

LIST OF FIGURES (Continued)

Figure Page

Figure 6.1. Strict consensus tree computed from most parsimonious trees obtained from fossil inclusive fruit morphological data set showing relationships in Cornales...... 186

Figure 6.2. Strict consensus tree of most parsimonious trees obtained from fossil exclusive (i.e., living species only) fruit morphological data set...... 187

Figure 6.3. Time-scaled phylogenetic tree of Cornales...... 188

Figure 6.4. Two-dimensional morphospace visualization of drupaceous cornalean fruits...... 189

Figure 6.5. Drupaceous cornalean fruit disparity through time...... 191

LIST OF TABLES

Table Page

Table 2.1. Comparison of endocarp structure among similar cornalean taxa...... 57

Table 3.1. General endocarp comparisons of dogwood fruits and close relatives...... 82

Table 3.2. Endocarp morphology of cornelian cherries, Cornus subgenus Cornus...... 83

Table 4.1. Comparison of endocarp structure among similar cornalean taxa ...... 116

Table 5.1. Comparison of endocarp structure among similar fossil and extant cornalean species...... 157

Table 6.1. Non-parametric PERMANOVA test for statistical significance among time bins...... 190

DEDICATION

To my ancestors.

1

CHAPTER 1: INTRODUCTION

2 The Cretaceous (145-66 Ma) was a critical time for the initial diversification of flowering plants (Hickey and Doyle, 1977; Hughes et al., 1991; Hughes, 1994; Friis et al., 2006, 2011; Doyle, 2015; Herendeen et al., 2017). During this period, many extant angiosperm lineages radiated rapidly. Unfortunately, these ancient evolutionary radiations have made it difficult for molecular phylogenetic analyses to resolve deep- node relationships within several flowering plant groups (Bremer et al., 2002; Xiang et al., 2002; Fan and Xiang, 2003; Judd and Olmstead, 2004; Soltis and Soltis, 2004;

Schönenberger et al., 2005; Lens et al., 2007; Wang et al., 2009; Barrett et al., 2014; Stull et al., 2015; Sun et al., 2015). One method to clarify ancient rapid radiations within angiosperms is to significantly increase the amount of genomic data used for phylogenetic analysis. While molecular studies using large scale genomic data sets have had some success in resolving deep nodes (e.g. Jian et al., 2008; Wang et al., 2009), there are still several diverse flowering plant clades in which early evolutionary relationships remain uncertain (e.g., Schönenberger et al., 2005; Xiang et al., 2011; Barrett et al., 2014;

Stull et al., 2015; Sun et al., 2015), limiting our understanding of the overall pattern of angiosperm phylogeny.

The fossil record is an essential data source for elucidating ancient evolutionary patterns and relationships of angiosperms (Donoghue et al., 1989; Lupia, 1999; Crepet et al., 2004; Rothwell and Nixon, 2006; Friis et al., 2006, 2011; Manos et al., 2007; Crepet,

2008; Martínez-Millán, 2010; Doyle and Upchurch, 2014; Doyle, 2015). Basal relationships can be resolved by directly including fossil species represented by systematically informative organs that contain data reflecting ancient and rapid

3 evolutionary patterns of divergence (Donoghue et al., 1989; Manos et al., 2007).

Furthermore, placing fossils within a phylogenetic context provides an opportunity to independently test phylogenetic hypotheses based solely on molecular data of extant taxa

(Donoghue et al., 1989; Crepet et al., 2004; Rothwell and Nixon, 2006; Manos et al.,

2007; Lee and Palci, 2015; Gernandt et al., 2016). Therefore, by thoroughly sampling

Cretaceous deposits, it is possible to recover fossils that provide vital information for reconstructing the early evolutionary history of angiosperms.

The Cretaceous record: Initial phases of angiosperm diversification

The earliest flowering plants are represented by monoaperturate pollen from the

Valanginian (~ 133 Ma) of Israel (Brenner, 1996) and upper Hauterivian (~ 132 Ma) of southern England (Hughes et al., 1991; Herendeen et al., 2017). By the Aptian (~ 124-

113 Ma) and Albian (113-100.5 Ma), macrofossils representing all major flowering plant groups appear in the fossil record: ANITA (basal) grade angiosperms, eumagnoliids, monocots, and eudicots (Doyle, 1992, 2015, Friis et al., 1994, 1999, 2004, 2009, 2015,

2016a; Hughes, 1994; Sun et al., 1998; Sun, 2002; Heimhofer et al., 2007; Doyle and

Endress, 2010; Jud, 2015; Herendeen et al., 2017). These fossils indicate that the initial phase of angiosperm diversification took place during the Early Cretaceous (145-100.5

Ma) and involved all major flowering plant groups (Crepet, 2008; Crepet and Niklas,

2009; Friis et al., 2011; Doyle, 2015; Herendeen et al., 2017).

The Late Cretaceous (100.5-66 Ma) was an important time for the evolution of the core eudicot clade (Friis et al., 2006, 2016b). Core eudicots comprise over 70% of extant angiosperm species including the diverse rosid (70,000 extant spp.) and asterid (80,000

4 extant spp.) clades, as well as several smaller lineages (Magallón et al., 1999; Judd and

Olmstead, 2004; Moore et al., 2010). Early diverging core eudicot lineages (e.g.,

Gunnerales and Dilleniaceae) and rosids diverged by the Cenomanian (100.5-93.9 Ma) and subsequently radiated throughout the Late Cretaceous (Doyle and Robbins, 1977;

Basinger and Dilcher, 1984; Nishida, 1994; Friis et al., 2006, 2011, 2016b). These fossils clearly indicate that the initial diversification of core eudicots was well underway by the

Turonian (93.9-89.8 Ma), hailing the second stage of the initial diversification of angiosperms. However, fossil data on the early evolution of asterids from this time interval is limited (Friis et al., 2011).

Asterids

With over 80,000 species, the asterid clade encompasses nearly one-third of extant angiosperm species diversity (Magallón et al., 1999; Albach et al., 2001), and therefore elucidating the early diversification of asterids is essential for furthering our understanding of the evolution of eudicots and flowering plants as a whole. The asterid clade comprises two monophyletic groups, the campanuliids and the lamiids (euasterids), as well as a basal grade consisting of the orders Cornales and Ericales (Magallón et al.,

1999; Albach et al., 2001; Bremer et al., 2002; Judd and Olmstead, 2004; Angiosperm

Phylogeny Group, 2016). Due to ancient rapid radiations, the early evolutionary relationships and patterns within lamiids, Ericales, and Cornales are not well resolved or supported (Magallón et al., 1999; Fan and Xiang, 2003; Judd and Olmstead, 2004;

Schönenberger et al., 2005; Xiang et al., 2011; Stull et al., 2015), thus our understanding of the initial diversification of asterids is poor. Cornales in particular, holds an important

5 phylogenetic position as the earliest diverging asterid lineage (Albach et al., 2001;

Bremer et al., 2002; Judd and Olmstead, 2004; Angiosperm Phylogeny Group, 2016).

Therefore, elucidating early cornalean evolution is essential for understanding the initial diversification of asterids.

Cornales: The dogwood order

The order Cornales is a relatively small, but morphologically diverse clade consisting of 605 living species many of which are commonly known as dogwoods

(Eyde, 1963, 1988; Hufford, 2001; Weigend et al., 2004; Yembaturova et al., 2009;

Xiang, 2013). Molecular studies have significantly improved our understanding of the systematics and evolution of the order (Xiang et al., 1998, 2002, 2011; Fan and Xiang,

2003; Schenk and Hufford, 2010) and suggest that Cornales contains ten families within five major clades: 1) Cornaceae, Alangiaceae; 2) Nyssaceae, Mastixiaceae, Davidiaceae;

3) Grubbiaceae, Curtisiaceae, 4) Hydrangeaceae, Loasaceae; 5) Hydrostachyaceae (Xiang et al., 1993, 2002, 2011; Fan and Xiang, 2003). However, deep-node relationships among these major clades are inconsistent and/or weakly supported in the results of molecular analyses (Magallón et al., 1999; Fan and Xiang, 2003; Judd and Olmstead, 2004; Xiang et al., 2011; Xiang, 2013).

Molecular divergence-time estimates indicate that crown-group Cornales diverged during the Cretaceous (Bremer et al., 2004; Magallón and Castillo, 2009; Schenk and

Hufford, 2010; Xiang et al., 2011; Magallón et al., 2015; Wikström et al., 2015). Many of these estimates range from the Early Cretaceous (~ 112 Ma) to well into the Late

Cretaceous, spanning the time interval from ~100 to ~72 Ma. Xiang et al. (2011)

6 conducted the most recent and comprehensive analysis of Cornales estimating that the order diverged and rapidly radiated into its five major clades between 100 to 84 Ma

(Xiang et al., 2011), thus supporting the hypothesis of an initial rapid radiation early in the Late Cretaceous. This rapid radiation, however, has made it difficult to resolve basal relationships within Cornales (Fan and Xiang, 2003; Xiang et al., 2011) and molecular data from extant taxa alone appear to be insufficient for reconstructing the initial diversification of the order (Xiang et al., 2002).

The reported fossil record of Cornales is extensive, but it is mostly concentrated in Cenozoic (66 Ma to Recent) deposits (Reid and Chandler, 1933; Kirchheimer, 1939;

Eyde and Barghoorn, 1963; Dilcher and Quade, 1967; Eyde et al., 1969; Eyde, 1988,

1997; Mai, 1993; Manchester, 1994, 2002, Manchester et al., 1999, 2009, 2010;

Manchester and Hickey, 2007; Pavlyutkin, 2009; Noll, 2013). Members from all major groups, excluding Hydrostachyaceae, are present by the end of the Paleogene (66-56

Ma). More specifically, Cornus L. (Cornaceae), Alangium Lamk. (Alangiaceae), Davidia

Baillon (Davidiaceae), Nyssa L. (Nyssaceae), Mastixia Blume (Mastixiaceae), Curtisia

Aiton (Curtisiaceae), and Hydrangea L.(Hydrangeaceae) are present at this time (Reid and Chandler, 1933; Dilcher and Quade, 1967; Manchester, 1994, 2002; Tiffney and

Haggard, 1996; Manchester et al., 2009, 2010). There are several other fossils representing extinct genera that have been described from concordant deposits (Reid and

Chandler, 1933; Mai, 1993; Manchester, 1994; Manchester et al., 1999; Manchester and

Hickey, 2007); and those are discussed thoroughly in the appropriate chapters of this dissertation.

7 The Cretaceous record of Cornales is relatively less extensive (Knobloch and Mai,

1986; Takahashi et al., 2002; Manchester et al., 2015; Stockey et al., 2016). The earliest and most accepted evidence of Cornales consists of fusainized (charcoalified) fruits of

Hironoia fusiformis Takahashi, Crane & Manchester (2002) from the lower Coniacian

(89 Ma) of Japan, and permineralized (anatomically preserved) fruits of Eydeia hokkaidoensis Stockey, Nishida & Atkinson (2016) from the Santonian (~84 Ma) of

Japan. Currently, it is uncertain as to which cornalean families Hironoia and Eydeia belong. The earliest evidence of extant cornalean families are fossils of unnamed davidiaceous and nyssaceous/mastixiaceous permineralized fruits from the upper

Campanian (~73 Ma) of North America (Manchester et al., 2015); however those fruits have yet to be thoroughly characterized and named. In addition, Knobloch and Mai

(1986) described several fossil fruits representing Mastixiaceae from the Maastrichtian

(72-66 Ma) of central Europe. These reported paleontological data confirm that the initial diversification of Cornales did occur during the Late Cretaceous, which is roughly concordant with the hypothesis of Xiang et al. (2011). Therefore, fossils from this time interval contain information essential for clarifying deep-node relationships within the order.

Fortunately, many cornalean fossils are fruits, which display numerous systematically diagnostic characters useful for the delineation of major clades, families, genera, and even sub-genera (Horne, 1909, 1914; Smith and Smith, 1942; Eyde, 1963,

1967, 1968, 1988; Leins and Erbar, 1990; Hufford, 2001; Weigend et al., 2004;

Yembaturova et al., 2009; Morozowska et al., 2013; Woźnicka et al., 2015; Morozowska

8 and Wysakowska, 2016). Furthermore, cornalean fruits reflect both floral diversity and different dispersal mechanisms of the plants they represent. Based on general fruit morphology, Cornales can be artificially divided into two groups: those with capsular fruits (Hydrangeaceae, Loasaceae, and Hydrostachyaceae [clades 4-5]) and those with drupaceous fruits (Cornaceae, Alangiaceae, Nyssaceae, Mastixiaceae, Davidiaceae,

Grubbiaceae, and Curtisiaceae [clades 1-3]). Drupaceous cornalean fruits, in particular, have a high preservation potential due to their thick-walled woody endocarps (Eyde and

Barghoorn, 1963; Eyde, 1997; Manchester et al., 2015; Stockey et al., 2016). Therefore, with the combination of systematically informative characters and great preservation potential, fossil cornalean drupaceous fruits have great promise for elucidating early evolutionary patterns and relationships within Cornales.

Since the late 19th century cornalean drupaceous fruits of living species have been the subject of many studies (Harms, 1898; Horne, 1909, 1914; Smith and Smith, 1942;

Wilkinson, 1944; Eyde, 1963, 1967, 1968, 1988; Yembaturova et al., 2009; Woźnicka et al., 2015; Morozowska and Wysakowska, 2016). Cornalean endocarps can be identified by their apically opening dorsal germination valves, a typical lack of a central (ventral) vascular strand, and one apically attached unitegmic seed per locule (Hill, 1933;

Wilkinson, 1944; Eyde, 1963, 1967, 1968, 1988; Takhtajan, 2000). These characters have often been used to link Cornaceae, Alangiaceae, Nyssaceae, Mastixiaceae, and

Davidiaceae (see Eyde, 1963, 1967, 1988). However, it worth noting that there are two exceptions; Curtisiaceae and Grubbiaceae have fruits with a central vascular bundle

(Smith and Smith, 1942; Eyde, 1967, 1988; Yembaturova et al., 2009). Overall, these

9 characters are helpful for identifying cornalean fruits in the fossil record (e.g. Eyde and

Barghoorn, 1963; Eyde et al., 1969; Eyde, 1988; Manchester et al., 1999, 2015;

Takahashi et al., 2002; Stockey et al., 2016).

In summary, Cornales holds an important phylogenetic position as the earliest diverging asterid lineage. However, the relationships among the basal-most lineages within the order remain uncertain. Paleobotanical evidence and molecular divergence time estimates indicate that the Late Cretaceous was an important time for the initial radiation of Cornales (Knobloch and Mai, 1986; Takahashi et al., 2002; Schenk and

Hufford, 2010; Xiang et al., 2011; Magallón et al., 2015; Manchester et al., 2015;

Stockey et al., 2016). Fossils of systematically informative organs (e.g., fruits) representing extinct cornalean plants from the Late Cretaceous may contain data that is useful for resolving deep-node relationships. Therefore, by increasing the sampling density of cornalean fossil fruits and incorporating them into phylogenetic analyses along with living taxa, it is possible to clarify the earliest evolutionary patterns and relationships of Cornales.

Content of dissertation: Pursued goals and objectives

The over-arching goal of this dissertation is to shed light on the initial cornalean diversification using fruit morphological data from extinct and extant taxa. To achieve this goal, specific objectives include:

Objective 1: Increase sampling density of cornalean fossil fruits from Late

Cretaceous deposits. Chapters 2-5 characterize seven new Cretaceous cornalean taxa

10 based on permineralized (anatomically preserved) fruits from strata spanning the time interval from the upper Coniacian (~ 89 Ma) to the lower Campanian (~73 Ma).

Objective 2: Clarify fruit characters that delimit cornalean fossil taxa.

Within chapters 2-5, newly discovered fossils are compared to extant species and previously described fossil cornalean fruits to clarify relationships and develop new hypotheses of fruit evolution within the order.

Objective 3: Conduct phylogenetic and morphological diversity analyses.

Chapter 6 focuses on reconstructing the initial diversification of Cornales. A morphological matrix was constructed using data [that I] collected from extinct and extant cornaleans, which was used for phylogenetic and morphological diversity

(disparity) analyses. The phylogenetic analyses of this dissertation were implemented to assess the impacts of fossils on inferring deep-node evolutionary relationships. Disparity analyses were conducted to understand early patterns of the morphological diversification of Cornales through time.

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CHAPTER 2: EARLY DIVERGING ASTERIDS OF THE LATE CRETACEOUS: SUCIACARPA STARRII GEN. ET SP. NOV. AND THE INITIAL RADIATION OF CORNALES.

Brian A. Atkinson

Journal: Botany Canadian Science Publishing (NRC Research Press), 65 Auriga Drive, Suite 203, Ottawa, ON K2E 7W6, Canada Article published September 1, 2016, vol. 95(9): 759-771. doi: 10.1139/cjb-2016-0035

21 Introduction

The order Cornales holds a critical phylogenetic position as the earliest diverging lineage within one of the most diverse clades of angiosperms (over 80,000 species), the asterids (APG 2016). Molecular phylogenetic studies have significantly improved our understanding of the evolutionary relationships within the order (Xiang et al., 1998, 2002,

2011; Fan and Xiang, 2003). These studies strongly support a cornalean phylogeny consisting of 10 families that form five major clades: 1) Cornaceae and Alangiaceae; 2)

Nyssaceae, Mastixiaceae, and Davidiaceae; 3) Grubbiaceae and Curtisiaceae; 4)

Hydrangeaceae and Loasaceae; and 5) Hydrostachyaceae (Xiang et al., 1998, 2002, 2011;

Fan and Xiang, 2003). However, phylogenetic relationships among the five major lineages are not well resolved. This obscures the early evolution of Cornales, thus hindering our ability to more fully understand the initial diversification of the asterids.

The source of difficulty for inferring the deep-node relationships within Cornales may be due to either: 1) a lack of sufficient molecular data, 2) an extremely rapid evolutionary radiation shortly after Cornales diverged (Xiang et al., 2002, 2011), and/or

3) highly restricted taxon sampling (i.e., including living taxa only). Molecular divergence time analyses using fossil calibrations suggest that all five major lineages rapidly diverged sometime during the middle to Late Cretaceous (Schenk and Hufford,

2010; Xiang et al., 2011). A rapid phylogenetic radiation during the Cretaceous, may be responsible for the lack of resolution near the base of the phylogeny. It is important to note that molecular divergence time studies are very sensitive to the tree topology used, the phylogenetic placement and number of fossil representatives used in calibration, and

22 the statistical parameters utilized (Schenk and Hufford, 2010; Xiang et al., 2011; Bell,

2015). Therefore, divergence time hypotheses should be tested with fossil data (Wilf and

Escapa, 2016).

The most widely accepted earliest record of Cornales, Hironoia fusiformis

Takahashi, Crane, et Manchester, is based on charcoalified fruits from the early

Coniacian of Japan (Takahashi et al., 2002). Other Cretaceous cornaleans are mastixioid fossil fruits (Mastixiaceae) from the Maastrichtian of Europe (Knobloch and Mai, 1986;

Mai, 1993) and a Cornus L. endocarp (Cornaceae) from the K/Pg boundary of India reported by Manchester and Kapgate (2014). Manchester et al. (2015) figured two cornalean fruits from the Late Campanian of Alberta, Canada that may be assignable to

Davidia Baill. and Nyssa L., but these have yet to be formally described. Thus far, data from the fossil record appears to support a Late Cretaceous radiation of Cornales, but additional Cretaceous cornalean fossil data are critical for further resolving the early evolutionary patterns of Cornales.

Herein, I characterize a new cornalean genus and species, Suciacarpa starrii

Atkinson gen. et sp. nov., based on permineralized fruits from the Campanian of western

North America. This new species increases our understanding of the Cretaceous phylogenetic and morphological diversity of Cornales. Furthermore, the fruits of

Suciacarpa starrii have a unique combination of characters that do not conform to a single extant family. In addition, this study sheds light on the deep node relationships within the order.

Materials and methods

23 Two permineralized fossil fruits within calcium-carbonate concretions were recovered from Sucia Island and Little Sucia Island west of the mainland of the state of

Washington, USA. The specimen from Sucia Island (specimen number UF 19276-54286) was found on the south side of Fossil Bay as float among other concretions that are in a sandstone matrix exposed along the beach terrace and bluffs. The other specimen (UF

19304-54982) was collected from the northwest end of Little Sucia Island.

Sediments from which the study specimens were recovered are part of the Cedar

District Formation of the Nanaimo Group. The depositional setting of the Sucia Islands localities is interpreted as a shallow marine environment (Ward et al., 2012). The permineralized fossil plant material from these sources is preserved in calcium carbonate concretions alongside ammonites, terrestrial gastropods, inoceramid bivalves, and a theropod femur (Ward, 1978; Roth 2000; Ward et al., 2012; Peecook and Sidor, 2015).

Combined biostratigraphy, magnetostratigraphy, and preliminary strontium isotopic data were used to infer a lower-middle Campanian (~80 Ma) date for the Sucia and Little

Sucia Island section of the Cedar District Formation (Ward et al., 2012).

Concretions were cut with a diamond blade on a water-cooled saw. Slabs were then peeled using the cellulose acetate peel technique (Joy et al., 1956). Peels were mounted on microscope slides using Eukitt (O. Kindler GmbH, Freiberg, Germany) mounting medium. Images were captured using a Better Light (Precision Digital Imaging system, Placerville, CA) focused through a Leitz Aristophot large format camera using either Summar lenses or a Zeiss WL compound microscope, and processed using

24 Photoshop CS 5.0 (Adobe, San Jose, California, USA) and Pixelmator 4.0 (Vilnius,

Lithuania).

One specimen was reconstructed three-dimensionally. Serial sections of the specimen were photographed at the same magnification. Subsequently, each photograph was traced in Photoshop CS 5.0 and Pixelmator 4.0. Serial traces were uploaded into

ImageJ as a single stack (Rasband 1997-2014). ImageJ plugins, StackReg (Thévenaz et al., 1998) and TurboReg (Thévenaz et al., 1998), were used to align the serial traces.

Spacing between each trace (or slice) was slightly modified in ImageJ to accurately reflect the shape of the fruit. ImageJ 3D viewer plugin (Schmid et al., 2010) was used to visualize the three dimensional reconstruction.

Studied fossil specimens and microscope slides are housed in the Florida Museum of Natural History. Prepared microscope slides of living material were borrowed from the

R.H. Eyde collection in the Botany Department, National Museum of Natural History

(Smithsonian Institution), Washington DC.

Systematic paleobotany

Order— Cornales Dumortier sensu Xiang et al. 2011

Family— indet.

Genus— Suciacarpa Atkinson gen. nov.

Generic diagnosis— Endocarp woody, thick walled, tetralocular. Locules crescent-shaped in cross section, each with apically opening dorsal germination valve.

Germination valves short, without conspicuous external longitudinal grooves. Valve tissue primarily consisting of isodiametric sclereids and secretory cavities. Septa

25 composed of elongate sclereids and secretory cavities. Inner-endocarp layer present, consisting of transversely elongated narrow fibers. Vascular bundles in two anastomosing longitudinal rows within each septum arm. One seed per locule.

Etymology— The generic name Suciacarpa (Sucia + carpa [=fruit]) refers to

Sucia Island where the fossil fruits were recovered.

Type species— Suciacarpa starrii Atkinson sp. nov., (Figs. 2.1-2.5)

Specific diagnosis— Fruit endocarp ellipsoidal, at least 16 mm long, 8 mm wide.

Germination valves at least 8 mm wide. Endocarp surface smooth with no major striations. Elongated sclereids of septum horizontally oriented perpendicular to long axis.

Secretory cavities globular, lacking epithelial lining. Inner-endocarp with ventral circum- locular fibers and dorsal longitudinal fibers.

Holotype hic designatus— UF 19276-54286.

Paratype— UF19304-54982.

Repository— Florida Museum of Natural History

Type locality— Fossil Bay, Sucia Island, Washington State, USA (48.749330°

N, 122.900798° W).

Stratigraphic position and age — Early to middle Campanian (~80 Ma).

Etymology— The species is named for David W. Starr (Bellevue, Washington,

U.S.A.) who helped collect the specimens and played a critical role in increasing the visibility and accessibility of Sucia Island fossils to the public and scientific community.

Description

26 Specimens consist of permineralized woody endocarps with enclosed seeds (Figs.

2.1A-D). Endocarps are ellipsoidal (Figs. 2.1C, 2.5) and are at least 16 mm long and 8 mm wide. Both fruits are tetra-locular (Figs. 2.1A, 2.1B). Each locule contains a single seed and is accompanied by a dorsal germination valve that is restricted to the apical half or third of the fruit (Figs. 2.1B, 2.1C, 2.2A, 2.5). The germination valve is 5-8 mm wide and tapers towards the apex (Figs. 2.1C, 2.5). Although the fruits are abraded, these endocarps appear to lack any significant striations or any conspicuous longitudinal grooves on the exterior surface of the germination valves (Figs. 2.1A-D) of the kind produced by many mastixioid species (Tiffney and Haggard, 1996).

Locules are crescent shaped in cross section (Figs. 2.1A, 2.1B, 2.2A, 2.2B; often referred to as “C-shaped”, see Tiffney and Haggard 1996) and are up to 5.5 mm wide and

1.5 mm thick. This locule shape is due to the germination valves having a broad, longitudinally elongate inner bulge or thickening (Figs. 2.1A, 2.1B, 2.2A, 2.2B). A thick central four-armed septum separates the locules and germination valves (Fig. 2.1B).

Near the base of the fruit the dorsal and septal tissues of the endocarp grade into one another (Figs. 2.1A, 2.1D, 2.2D). These tissues become more distinct towards the apex of the endocarp (Fig. 2.1B). However, distinct germination valves are not observable until their planes of weakness (Figs. 2.2A, 2.2E) are seen near the upper half to one-third of the endocarp (Figs. 2.1B, 2.1C, 2.2A). Specifically, this plane is marked by a dark line and distinct separation zone located where the germination valves contact the septum (Figs. 2.1B, 2.1C, 2.2A, 2.3B). Smaller cells with somewhat thinner walls mark the plane of separation for the valves (Fig. 2.2E). Cells in this area all have dark

27 contents and there is evidence of fungal hyphae following the margin of the germination valve. Therefore, the germination valves are most likely restricted to the apical half to one-third of the fruit and are interpreted as being short rather than elongate.

The endocarp including the germination valves and septa, is composed of three types of sclerenchyma cells and numerous prominent lacunae (Figs. 2.1A-D, 2.2A-H,

2.3A-C). Germination valves consist mainly of isodiametric sclereids (Figs. 2.2A, 2.2F), which are 60-84 m in diameter. Numerous globular secretory cavities, that measure up to 5 mm in diameter, are found in the valve tissue (Figs. 2.1B, 2.1C, 2.2A, 2.2B, 2.2F) as well as the septum (Figs. 2.2C, 2.2G, 2.3A). The septum is composed mostly of elongated sclereids that are horizontally oriented (Figs. 2.2C, 2.2G, 2.3A, 2.3B). These cells are 120-275 m long and 48-84 m wide. The endocarp has an anatomically distinctive inner layer that lines each locule, similar to the “inner-endocarp layer” observed in cornelian cherries (Morozowska et al., 2013). This inner-endocarp layer is a multiseriate zone of transversely elongated narrow fibers (9-15 m in diameter) that are more or less circum-locular (Figs. 2.2A, 2.2B, 2.2G). Inner-endocarp fibers of the germination valve are often longitudinal and run parallel to the long axis of the fruit (Fig.

2.2B, 2.2H) rather than being circum-locular.

There are two irregular rows of vascular bundles in each septal arm (Figs. 2.2C,

2.3A). These fruit bundles are often accompanied by longitudinal thick-walled fibers

(Figs. 2.1D, 2.2C, 2.3A, 2.3B). Bundles enter the base of the endocarp from the periphery of the ventral side of each carpel (Fig. 2.1D). Also at this level there are minor scattered vascular bundles in the dorsal area (Fig. 2.1D). Ventral bundles divide to form the two

28 anastomosing longitudinal rows in each septal arm (Figs. 2.2C, 2.3A). At the apex of the locules the bundles converge to enter the seeds (Fig. 2.1B). There is no major central vascular strand (Fig. 2.3C).

There is one apically attached seed per locule (Figs. 2.2C, 2.2B, 2.3A). The integument is thin, and is somewhat obscured by pyrite and colonizing fungi (Figs. 2.4A,

2.4C). Endosperm cells appear to be preserved, however upon closer examination the cell pattern is seen to consist of fungi that are concentrated in the positions of unpreserved endosperm cell walls (Figs. 2.4A, 2.4B, 2.4C). The endosperm cells as deduced from the patterns of fungal hyphae are relatively small and rectangular (Figs. 2.4A, 2.4B). The fungal hyphae are septate and branched. No clamp connections were observed.

Discussion

Suciacarpa fruits consist of woody sclerenchymatous endocarps with apically opening germination valves, locules with one apically attached seed, and no central vascular bundles. This suite of characters clearly indicates affinities with Cornales (Eyde,

1963, 1988; Xiang et al., 2011). Cornales encompasses ten families that are commonly classified in five major groups: 1) Cornaceae, Alangiaceae; 2) Curtisiaceae, Grubbiaceae;

3) Nyssaceae, Mastixiaceae, and Davidiaceae (hereafter designated as NMD); 4)

Hydrangeaceae, Loasaceae; and 5) Hydrostachyaceae (Xiang et al., 1998, 2002, 2011;

Fan and Xiang, 2003). Two groups, Hydrangeaceae/Loasaceae and Hydrostachyaceae, have capsular fruits that have numerous seeds per locule and are typically less woody

(Brown and Kaul, 1981; Erbar and Leins 2004; Hufford 2004; Weigend, 2004;

Morozowska et al., 2012), which differ from the single seeded endocarps of Suciacarpa

29 starrii. Grubbiaceae can be distinguished from Suciacarpa starrii because fruits within that family lack germination valves and are usually fused together in a head (Carlquist,

1977).

The remaining families Cornaceae, Alangiaceae, Nyssaceae, Davidiaceae,

Mastixiaceae, and Curtisiaceae all have woody endocarps with germination valves. The fossil record of these families is extensive and is particularly well documented by fruits

(Reid and Chandler, 1933; Eyde and Barghoorn, 1963; Knobloch and Mai, 1986; Eyde,

1988, 1997; Mai, 1993; Tiffney and Haggard, 1996; Stockey et al., 1998; Manchester et al., 1999, 2010, 2015; Martinetto, 2011). The general endocarp morphology observed in these families is also seen in Suciacarpa.

Curtisiaceae contains one genus Curtisia Aiton with two species (Yembaturova et al., 2009), C. dentata (Burm. F.) C.A. Sm. from southern Africa and C. quadrilocularis

(Reid & Chandler) Manch., Q Y Xi & Q-P Xiang (Manchester et al., 2007) from the

Eocene of Europe. This genus differs from Suciacarpa in several respects. The germination valves of Curtisia fruits are elongate (Manchester et al., 2007; Yembaturova et al., 2009) while those of Suciacarpa are short, confined to the apical part of the fruit.

The endocarp of Curtisia is mostly made up of isodiametric sclereids (Manchester et al.,

2007; Yembaturova et al., 2009), while in Suciacarpa the sclereids may be isodiametric in the valve tissue, but they are elongate in the septum. The most striking character that separates Curtisia from Suciacarpa and the remaining Cornales is the presence of a central vascular strand in the endocarps of Curtisia.

Cornaceae/Alangiaceae and the NMD group

30 Based on morphology and anatomy Cornaceae, Alangiaceae, Nyssaceae,

Mastixiaceae, and Davidiaceae have been considered a monophyletic group designated as

Cornaceae sensu lato (e.g. Eyde, 1967, 1988). However, recent molecular phylogenetic analyses have suggested that Cornaceae/Alangiaceae and NMD are a polyphyletic assemblage within Cornales (Xiang et al., 1998, 2002, 2011). Xiang et al. (2011) found strong support for this hypothesis using cpDNA, however it was not strongly supported in their total evidence tree.

The fruit characters of the Cornaceae/Alangiaceae and the NMD lineages display a wide range of variation (Figs. 2.6A-D, 2.7-D). However, all of them have woody endocarps with dorsal germination valves that lack a single central vascular strand (Eyde,

1963, 1967, 1988; Takahashi et al., 2002). Therefore, Suciacarpa starrii is most similar to these taxa (Table 2.1).

Cornaceae/Alangiaceae

Alangiaceae has a single genus, Alangium Lam. of 24 species (Feng et al., 2009).

Fruits of Alangium differ from those of Suciacarpa in several ways. Germination valves are elongate in Alangium (Eyde, 1968), which differ from the short valves seen in the endocarps of Suciacarpa. There are one to two locules in Alangium fruits (Eyde, 1968) while there are four in the Suciacarpa fruits. Alangium endocarps consist of isodiametric sclereids, which contrast with the elongated sclereids and fibrous inner-endocarp layer seen in Suciacarpa. Furthermore, endocarps of Alangium lack the secretory cavities that are present in the endocarps of Suciacarpa (Table 2.1).

31 Cornaceae consists of a single genus, Cornus L., which contains about 58 species

(Xiang et al., 2006). The genus is divided into four monophyletic groups: blue and white

(BW) fruited dogwoods, cornelian cherries, dwarf dogwoods, and big bracted (BB) dogwoods (Eyde, 1987, 1988, Xiang et al., 1996, 1998, 2006; Fan and Xiang, 2003). The

Cenozoic record of Cornus is rich and has been thoroughly reviewed by Eyde (1988), but the Cretaceous record is sparse. Eyde (1988) mentions an unpublished report that documents a BW Cornus fossil fruit from the Santonian of Sweden (found by E.M. Friis).

However, this fossil has yet to be formally characterized and named. Molecular clock analyses estimate that all four Cornus clades diverged by the end of the Cretaceous or early Paleocene (Xiang and Thomas, 2008; Xiang et al., 2011). This is supported by the occurrence of cornelian cherry fossils from the K/Pg boundary of India (Manchester and

Kapgate, 2014) and the Paleocene of North America (Manchester et al., 2010).

There are six extant and three extinct species within the cornelian cherry clade

(Xiang et al., 2005; Manchester et al., 2010). Cornelian cherries (Cornus subg. Cornus) are easily distinguished from other Cornus species by their distinctive endocarp anatomy.

The endocarps of cornelian cherries are characterized by elongated sclereids at maturity and by conspicuous sub-globose secretory cavities (Figs. 2.6A-D; Eyde 1988;

Manchester et al., 2010; Morozowska et al., 2013), which makes them distinct from all other species of Cornus. In addition, some species of Cornus subg. Cornus can have an inner-endocarp layer (Xiang et al., 2003; Morozowska et al., 2013) consisting of transversely elongated narrow fibers that are circum-locular. Endocarps of Suciacarpa

32 also possess these cell types and have secretory cavities that are strikingly similar to those of cornelian cherries. (Figs. 2.1A, 2.1B, 2.2F, 2.6A-D).

Although Suciacarpa and Cornus subg. Cornus endocarps are similar in many characters, there are also important differences between the two (Table 2.1). The germination valves found in Suciacarpa fruits are short, confined to the apical one-third, while those in Cornus are elongate extending nearly the full length of the endocarp

(Eyde, 1988; Manchester et al., 2010). The locule shape of Cornus endocarps is ellipsoidal to sub-triangular in cross section (Manchester et al., 2010) while those of

Suciacarpa are crescent-shaped. The vascular bundles in Cornus are few in number and are confined towards the periphery of the ventral area of the septum for much of the length of the endocarp (Figs. 2.6A, 2.6B), and near the apex they extend across the septum to connect with the seed bundles (Wilkinson, 1944; Eyde, 1967, 1988). In contrast, the bundles in the endocarps of Suciacarpa are numerous and form roughly two rows across each septal arm. Therefore, Suciacarpa cannot be placed within Cornus subg. Cornus and should be excluded from Cornaceae.

NMD group

Morphological studies have suggested a close relationship between Nyssaceae,

Mastixiaceae, and Davidiaceae (Eyde, 1963, 1967, 1988). This relationship is strongly supported by several molecular phylogenetic studies (Xiang et al., 2002, 2011; Fan and

Xiang, 2003). The morphological diversity of fruits within this lineage is striking (Eyde,

1963) and appears to have been even more diverse in the past according to the fossil record (Eyde and Barghoorn, 1963; Eyde, 1997; Manchester et al., 2015).

33 Nyssaceae consists of two extant genera, Nyssa L. (7 extant spp.) and

Camptotheca Decne. (1 sp.), as well as two extinct genera, Amersinia Manchester, Crane, et Golovneva (1 sp.), and Browniea Manchester and Hickey (1 sp.) with a Northern

Hemisphere distribution (Eyde, 1963; Wen and Stuessy, 1993; Fan and Xiang, 2003;

Xiang et al., 2011; Wang et al., 2012). The fossil record of Nyssaceae dates back to at least the Paleocene (Manchester et al., 1999). With the exception of Browniea, nyssaceous fruits are unique within Cornales in having short germination valves confined to the apical half of the fruit (Eyde, 1963; Manchester and Hickey, 2007). This valve morphology is also seen in the endocarps of Suciacarpa.

Amersinia, Camptotheca, and Browniea show similarities with one another in fruit characters (Eyde, 1963; Manchester et al., 1999; Manchester and Hickey, 2007).

These include the structure of the mesocarp, which is not preserved in the fruits of

Suciacarpa described here. The walls of the endocarps of Amersinia, Camptotheca, and

Browniea are relatively thin while those of Suciacarpa are thick. Another significant difference is that the endocarps of Amersinia, Camptotheca, and Browniea consist of slender fibers while those of Suciacarpa have elongated sclereids (Table 2.1). In addition, endocarps of Amersinia, Camptotheca, and Browniea lack the secretory cavities seen in the endocarps of Suciacarpa. Endocarp vascular bundles of Amersinia and Camptotheca are like those of Cornus (see above), where they are confined towards the periphery of the septa for most of the endocarp length and then traverse the septal tissue towards the apex (Eyde, 1963; Manchester et al., 1999). By contrast, the endocarps of Suciacarpa have two anastomosing rows of bundles within the septa.

34 Nyssa has an outstanding Cenozoic fossil record (reviewed in Eyde and

Barghoorn, 1963; Eyde, 1997; Noll, 2013). It is worth noting that there is a late

Campanian fossil that is thought to represent Nyssa (Figs. 5C, 5D in Manchester et al.

2015), however it has yet to be thoroughly described. Endocarps of Nyssa are similar to those of Suciacarpa in having short germination valves. Some species of Nyssa have endocarps with crescent-shaped locules in which there is a broad thickening of the inner side of the valve (Table 2.1; Hammel and Zamora, 1990; Noll, 2013) as observed in the endocarps Suciacarpa. Furthermore, both Nyssa and Suciacarpa have endocarps that lack a deep dorsal groove along the germination valve surface.

Nyssa fruits differ from those of Suciacarpa in that the endocarp is mostly made up of long and narrow fibers that may aggregate into chaotically oriented groups (Eyde,

1963; Noll, 2013) as opposed to the shorter and broad sclereids and circum-locular fibers in the endocarps of Suciacarpa. Furthermore, the germination valves are made up of fibers in Nyssa (Eyde, 1963) while those of Suciacarpa consist of isodiametric sclereids.

The secretory cavities prominent in the endocarps of Suciacarpa are absent in the endocarps of Nyssa (Fig. 2.7A; Table 2.1). The endocarp vasculature of Nyssa, like

Amersinia, Camptotheca, and Cornus, consists of peripheral vascular bundles for most of the endocarp length (Fig. 2.7A; Table 2.1; Eyde, 1967) and towards the apex the bundles enter the septum to connect with ovular bundles. In contrast, Suciacarpa has endocarps with numerous longitudinal anastomosing vascular bundles in two rows along the septa.

Mastixiaceae has two extant genera, Mastixia Blume and Diplopanax Hand.-

Mazz., and has an extensive fossil record consisting of about eight fossil genera (see

35 Tiffney and Haggard, 1996; Stockey et al., 1998). The oldest fossils of the family come from the Maastrichtian of Europe (Knobloch and Mai, 1986; Mai, 1993). Similar to

Suciacarpa, mastixiaceous endocarps have crescent-shaped locules in cross section (Fig.

2.7B). This is due to a ventral “intrusion” of the germination valve (Eyde, 1963; Tiffney and Haggard, 1996). This intrusion is often accompanied by a shallow to deep furrow on the dorsal surface of the germination valve in Mastixia (Fig. 2.7B, at bottom). Similar to

Suciacarpa, Diplopanax and Mastixicarpum Chandler lack such external furrows (Eyde and Xiang, 1990; Mai, 1993; Tiffney and Haggard, 1996; Stockey et al., 1998).

Mastixioid germination valves are elongate rather than short like those of Suciacarpa

(Table 2.1). Furthermore, the endocarps of Diplopanax, Mastixicarpum, Retinomastixia

Kirchh., and some species of Mastixia have resin canals or cavities with an epithelium

(Eyde, 1963; Mai, 1993; Tiffney and Haggard, 1996; Stockey et al., 1998) whereas

Suciacarpa has relatively large cavities that lack an epithelium.

Davidiaceae consists of a single genus, Davidia, and one extant species, D. involucrata Baillon, native to China. Two extinct species, D. antiqua Manchester (2002) from the Paleocene of western North America and East Asia and D. paleoinvolucrata

Pavlyutkin (2009), from the Miocene of Russia have been described. In addition, there are several unnamed fossil fruits of Davidia that have been described from the Late

Eocene John Day Formation of Oregon (Manchester and McIntosh, 2007) and the late

Campanian of Alberta, Canada (Figs. 5A, 5B in Manchester et al. 2015). A significant similarity between Davidia and Suciacarpa is the unique vascular architecture of the endocarp. In the endocarps of both of these plants there are roughly two longitudinal

36 rows of anastomosing vascular bundles in each septal arm (Figs. 2.7C, 2.7D; Horne,

1909; Eyde and Barghoorn, 1963; Manchester, 2002; Manchester et al., 2015). However, there are important differences between Davidia and Suciacarpa. Fruits of Davidia are characterized as having five to nine locules (Horne, 1909; Eyde, 1963; Manchester, 2002;

Manchester et al., 2015) while those of Suciacarpa are tetralocular. Locule shape also differs, being sub-triangular in Davidia (at maturity) and crescent-shaped in the endocarps of Suciacarpa (Table 2.1; Morozowska et al., 2013). Germination valves are elongate in Davidia in contrast to the short valves of Suciacarpa. Furthermore, Davidia endocarps lack the secretory cavities that characterize the endocarps of Suciacarpa.

Fossils of uncertain affinities

Hironoia fusiformis Takahashi, Crane, & Manchester (2002) from the Late

Cretaceous of Japan is the oldest representative of Cornales to date. Takahashi et al.

(2002) placed Hironoia, within the NMD group (nyssoids) because of the fibrous anatomy of the endocarp (Takahashi et al., 2002). However, there are other taxa with fiber-like elements within the endocarp such as Suciacarpa and Cornus subg. Cornus

(Table 2.1), thus the phylogenetic position of Hironoia may be uncertain.

Hironoia and Suciacarpa have several differences (Table 2.1). The endocarps of

Hironoia have a fusiform shape (Takahashi et al., 2002) while those of Suciacarpa are ellipsoidal. Hironoia has endocarps with elongate germination valves while those seen in the endocarps of Suciacarpa are short. Locule shape within the fruits of Hironoia is sub- triangular in cross section rather than crescent-shaped (Table 2.1). Furthermore,

37 endocarps of Hironoia lack the secretory cavities that are present in the endocarps of

Suciacarpa.

Evolutionary patterns within Cornales

Contrary to the hypotheses of past morphological studies (Eyde, 1967, 1988), most molecular phylogenetic studies suggest that within Cornales Cornaceae,

Alangiaceae, and the Nyssaceae-Mastixiaceae-Davidiaceae clade (NMD group) form a polyphyletic assemblage (Xiang et al., 1998, 2002, 2011). The molecular studies utilized a significantly larger number of characters and more robust methodologies as compared to the morphological studies, but the inferred relationships for the five major cornalean lineages consistently have low support (Xiang et al., 1998, 2002, 2011; Fan and Xiang,

2003). This may be attributed to: 1) insufficient molecular data, 2) initial rapid evolutionary radiation, (Xiang et al., 2002, 2011; Fan and Xiang 2003), and/or 3) exclusion of important fossil data from the analyses (i.e., restricted taxon sampling).

Divergence time estimations based on molecular clocks with fossil calibrations suggest that Cornales underwent a rapid radiation sometime during the mid to Late

Cretaceous (Schenk and Hufford, 2010; Xiang et al., 2011; Magallón et al., 2015), resulting in the divergence of all five major clades. This is supported by the relatively small number of reported Late Cretaceous cornalean fossils including the earliest occurrence of Cornales, Hironoia fusiformis, from the early Coniacian of Japan

(Takahashi et al., 2002). Other described Cretaceous cornalean fossils are mastixiaceous fruits from the latest Maastrichtian of Europe (Knobloch and Mai, 1986; Mai, 1993).

There are Davidia and Nyssa-like fruits from the Late Campanian of Alberta, Canada

38 (Figs. 5A-D, in Manchester et al., 2015) as well as a cornaceous fruit (cornelian cherry) from the K/Pg boundary of India (Manchester and Kapgate, 2014). All of these sources of data support the hypothesis that the Cornaceae/Alangiaceae and NMD clades diverged and subsequently diversified by the end of the Cretaceous.

Evolutionary implications of Suciacarpa

Suciacarpa starrii from the Campanian of western North America has a mosaic of characters that are representative of the Cornaceae/Alangiaceae and NMD clades. More specifically, the valve morphology in the endocarps of Suciacarpa is comparable to the fruits of Nyssa, Amersinia, and Camptotheca (Nyssaceae). The locule shape in endocarps of Suciacarpa is similar to those of some species in Mastixiaceae and Nyssaceae.

However, the similarities that Suciacarpa shares with Davidia (Davidiaceae) and cornelian cherries (Cornaceae: Cornus subg. Cornus) are of particular interest.

Eyde (1967) inferred that the unique gynoecial/fruit vascular pattern observed in

Davidia is ancestral within the NMD lineage, and that Davidia is the closet living taxon to a common ancestor of Cornaceae and Nyssaceae. Furthermore, Eyde (1967) hypothesized that the relatively reduced gynoecial vascular pattern of like that illustrated here for Cornus, Mastixia, and Nyssa (Figs. 2.6A, 2.6B, 2.7A-D), evolved from the more complex state observed in Davidia (Figs. 2.7C, 2.7D). Endocarps of Suciacarpa have a vascular pattern similar to what is seen in those of Davidia, supporting the hypothesis that this type of vascular pattern is ancestral within Cornales (Eyde 1967).

The occurrence of secretory cavities within the endocarp was once thought to be a unique character only found in cornelian cherries (Cornus subg. Cornus). However, there

39 are secretory cavities in the endocarps of Suciacarpa as well. Thus, it is possible that secretory cavities within endocarps is a relatively more plesiomorphic character state within Cornales that has been retained in cornelian cherries, but lost in other taxa, or could be a synapomorphy linking Suciacarpa and Cornus subg. Cornus.

Suciacarpa starrii represents a new genus and species of Cornales from the Late

Cretaceous of western North America. The stratigraphic age of Suciacarpa and its possession of endocarp characters that were once thought to be unique to Davidia (rows of vascular bundles in the septa), Nyssaceae (short germination valves), and cornelian cherries (secretory cavities within the walls of the endocarps) supports the hypotheses that Cornaceae/Alangiaceae and the NMD lineages represent a single monophyletic clade

(Eyde, 1963, 1967, 1988). It is possible that Suciacarpa is representative of an ancestral complex from which the Alangiaceae/Cornaceae and NMD clades evolved. These hypotheses can only be tested with the addition of more fossil data from Late Cretaceous deposits and rigorous evolutionary analyses.

Acknowledgements

The author is most grateful to David W. Starr (Bellevue, Washington, U.S.A.) and

Jim Goedert, Burke Museum, University of Washington for collection of fossils as well as Steven R. Manchester, Florida Museum of Natural History, University of Florida, for preliminary fossil preparations, loaning the specimens for study and a critical review of the manuscript. Furthermore, the author would like to thank Stan Yankowski, Botany

Department, National Museum of Natural History, Smithsonian Institution for aid in obtaining microscope slides of living material. Thanks to Gar W. Rothwell and Ruth A.

40 Stockey, Oregon State University for their valuable comments on the manuscript, and to

Ashley Klymiuk, University of Kansas, Stefan Little Université Paris Sud, and Selena

Smith, University of Michigan for organizing this volume to honor Ruth A. Stockey, and for the invitation to contribute. This work was supported by NSF grant DGE-1314109.

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46

Figure 2.1. Suciacarpa starrii gen. et sp. nov. A, Cross section of endocarp showing four locules (arrows). Note left locule is abraded. Holotype UF-19276-54286 B bot, #2 x11. B, Cross section of endocarp near apex displaying germination valves (v) and septum (s). Note single seed in left locule and that the bottom locule is abraded. Holotype UF-19276- 54286 E top, #7 x10. C, Tangential longitudinal section of fruit showing ellipsoid shape and germination valve (v) confined to the apical third of the endocarp. UF19304-54982 #11 x8. D, Cross section of fruit near base showing ventral vascular bundles diverging into endocarp (arrows). Note vascular bundles in dorsal area of the endocarp (arrowheads). Holotype UF19276-54286 A bot #45 x10.

47

Figure 2.1

48

Figure 2.2. Suciacarpa starrii gen. et sp. nov., Holotype. Specimen no. UF19276-54286. A, Cross section of endocarp showing crescent shaped locule, single seed (sd) in locule cavity, germination valve (v) with numerous secretory cavities. E top #7 x12.5. B, Transverse section towards base of locule displaying surrounding inner-endocarp layer (ie) and seed within locule cavity. C top #4 x25. C, Cross section of septum arm showing horizontally oriented elongated sclereids, secretory cavities, and vascular bundles (arrows). E top #7 x26. D, Transverse section near basal third of endocarp showing contact zone of dorsal tissue and septum. Note absence of discrete zone of weakness. C top #6 x60. E, Cross section towards apical third of endocarp showing separation suture of valve. E top #7 x133. F, Cross section of germination valve showing isodiametric sclereids and secretory cavities. Note shrunken substance in cavity and lack of an epithelium. A Bot #45 x92. G, Cross section of septum (s) and inner-endocarp (ie) layer with narrow fibers. C top, #14 x43. H, Cross section of interior surface of germination valve detailing longitudinal fibers of inner-endocarp layer. Dorsal side is towards left and ventral side is to the right. C top, #14 x150.

49

Figure 2.2

50

Figure 2.3. Suciacarpa starrii gen. et sp. nov., Holotype. Specimen no. UF19276-54286. A, Cross section of septum arm showing two rows of vascular bundles (arrows) that each correspond to one locule (l). D bot #13 x17. B, Cross section of fruit at apex of locule showing vascular bundles (arrows) converging. E top #77 x23.5. C, Cross section of septum central axis. Note absence of central vascular strand. C top, #14 x24.

51

Figure 2.3

52

Figure 2.4. Suciacarpa starrii gen. et sp. nov., Holotype. Specimen no. UF19276-54286 D bot # 13. A, Cross section of seed showing thin integument (in) colonized by fungi and obscured by pyrite and traces of endosperm tissue (arrow) in which cell walls are replaced by fungal hyphae. x333. B, Close up of fungal mycelium found in seed. x1000. C, Branching fungal hyphae. x1,333.

53

Figure 2.4

54

Figure 2.5. Three-dimensional reconstruction of Suciacarpa starrii gen. et sp. nov., Holotype. Specimen no. UF19276-54286. Side view of fruit showing outer endocarp wall (purple) and germination valve (green). Note the acute apex of the germination valve.

55

Figure 2.6. Structure of extant Cornus fruits representing the cornelian cherry clade. A, Cross section of young Cornus mas fruit showing mesocarp (m) and two-loculed endocarp with numerous secretory cavities. x18. B, Cross section of immature Cornus sessilis fruit showing mesocarp (m) and two-loculed endocarp with secretory cavities. x43. C, Close up view of Fig. 6A detailing secretory cavities. x63. D, Magnified view of Fig. 6B showing secretory cavities. x183.

56

Figure 2.7. Structure of extant fruits representing Nyssaceae, Mastixiaceae, and Davidiaceae. A, Cross section two-loculed Nyssa sylvatica fruit showing ventral trans- septal bundles (arrows) and bundles transversing the septum (arrowheads). #11 x14. B, Cross section of Mastixia philippinensis showing crescent/U-shaped locule and ventral vascular bundles (arrows). X4. C, Cross section of immature Davidia involucrata fruit showing nine locules and numerous vascular bundles in septum arms. x20. D, Tangential section of young Davidia involucrata fruit showing anastomosing bundle (arrow). x23.

57

Table 2.1. Comparison of endocarp structure among similar cornalean taxa. Data from: aEyde 1963; bNoll 2013; cHammel and Zamora 1990; dManchester et al. 1999; eTakahashi et al. 2002; fManchester et al. 2010; gEyde 1988; hEyde 1968

58

Table 2.1

Number Exterior Exterior Locule Valve Septum Valve Endocarp Endocarp Taxon Age of groove(s) ridge(s) shape length histology histology lacunae vasculature locules on valve on valve

Isodiametric Longitudinal Suciacarpa Elongate Secretory Campanian 4 C-shaped Absent Absent Short sclereids, rows in starrii sclereids cavities lacunae septum Isodiametric Isodiametric Hironoia Sub- Septum and Coniacian (3)-4 Absent Present Elongate sclereids, Sclereids, Absent fusiformise triangular axis periphery fibers fibers Isodiametric Isodiametric Amersinia Septum and Paleocene 3 Ellipsoidal Absent Absent Short sclereids, sclereids, Absent obtrullatad axis periphery fibers fibers Nyssa Septum Extant 1-(2) Ellipsoidal Absent Absent Short Fibers Fibers Absent sylvaticaa periphery Nyssa Septum Extant 1 C-shaped Absent Present Short Fibers Fibers Absent ogechea,b periphery Nyssa Septum talamancanab, Extant 2-3 C-shaped Absent Absent Short Fibers Fibers Absent periphery c Nyssa Septum Extant 1 W-shaped Absent Absent Short Fibers Fibers Absent javanicaa,b periphery

59 Table 2.1 (Continued)

Camptotheca Septum Extant 1 Ellipsoidal Absent Absent Short Fibers Fibers Absent acuminataa periphery

Varying Longitudinal Davidia Sub- Extant 6+ Absent Present Elongate sclereids, Fibers Absent rows in involucrataa triangular fibers septum

Mastixia U or C- Septum Extant 1 Present Absent Elongate Fibers Fibers Absent philippinensis shaped periphery

Scattered in Diplopanax U or C- Fibers, Resin Extant 1 Absent Absent Elongate Fibers ventral stachyanthus shaped parenchyma canals endocarp wall

Elongate, Isodiametric Cornus Secretory Septum Extant 2-(3) Ellipsoidal Absent Absent Elongate isodiametr sclereids, masf,g cavities periphery ic sclereids lacunae

Alangium Isodiametri Isodiametric Septum Extant 2 Ellipsoidal Absent Absent Elongate Absent chinenseh c sclereids Sclereids periphery

60

CHAPTER 3: CRETACEOUS ORIGIN OF DOGWOODS: AN ANATOMICALLY PRESERVED CORNUS (CORNACEAE) FRUIT FROM THE CAMPANIAN OF VANCOUVER ISLAND.

Brian A. Atkinson, Ruth A. Stockey, and G.W. Rothwell

Journal: PeerJ PeerJ, Inc. PO Box 910224, San Dieago, CA 92191 USA Article published December 21, 2016 vol. 4:e2808. doi: 10.7717/peerj.2808

61 Introduction

The family Cornaceae comprises 58 species of trees, shrubs, and rhizomatous herbs, commonly known as dogwoods, within the genus Cornus L. (Eyde, 1987, 1988;

Murrell, 1993; Xiang et al., 1993, 1998, 2006). Dogwoods are broadly distributed across

Eurasia, North America, northern South America, and sub-Saharan eastern Africa. (Eyde,

1988; Murrell, 1993, 1996; Xiang et al., 2003). There are four major clades within

Cornus supported by both morphological and molecular characters: the blue- or white- fruited dogwoods, big-bracted dogwoods, dwarf dogwoods, and cornelian cherries (Eyde,

1987, 1988; Xiang et al., 2006). Each of these groups can be distinguished based on fruit morphology and anatomy (see Eyde, 1988).

Throughout the past decade, molecular phylogenetic studies have made significant progress towards our understanding of the evolutionary patterns and relationships of Cornus (Xiang et al., 1993, 1996, 1998, 2002, 2005, 2006, 2008; Xiang and Thomas, 2008; Feng et al., 2011). Molecular divergence-time analyses, that used

Cenozoic fossil calibrations, suggested a Late Cretaceous origin for Cornaceae, and that the four major clades of Cornus diverged and diversified by the latest Cretaceous or early

Paleogene (Xiang et al., 2005, 2006, 2011). However, there have been no Cornus fossils formally described from Cretaceous deposits to date (Manchester et al., 2009).

Cornus has an extensive Cenozoic record (Eyde, 1988; Manchester et al., 2010).

The most widely accepted, earliest described fossils of the genus consist of leaves of

Cornus swingii Manchester, Xiang, Kodrul & Akhmetiev (2009) and C. krassilovii

Manchester, Xiang, Kodrul & Akhmetiev (2009) from the Paleocene of North America

62 and Asia, respectively, and fruits described as Cornus piggae Manchester, Xiang &

Xiang (2010) from the Paleocene of North Dakota, USA. In addition, Manchester &

Kapgate (2014) recently reported a fruit resembling a cornelian cherry (Cornus subg.

Cornus) from the K/Pg boundary of India, which is currently being studied (S.R.

Manchester pers. comm., 2016). Given that fruit characters of Cornus are systematically informative (Eyde, 1988; Xiang et al., 2003; Manchester et al., 2010; Morozowska et al.,

2013; Woźnicka et al., 2015), fossil fruits of this genus have great potential for revealing ancient evolutionary patterns and relationships.

As part of a broader initiative to understand the earliest evolutionary patterns and relationships of the order Cornales (Atkinson, 2016; Stockey et al., 2016), we describe a permineralized fruit assigned to Cornus cf. piggae from the Cretaceous (late Campanian

~73 Ma) of Vancouver Island. This fruit is the oldest known dogwood fossil to date and provides a new minimum age for the diversification of crown-group Cornus. The presence of an Upper Cretaceous Cornus has important implications for our understanding both the plesiomorphic characters of Cornaceae and the early diversification of Cornales.

Materials and methods

A single permineralized fruit was recovered from the Shelter Point locality on

Vancouver Island, British Columbia, Canada. The exposure at Shelter Point consists of six units, two of which contain permineralized fossil plants (Richards, 1975). Sediments are part of the Upper Cretaceous Spray Formation of the Nanaimo Group, which is considered upper Campanian based on the presence of Longusorbis decapod fossils

63 (Richards, 1975). Plant material at Shelter Point is rare, and preserved in calcareous concretions. Pinaceous seed cones of Pityostrobus beardii (Smith and Stockey, 2002) and cyatheaceous tree fern remains of Rickwoodopteris hirsuta (Stockey and Rothwell,

2004)) have been described from this locality.

The concretion containing the study specimen was cut into slabs using a water- cooled saw with a diamond-edged blade. The fossil fruit was exposed on one face of a slab, and was subsequently sectioned using the cellulose acetate peel technique (Joy et al., 1956). Peels were mounted on microscope slides using Eukitt (O. Kindler GmbH,

Freiberg, Germany) xylene soluble mounting medium. Photographs were taken with a digital Better Light (Placerville, CA) scanning camera mounted on a Leitz Aristophot large format camera, and focused through either Summar lenses or a Zeiss WL compound microscope. Images were processed with Adobe Photoshop CS 5.0 (Adobe, San Jose,

California, USA). Microscope slides are housed in the paleontology collections of the

Royal British Columbia Museum, Victoria, British Columbia, Canada.

Systematic paleobotany

Order— Cornales

Family— Cornaceae (sensu Xiang et al., 2002)

Genus— Cornus L.

Subgenus— Cornus (Xiang et al., 2005, 2006)

Species— Cornus cf. piggae Manchester, Xiang & Xiang 2010

Repository— Royal British Columbia Museum, Victoria, British Columbia,

Canada.

64 Locality— Beach at Shelter Point, Vancouver Island, British Columbia, Canada

(49° 56’ 39’’N, 125° 11’ 10’’ W).

Stratigraphic position and age — Spray Formation, Late Campanian (~73 Ma).

Description

The fossil fruit consists of a tri-locular woody endocarp with preserved seeds

(Fig. 3.1). One of the locules contains a fungal structure (Fig. 3.1), and hyphae can be seen in several places within the locule. Although the apex of the fruit was lost in the saw cut, the remaining endocarp is 1.3 mm long and 4.0 mm wide. The exterior surface of the endocarp is smooth, without conspicuous grooves or ridges. Locules are ellipsoidal to sub-triangular in cross section (Fig. 3.1) and at least 2.0 mm in diameter. Each locule has a dorsal germination valve, 0.4 - 0.5 mm thick, that extends the length of the fruit

(Fig. 3.2A). Septa are relatively thin, 0.2 – 0.5 mm thick (Figs. 3.1, 3.2A, 3.2D).

The ground tissue of the endocarp consists of sclerenchyma in the form of isodiametric and elongated sclereids (Figs. 3.2A-D). The elongated sclereids, 120 µm long and 12-24 µm wide, are often circum-locular (sometimes longitudinally elongated), and form a distinct multiseriate layer (Figs. 3.1, 3.2B-D). This layer is interpreted as the inner endocarp (Morozowska et al., 2013; Morozowska and Wysakowska, 2016). Outside of the inner endocarp is a zone of isodiametric sclereids, 18-30 µm wide, and secretory cavities, 50-100 µm wide, that form a uniseriate cycle around each locule (Figs. 3.1,

3.2A-3.2D). This tissue is designated as the outer endocarp (Morozowska et al., 2013;

Morozowska and Wysakowska, 2016).

65 There is no central vascular bundle or any other internal vascular tissue that can be identified within the endocarp. Many taxa within Cornales, including Cornus, have endocarps with no internal vasculature for much of their length; however, bundles run in the mesocarp along the outer periphery of the septa, for some distance before entering the endocarp towards the apex and traversing the septa to supply the seeds (Horne, 1914;

Wilkinson, 1944; Eyde, 1988; Manchester et al., 2010; Woźnicka et al., 2015; Atkinson,

2016; Stockey et al., 2016). Although the apex of the fossil fruit is missing, due to the conspicuous absence of any internal vascular tissue, it is most likely that this endocarp had a similar vascular pattern.

Each locule has one seed enclosed with a membranous seed coat that is one cell layer thick (Figs. 3.1, 3.3A). Integumentary cells are short and irregularly shaped

(cuboidal to polygonal) in paradermal section (Fig.3. 3B). One seed in the fruit has a dicotyledonous embryo that is surrounded by remains of endosperm (Figs. 3.1, 3.3C).

Cotyledons are spathulate, and measure 900 µm wide by 180 µm thick in transverse section.

One seed is heavily colonized by fungi (Fig. 3.1, at upper right). Endosperm and embryo tissues are not preserved in this seed. The fungal structure has a hollow center; towards the outside the fungal hyphae form pseudoparenchyma, and towards the inside cellular patterning becomes disorganized.

Discussion

The fossil fruit from the Campanian Shelter Point locality on Vancouver Island consists of a sclerenchymatous endocarp with dorsal germination valves, without a

66 central vascular bundle, and one seed per locule. This suite of fruit characters is characteristic of several taxa within Cornales (Eyde, 1988; Takahashi et al., 2002;

Manchester et al., 2010; Xiang et al., 2011; Atkinson, 2016; Stockey et al., 2016). More specifically, the endocarp of the Shelter Point fruit contains isodiametric and elongated sclereids, as well as secretory cavities, which are characteristic of some species within

Cornaceae (Eyde, 1988; Takahashi et al., 2002; Manchester et al., 2010; Atkinson, 2016;

Stockey et al., 2016), and the extinct taxon Suciacarpa starrii Atkinson (2016).

Living species of Cornaceae belong to four major clades within the genus, Cornus

L.: cornelian cherries, big-bracted dogwoods, dwarf dogwoods, and blue- or white-fruited dogwoods, (Eyde, 1988; Xiang et al., 2006). All four of these clades have endocarps with isodiametric sclereids, ellipsoidal to sub-triangular locules in cross section, and vascular bundles located at the periphery of the septa (Wilkinson, 1944; Eyde, 1988). The cornelian cherry clade, Cornus subg. Cornus (sensu Xiang et al., 2005), is the only lineage, however, with endocarps that contain both secretory cavities and elongated sclereids (Eyde, 1987, 1988; Xiang et al., 2003; Manchester et al., 2010). These characters are shared by the Shelter Point fruit, and confirm its affinity with the cornelian cherry clade.

There are six extant species of cornelian cherries: C. chinensis Wangerin, C. eydeana Xiang & Shui, C. mas L., C. officianalis Sieb. et Zucc., C. sessilis Torr. ex

Durand, C. volkensii Harms (Eyde, 1988; Xiang et al., 2005; Manchester et al., 2010). In addition, there are three previously described extinct species of cornelian cherries based on silicified and pyritized endocarps: C. piggae Manchester, Xiang & Xiang from the

67 Paleocene of North America, and C. ettingshausenii (Gardner) Eyde and C. multilocularis Gardner (Eyde) from the Eocene London Clay localities of England (Reid and Chandler, 1933; Eyde, 1988; Manchester et al., 2010).

One of the more obvious differences between endocarps of extant and extinct species of Cornus subg. Cornus is the number of locules. Living species commonly have one to two locules per endocarp (rarely three); whereas extinct species, including the fossil described in this study, frequently have more than two locules per endocarp (Table

3.2; Eyde, 1988). Another character that has been used in comparing endocarps of cornelian cherries is the wall thickness percentage (Wtp in Table 3.2), which is the thickness of the endocarp wall (germination valve) divided by the diameter of the endocarp, multiplied by 100 (Manchester, Xiang & Xiang, 2010). It is worth noting that caution should be taken while using this character for comparative analysis of fossils because of the possibility of measuring heavily eroded endocarps, which could lead to inaccurate wall thickness percentages. The fossil endocarp in this study has a more or less uniform wall thickness: thus we do not suspect that there was significant abrasion. While observing wall thickness percentages across the cornelian cherry clade, it appears that the majority of endocarps have relatively high values (Manchester et al., 2010), while C. piggae, C. multilocularis, and the Shelter Point fruit have low values (Table 3.2).

The endocarps of C. multilocularis can be distinguished from those of the Shelter

Point fruit by endocarp size and locule numbers (Table 3.2). Cornus multilocularis has large endocarps with a diameter of at least 8.0 mm and a valve thickness of at least 0.9 mm, almost double that of the Shelter Point endocarp (Table 3.2). Furthermore, the septa

68 of C. multilocularis are thicker than those of the Shelter Point fruit (Manchester et al.,

2010). The endocarps of C. multilocularis typically have four to six locules, but rarely three (Manchester et al., 2010) as in the Shelter Point endocarp.

The endocarp of the Shelter Point fruit is most similar to those of C. piggae from the Paleocene of North Dakota (Table 3.2). Similar to the Shelter Point fruit, endocarps of C. piggae often have three locules (two is less common) (Table 3.2).. The germination valves of both the Shelter Point fruit and those of C. piggae are 0.5 mm thick (Table 3.2).

Furthermore, the wall thickness percentages of the Shelter Point fruit and C. piggae are indistinguishable given the sample size (Table 3.2). Although the apex of the Shelter

Point fruit is missing, the available data reveals striking similarities between the Shelter

Point endocarp and those of C. piggae. The presence or absence of an apical depression in the endocarp, the length of the endocarp, and the amount of exposure of vascular bundles on the outer periphery of the septa are important taxonomic characters that may distinguish the Shelter Point fossil from C. piggae, but cannot be determined at this time

(Table 3.2). Additional specimens are needed to confidently assign the Shelter Point fruit to either Cornus piggae or to a new species of Cornus. Although our assignment of the

Shelter Point fossil to Cornus cf. piggae remains tentative, we nonetheless document that the C. piggae-type fruits are, so far, the most ancient of Cornaceae.

Suciacarpa starrii Atkinson (2016) from the Campanian of North America is the only other known cornalean outside of the cornelian cherry clade that has endocarps with secretory cavities (Table 3.1), and should be discussed briefly. Endocarps of S. starrii can be distinguished from those of C. piggae by several characters. While fruits of

69 Suciacarpa have four crescent-shaped locules and rows of vascular bundles within the septa (Atkinson, 2016), those of C. piggae (including the fossil in this study) have three ellipsoidal to sub-triangular locules and lack rows of vascular bundles within the septa

(Manchester, Xiang & Xiang, 2010). Thus, in number of locules, shape of locules, and position of vascular bundles, fruits of Suciacarpa differ from C. piggae and also from the

Shelter Point fruit (Table 3.1). Atkinson (2016) speculated that Suciacarpa may represent an extinct member of Cornaceae, however its phylogenetic position within the order Cornales is uncertain at this time.

Early evolutionary patterns of dogwoods and other cornaleans.

Cornus cf. piggae from the late Campanian of western North America is the oldest known fossil of Cornus subg. Cornus and crown group Cornaceae to date. Cornus piggae was first described from silicified endocarps preserved in upper Paleocene deposits of central North America (Manchester et al., 2010) and was previously recognized as the oldest representative of the cornelian cherry clade. The occurrence of

C. cf. piggae on Vancouver Island, Canada documents that the cornalean cherries originated well before the K/Pg boundary. The endocarp described in this study provides a minimum age for the subgenus Cornus of 73 Ma, and extends the fossil record of

Cornus and Cornaceae by at least 12 million years. This minimum clade age for Cornus subg. Cornus is congruent with the latest divergence time estimate of 73.4 Ma for the split between the cornelian cherry clade and the big-bracted and dwarf dogwood clade

(Xiang and Thomas, 2008).

70 Molecular divergence-time calculations predict that Cornaceae diverged from their sister group, Alangiaceae, around 80 Ma (Xiang and Thomas, 2008; Xiang et al.,

2011) and that the family radiated into its four major clades by the end of the Cretaceous

(Xiang et al., 2008). The fossil cornelian cherry described in this study is the first fossil evidence for crown-group Cornaceae during the Cretaceous, providing empirical support for the clade age calculation of previous studies (Xiang and Thomas, 2008; Xiang et al.,

2011) and rejecting the hypothesis of a Paleogene origin for the family and sub-clade

(Xiang et al., 2006; Xiang & Thomas, 2008).

The geographic distribution of Cornus is characterized by several intercontinental disjunctions which have been the subject of a number of studies and biogeographic analyses (Eyde, 1988; Xiang et al., 1996 p. 199, 2000, 2005, 2006; Xiang and Thomas,

2008; Manchester et al., 2009, 2010). Previous analyses have cautiously (Xiang and

Thomas, 2008) concluded that Europe was either the ancestral area or the site of initial diversification of Cornus (Xiang et al., 1996, 2005; Xiang and Thomas, 2008) because of the presence of Cenozoic fossils representing each sub-group. The intercontinental disjunctions of extant Cornus are often thought to be products of migrations over high latitude land bridges during the Paleogene and long distance dispersal events from

Europe. However, recent discoveries and descriptions of Cornus fossils offer a different perspective. The oldest fossils of Cornus subg. Cornus, are now known from the

Campanian and Paleocene of North America (Manchester et al., 2010). In addition,

Manchester & Kapgate (2014) reported a bi-locular cornelian cherry endocarp from the

K/Pg boundary of India, demonstrating that the clade was geographically widespread

71 early on in its evolutionary history. As of now, these ancient distributions indicate that

Europe was not an ancestral area for cornelian cherries. Cornus subg. Cornus, and

Cornaceae as a whole were probably more diverse and widely distributed in the past.

These insights suggest that what remains of these clades may be ancient lineages with relictual distributions. We anticipate that as more fossils are recovered from Cretaceous and Paleocene deposits the paleogeographical distributions of cornaceous lineages will become more apparent, and that the evolutionary history of Cornaceae may prove to be more complex than previously thought.

The Late Cretaceous was a critical time for the initial phylogenetic radiation of the basal asterid order, Cornales (Knobloch and Mai, 1986; Mai, 1993; Takahashi et al.,

2002; Schenk and Hufford, 2010; Xiang et al., 2011; Manchester et al., 2015; Atkinson,

2016; Stockey et al., 2016). The most accepted earliest known cornalean fossils are fruits of Hironoia fusiformis Takahashi, Crane & Manchester (2002) from the early Coniacian of Japan. Recently, additional anatomically preserved cornalean fruits were recovered from Late Cretaceous deposits including those of Eydeia hokkaidoensis Stockey, Nishida

& Atkinson (2016) from the Santonian of Japan and the aforementioned Suciacarpa starrii (Atkinson, 2016) from the Campanian of North America. The phylogenetic relationships of these Coniacian, Santonian, and Campanian taxa are uncertain and at this point it appears that they probably represent stem lineages within Cornales (Atkinson,

2016; Stockey et al., 2016).

Until recently the only reported evidence of crown cornalean lineages in the

Cretaceous were fruits of Mastixiaceae from the Maastrichtian of Europe (Knobloch &

72 Mai 1986; Mai 1993). However, in the past few years paleobotanical studies, including this one, have documented more cornalean fossil fruits from the upper Campanian of

North America that represent crown-group families and genera. These include fossil fruits of Cornus (Cornaceae), Davidia Baill. (Davidiaceae), and Nyssaceae (Manchester et al., 2015). This enhanced fossil record of Cornales documents that the primary diversification of extant cornalean families did indeed occur before the end of the

Campanian, emphasizing the antiquity of some taxa.

Conclusions

The current study is part of an ongoing series of investigations seeking to elucidate the early evolutionary patterns and relationships of the early diverging asterid order, Cornales (Atkinson, 2016; Stockey et al., 2016). Each new fossil provides an empirical test for clade age and biogeographic hypotheses that are based on molecular trees of living species. The Campanian fruit described in this study is assigned to Cornus subg. Cornus cf. piggae, represents the oldest occurrence of Cornaceae to date and provides a minimum age of 73 Ma for the origin of subgenus Cornus. The discovery of this Cretaceous fossil and concurrent paleontological data suggest that the early biogeographic history of cornelian cherries as well as Cornaceae was probably more complex than previously realized, and that the ancestral area for the family remains uncertain.

Acknowledgements

The authors are most grateful to Graham Beard, Qualicum Beach Museum,

Qualicum Beach, British Columbia, for collecting and loaning the concretion that

73 contained the specimen for study. We thank Nathan Jud, Cornell University for a thorough review of the manuscript.

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Figure 3.1. Permineralized fruit of Cornus. Cross section of endocarp with three locules and one seed per locule. Note numerous secretory cavities within endocarp tissue. SH 790 B1 Bot #10. Scale = 1.0 mm.

79

Figure 3.2. Permineralized fruit anatomy of Cornus. A, Cross section of endocarp wall towards base showing planes of weakness of two germination valves (V) separated by septum (S), and locule (L). SH 790 B1 Bot #34. Scale = 100 µm. B, Cross section of valve showing isodiametric sclereids and secretory cavities (arrows). SH 790 B1 Bot #34. Scale = 60 µm. C, Cross section of valve showing inner endocarp (IE) with elongated sclereids tangential to locule (L), outer endocarp (OE) with isodiametric sclereids and secretory cavities (arrows). SH 790 B1 Bot #4. Scale = 50 µm. D, Cross section of endocarp showing locules (L) and central axis with inner endocarp tissue (elongated sclereids tangential to locules) and outer-endocarp tissue (secretory cavities and isodiametric sclereids). Note absence of central vascular bundle. SH 790 B1 Bot #33. Scale = 230 µm.

80

Figure 3.2

81

Figure 3.3. Seed anatomy of Cornus. A, Oblique cross section of seed with membranous seed coat (arrow). Sh 790 B1 Bot #15. B, Peridermal section of integument. SH 790 B1 Bot #1. C, Cross section of seed with endosperm (e) surrounding embryo with two cotyledons (c). Sh. 790 B1 Bot #25.

82

Table 3.1. General endocarp comparisons of dogwood fruits and close relatives. Data from: Wilkinson (1944), Eyde (1967,1988), Manchester, Xiang, & Xiang (2010), Atkinson (2016). 1Specimen described in this paper.

Ridges and/or Locule shape in Elongate Secretory grooves on Vasculature x.s. sclereids cavities valve surface Cornus cf. Ellipsoidal/ Periphery of Absent Present Present piggae1 subtriangular septum? Blue/White- Ellipsoidal/ Periphery of fruited Present Absent Absent subtriangular septum dogwoods Big-bracted Ellipsoidal/ Periphery of Present Absent Absent dogwoods subtriangular septum Dwarf Ellipsoidal/ Periphery of Absent Absent Absent dogwoods subtriangular septum Cornelian Ellipsoidal/ Periphery of Present/absent Present Present cherries subtriangular septum Rows of Suciacarpa bundles Absent Crescent Present Present starrii within septum

83

Table 3.2. Endocarp morphology of cornelian cherries, Cornus subgenus Cornus. Modified from Manchester, Xiang & Xiang (2010). 1Specimen described in this paper. *Wall (germination valve) thickness percentage (Wtp) is calculated by the thickness of the germination valve divided by the diameter of the endocarp X 100.

Germination Vascular Locule Length Width Length/w Apical valve (wall) Age bundle Wtp* No. (mm) (mm) idth ratio depression thickness exposure Taxa (mm) Cornus cf. Campanian 3 1.3+ 4 ? ? ? 0.5 12.5 piggae1 Cornus piggae Paleocene 2-3 5-10 5-7 1-1.6 Absent Apical half 0.5 13 Cornus Eocene 3(-5) 14 13 1 Present Apical half 2.3 27 ettingshausenii Cornus Eocene 3-6 5-17 8-12.5 1.4-1.6 Present Apical half 0.9 12 multilocularis Cornus Recent 2 7.5 4 1.8 Present Basal half 0.6 23 chinensis Cornus eydeana Recent 2 20-25 7-8 3 Present Basal half 1.8 24 Cornus mas Recent 2 (1-3) 9-20 4-7.3 2.2-2.7 Present Apical half 1.2 20 Cornus Recent 1 11-18 6-11.5 1.5-1.8 Present Apical half 0.9 34 officianalis Cornus sessilis Recent 2 11 4.2 2.6 Absent Apical half 1.2 28 Cornus volkensii Recent 2 8 4 2 Present Basal half 0.9 21

84

CHAPTER 4: THE EARLY DIVERSIFICATION OF CORNALES: PERMINERALIZED CORNALEAN FRUITS FROM THE CAMPANIAN (UPPER CRETACEOUS) OF WESTERN NORTH AMERICA.

Brian A. Atkinson, Ruth A. Stockey, and Gar W. Rothwell

Journal: International Journal of Plant Sciences The University of Chicago Press Journals, 1427 East 60th St., Chicago, IL 6037, USA In press.

85 Introduction

The order Cornales holds an important phylogenetic position as the earliest diverging lineage within the largest clade of eudicots, the asterids

(>80,000 spp.) (Magallón et al., 1999; Albach et al., 2001); APG 2016). The order is morphologically diverse and consists of approximately 605 species within ten families that are often circumscribed into five major lineages: 1) Cornaceae +

Alangiaceae; 2) Nyssaceae + Mastixiaceae + Davidiaceae; 3) Curtisiaceae +

Grubbiaceae; 4) Hydrangeaceae + Loasaceae; and 5) Hydrostachyaceae (Xiang et al., 1993, 1998, 2002, 2011; Fan and Xiang, 2003). Despite significant advances in molecular phylogenetics, early evolutionary patterns and deep-node relationships among the major clades remain problematic (Magallón et al., 1999;

Albach et al., 2001; Xiang et al., 2002, 2011; Fan and Xiang, 2003; Judd and

Olmstead, 2004).

Paleobotanical studies have identified a number of cornalean fossils from

Upper Cretaceous deposits (Knobloch and Mai, 1986; Takahashi et al., 2002;

Manchester et al., 2015; Atkinson, 2016; Stockey et al., 2016). Many of these fossils consist of fruits with thick-walled woody endocarps that preserve fairly well and contain many systematically informative characters (Eyde, 1988;

Atkinson, 2016; Stockey et al., 2016). The oldest described cornalean fossils are fruits of Hironoia fusiformis Takahashi, Crane, & Manchester (2002) from the

Coniacian of Honshu, Japan. More recently, permineralized fruits of Eydeia hokkaidoensis Stockey, Nishida, & Atkinson (2016) have been described from the

86 Santonian of Hokkaido, Japan. An additional extinct taxon, Suciacarpa starrii

Atkinson (2016) occurs in the Campanian of western North America. Recently, the oldest representative of Cornus L. (Cornaceae) has been described from the late Campanian of western North America (Atkinson et al., 2016). Manchester et al. (2015) have figured additional cornalean fruits from the Campanian of North

America; however, these fossils have yet to be formally described. In addition,

Knobloch and Mai (1986) have named several mastixiaceous taxa represented by fruits from the Maastrichtian of Europe. All of these fossil remains clearly indicate that the initial diversification of Cornales was well underway during the

Late Cretaceous and are providing essential data for inferring early evolutionary patterns and relationships within the order.

As part of a broader research agenda to elucidate the initial diversification patterns and relationships of Cornales (Atkinson et al., 2016; Atkinson, 2016;

Stockey et al., 2016), herein we characterize two new cornalean taxa based on permineralized fruits from the Late Cretaceous (Campanian) of Vancouver Island,

British Columbia, Canada. A new species, Suciacarpa xiangae Atkinson,

Stockey & Rothwell is established based on a well preserved endocarp. An additional unique cornalean endocarp, while incompletely preserved is characterized as a new taxon, Sheltercarpa vancouverensis gen. et sp. nov. based on a novel combination of characters. These cornalean fossils display character mosaics that are reminiscent of several families from different major cornalean lineages and provide insights into the initial radiation of the order.

87 Material and methods

Two anatomically preserved cornalean fruits have been recovered at the

Shelter Point Locality of Vancouver Island, British Columbia, Canada. The fruits are permineralized within a single calcareous concretion from the Shelter Point locality of Vancouver Island, British Columbia, Canada. Deposits at this locality are part of the Spray Formation, Nanaimo Group. Based on the presence of

Longusorbis decapod fossils, this locality is considered to be late Campanian

(Late Cretaceous, ~73Ma) in age (Richards, 1975). Plant fossils that have been described previously from Shelter Point include pinaceous seed cones of

Pityostrobus beardii (Smith and Stockey, 2002), a tree fern stem with attached roots, Rickwoodopteris hirsuta (Stockey and Rothwell, 2004), and a fruit assignable to Cornus cf. piggae (Atkinson et al., 2016).

Less than 5% of the concretions from the Shelter Point locality contain plant fossils, and those are consistently at a very low density. Nevertheless, fossils from this source frequently represent previously unknown species. The concretion containing the cornalean fruits was cracked open exposing one fruit in oblique longitudinal section (Figs. 4.1A, 4.1B). In order to obtain cross sections of the fruit the concretion was epoxied back together and cut into slabs using a water cooled saw with diamond edged blade. In doing so, another fruit type was exposed. Both fruits were sectioned using the cellulose acetate peel technique

(Joy et al., 1956). Microscope slides of the peels were prepared with Eukitt (O.

Kindler GmbH, Freiberg, Germany) xylene-soluble mounting medium.

88 Specimens mounted on microscope slides were photographed with transmitted light using a Better Light camera (Precision Digital Imaging system, Placerville,

CA) mounted on a Leitz Aristophot stand. The camera was focused using

Summar lenses or a Zeiss WL compound microscope. Images were processed in

Photoshop CS 5.0 (Adobe, San Jose, California, USA). Peels and microscope slides of the specimens are housed in the paleontology collections of the Royal

British Columbia Museum, Victoria, British Columbia, Canada.

Systematic paleobotany

Order— Cornales

Family— Incertae sedis

Genus— Suciacarpa Atkinson

Species— Suciacarpa xiangae sp. nov. Atkinson, Stockey, & Rothwell.

(Figs.4.1-4.4)

Species diagnosis— Fruit endocarp ovoid, at least 1.5 x 1.0 cm. Endocarp surface smooth, lacking ridges or grooves. Locules C-shaped in transverse section, some may be abortive. Germination valves with small isodiametric sclereids and densely packed secretory cavities. Septa thick, club-shaped in transverse section, consisting of transversely and chaotically oriented fibers, elongate sclereids, and small secretory cavities. Vascular bundles small, associated with large longitudinally oriented bundles of fibers, scattered throughout septa and central axis.

89 Holotype hic designatus— Suciacarpa xiangae sp. nov. Atkinson,

Stockey, et Rothwell sp. nov. SH. 790 B1 Bot

Repository— Royal British Columbia Museum, Victoria, British

Columbia, Canada.

Type locality— Shelter Point (49o56’39”N, 125o11’10”W), Vancouver

Island, British Columbia, Canada.

Stratigraphic position and age — Spray Formation, Nanaimo Group; Late

Cretaceous (late Campanian, ~73 Ma).

Etymology— The specific epithet is proposed in honor of Dr. Jenny Q-Y

Xiang, North Carolina State University, for her meaningful contributions towards elucidating the evolution, biogeography, and developmental biology of Cornales.

Description.

The permineralized fruit is represented by a thick-walled woody endocarp, ovoid in shape (Figs. 4.1A, 4.1B), 1.5 x 1.0 cm, circular in cross section, and tetra-locular (Figs. 4.2A, 4.2B). The surface of the endocarp is smooth, lacking conspicuous ridges or furrows. The apex of the endocarp appears to be slightly acuminate (Fig. 4.1B). Locules are crescent-shaped, or more specifically, C- shaped in cross section, and two of the four are abortive and distorted (Fig. 4.2B).

Each locule has a germination valve that in cross section, is only discernable within the apical third to half of the fruit (Figs. 4.2A, 4.2B, 4.3A). Therefore, the valves are interpreted to be short rather than elongate. The septa are club-shaped

90 in cross section, and intersect to form a relatively thin central axis (Figs. 4.2A,

4.2B).

The endocarp tissue is composed of different types of sclerenchymatous cells and secretory cavities. Valves consist of small isodiametric sclereids and numerous densely spaced secretory cavities (Figs. 4.2A, 4.2B, 4.3A, 4.3B). In cross section the septa and central axis contain elongated sclereids and interwoven fibers as well as scattered small vascular bundles that are accompanied by larger bundles of thick-walled fibers (Figs. 4.3C, 4.3D, 4.3E). In addition, there are a few secretory cavities found in the septa and central axis (Fig. 4.3E), but far fewer than are in the valves (Figs. 4.2, 4.3A-4.3C).

A distinct inner endocarp tissue zone can be found tangential to the locules

(Figs. 4.3F, 4.3G). Within the septum and central axis, the inner endocarp consists of a thin layer of circum-locular fibers (Fig. 4.3F). The inner-endocarp of the germination valve is made up of longitudinally oriented fibers (Fig. 4.3G).

There is one apically attached seed per locule (Figs. 4.3A, 4.4A). The seed coat is membranous, uniseriate, and consists of rectangular cells in paradermal section (Figs. 4.4A, 4.4B). One seed contains the remains of a copious endosperm with ghosts of cell outlines (Fig. 4.4A). A single embryo is preserved in the same seed showing the hypocotyl in cross section (Fig. 4.4A), but cotyledons are not preserved. Tissues of the embryo are incompletely preserved, but a central vascular cylinder is probably represented by a tissue gap in the center of the hypocotyl.

91 Order— Cornales

Family— Incertae sedis

Genus— Sheltercarpa Atkinson, Stockey & Rothwell gen. nov.

Generic diagnosis— Endocarp woody, tetra-locular; external surface smooth, lacking ridges or grooves. Locules ellipsoidal to sub-triangular in cross section with uniseriate locule lining. One seed per locule. Germination valves short, with isodiametric sclereids. Septa with isodiametric and elongate sclereids.

Central vascular bundle lacking.

Species— S. vancouverensis Atkinson, Stockey & Rothwell sp. nov. (Fig.

4.5)

Specific diagnosis— As per genus.

Holotype hic designatus— Suciacarpa xiangae sp. nov. Atkinson,

Stockey, et Rothwell sp. nov. SH. 790 B1 Bot.

Repository— Royal British Columbia Museum, Victoria, British

Columbia, Canada.

Type locality— Shelter Point (49o56’39”N, 125o11’10”W), Vancouver

Island, British Columbia, Canada.

Stratigraphic position and age — Spray Formation, Nanaimo Group; Late

Cretaceous (latest Campanian, ~73 Ma).

Description.

The fossil fruit consists of a tetra-locular woody endocarp with sub- triangular to ellipsoidal locules in cross section (Fig. 4.5A). The endocarp is at

92 least 1.0 mm long and 2.0 mm wide. Sections of the endocarp are transversely oblique showing carpels and locules at varying levels. Separation tissue of the associated germination valves for three of the basal-most locules is difficult to identify (one is heavily abraded). However, the fourth locule, sectioned near the fruit apex, shows a germination valve with well-defined zones of separation (Fig.

4.5A, arrows). Therefore, the valves are interpreted to be short rather than elongate. The endocarp consists primarily of isodiametric sclereids (Figs. 4.5A-

C). However, some sclereids appear slightly elongate in the septa and central axis zone. There is an incomplete uniseriate to biseriate layer of fiber-like cells lining each locule (Fig. 4.5C, at arrow). Due to the preservation of the endocarp, it was difficult to determine its vascular pattern. However, there is no central vascular bundle. Details on seed and morphology were also not preserved, but the remains of a single seed are present in two of the locules (Fig. 4.5A).

Discussion

The two fruits described in this study are characterized by thick-walled woody endocarps with dorsal germination valves and no central vascular strand.

This suite of characters is strongly indicative of several families within the order

Cornales, such as Cornaceae, Alangiaceae, Nyssaceae, Mastixiaceae, and

Davidiaceae (Harms, 1898; Horne, 1909, 1914; Wilkinson, 1944; Eyde, 1963,

1967, 1988; Hammel and Zamora, 1990; Takahashi et al., 2002; Noll, 2013;

Atkinson, 2016; Stockey et al., 2016). Moreover, Suciacarpa xiangae has one apically attached seed per locule, which is also characteristic of these families.

93 Seed attachment is not preserved in Sheltercarpa. Other cornalean families such as Hydrangeaceae, Loasaceae, and Hydrostachyaceae have thin-walled capsular fruits with many seeds per locule (Leins and Winhard, 1973; Brown and Kaul,

1981; Hufford, 1988; Leins and Erbar, 1988, 1990; Weigend et al., 2004;

Morozowska et al., 2012), which obviously differ from the fruits described in this study. In addition, two other families within the order, Curtisiaceae and

Grubbiaceae, also have thick-walled woody endocarps, but those fruits have a central vascular strand (Smith and Smith, 1942; Eyde, 1988; Manchester et al.,

2007; Yembaturova et al., 2009), a character that is absent from the Shelter Point fruits.

Although the two Shelter Point fruits have a similar general morphology, they differ in several important characters. In transverse section, locules are crescent-shaped in the endocarp of Suciacarpa xiangae while those of

Sheltercarpa are ellipsoidal to subtriangular (Table 4.1). There are many secretory cavities within Suciacarpa xiangae endocarps that are absent from Sheltercarpa vancouverensis. Furthermore, the septa and central axis of the fruit of Suciacarpa. xiangae consist of fibers and elongate sclereids while those of the Sheltercarpa fruit mainly consist of isodiametric sclereids (Table 4.1). Due to these differences, it is clear that these Shelter Point fruits represent two distinct taxa.

Affinities of Suciacarpa xiangae.

The fruit of Suciacarpa xiangae has a thick-walled woody endocarp with four crescent-shaped locules, short germination valves consisting of isodiametric

94 sclereids and secretory cavities, septa composed of elongate sclerenchyma. The inner endocarp is composed of fibers, and there are a number of vascular bundles within the septa. The only other cornalean taxon with this combination of characters is Suciacarpa starrii Atkinson (2016) from the Campanian of western

North America (Table 4.1). Despite the striking similarities between these two species, there are important structural differences. Although the locules of both species are roughly crescent shaped in transverse sections, those of S. xiangae are

C-shaped while those of S. starrii are V-shaped (Atkinson, 2016). Furthermore, the septa and central axis of S. starrii endocarps consist mainly of transversely oriented elongated sclereids (Atkinson, 2016), while those of S. xiangae contain interwoven fibers (Table 4.1). The vascular bundles of S. starrii are conspicuous and are roughly in two parallel rows within the septa, while those of S. xiangae are small and scattered within the septa. Therefore, S. xiangae represents a new species of Suciacarpa that differs from S. starrii in several features.

Affinities of Sheltercarpa vancouverensis.

The fruit of Sheltercarpa vancouverensis has an endocarp with four locules that are sub-triangular to ellipsoidal-shaped in cross section, short germination valves, and lacks a central vascular bundle. The endocarp primarily consists of isodiametric sclereids. This endocarp displays of a suite of characters that occur in Mastixiaceae, Nyssaceae, Davidiaceae, Cornaceae, and Alangiaceae

(Eyde, 1963, 1967; 1968, 1988; Table 4.1).

95 Nyssaceae, Mastixiaceae, and Davidiaceae form a monophyletic group

(Fan and Xiang, 2003; Xiang et al., 2011) with a rich fossil record that extends back to the Late Cretaceous (Kirchheimer, 1939; Eyde and Barghoorn, 1963;

Dilcher and Quade, 1967; Knobloch and Mai, 1986; Eyde and Xiang, 1990; Mai,

1993; Manchester, 1994, 2002; Eyde, 1997; Stockey et al., 1998; Manchester and

McIntosh, 2007; Noll, 2013). Mastixiaceous endocarps have crescent-shaped locules (Eyde, 1963; Eyde and Xiang, 1990; Mai, 1993; Tiffney and Haggard,

1996; Stockey et al., 1998), which differ from the ellipsoidal to sub-triangular locules in Sheltercarpa (Table 4.1). Endocarps in Mastixiaceae and Davidiaceae have elongate germination valves while the endocarp of Sheltercarpa has short valves. Of these three families, Sheltercarpa is most similar to Nyssaceae due to the presence of short germination valves (Eyde, 1963, 1997; Eyde and Barghoorn,

1963; Noll, 2013; Table 4.1).

The family Nyssaceae contains two living genera, Nyssa L. and

Camptotheca Decne., distributed across the Northern Hemisphere (Eyde, 1963;

Wen and Stuessy, 1993; Fan and Xiang, 2003; Xiang et al., 2011). Nyssa, in particular, has a rich fossil record dating back to the Eocene (Reid and Chandler,

1933; Manchester, 1994). Despite the similarity between Nyssaceae and

Sheltercarpa, in having short germination valves (Table 4.1), they differ significantly in endocarp histology. Nyssaceous endocarps consist of narrow fibers (Eyde, 1963) while the endocarp of Sheltercarpa consists mainly of isodiametric sclereids.

96 Two extinct genera Amersinia Manchester, Crane & Golovneva (1999) and Browniea Manchester and Hickey (2007), both of which are Paleocene in age, have been assigned to Nyssaceae (Manchester et al., 1999; Manchester and

Hickey, 2007). Endocarps of Browniea differ from Sheltercarpa in having elongate germination valves (Table 4.1). Similar to Sheltercarpa, Amersinia has endocarps with short valves and isodiametric sclereids (Manchester et al. 1999).

However, Amersinia has endocarps with a distinct multiseriate zone of circum- locular fibers that we interpret as an inner endocarp (sensu Morozowska et al.,

2013) layer (Table 4.1), and that are absent from Sheltercarpa.

The monotypic families, Cornaceae (Cornus L.) and Alangiaceae

(Alangium Lam.), form a monophyletic group (Xiang et al., 2002, 2011; Fan and

Xiang, 2003) that has an extensive fossil record dating back to the Campanian

(Eyde et al., 1969; Eyde, 1988; Manchester, 1994; Feng et al., 2009; Manchester et al., 2010; Atkinson et al., 2016). Similar to Sheltercarpa vancouverensis, endocarps of Cornaceae and Alangiaceae usually consist of isodiametric sclereids

(Eyde, 1968, 1988). It is worth noting that species within Cornus subg. Cornus contain endocarps with elongated sclereids as well (Eyde, 1988; Manchester et al.,

2010; Morozowska and Wysakowska, 2016). However, cornaceous and alangiaceous endocarps have elongate valves while the valves of the Sheltercarpa endocarp are short (Eyde, 1968, 1988; Table 4.1).

Other than the genus Suciacarpa (see above comparison), there are two

Cretaceous cornalean fruits with uncertain familial affinities: Hironoia fusiformis

97 Takahashi, Crane & Manchester (2002) and Eydeia hokkaidoensis Stockey,

Nishida & Atkinson (2016), from the Coniacian and Santonian of Japan, respectively. Similar to Sheltercarpa, Hironoia and Eydeia have fruits with four locules. However, endocarps of Hironoia have an inner endocarp, which is lacking in the fruit of Sheltercarpa (Table 4.1). Moreover, endocarps of Hironoia and Eydeia have elongate germination valves rather than short valves that characterize the Sheltercarpa endocarp (Table 4.1).

Despite the incomplete preservation of the Sheltercarpa vancouverensis fruit, available data document that this species possesses a unique combination of characters (Table 4.1), revealing that the fossil endocarp represents a new taxon.

However, additional specimens will be required to more fully understand the vascular system of the fruit, and to clarify the systematic affinities of Sheltercarpa within Cornales.

Initial diversification of Cornales

Molecular phylogenetic studies resolve Cornales as ten families within five clades: 1) Cornaceae + Alangiaceae; 2) Mastixiaceae + Nyssaceae +

Davidiaceae (hereafter referred to as the NMD clade); 3) Curtisiaceae +

Grubbiaceae; 4) Hydrangeaceae + Loasaceae; and 5) Hydrostachyaceae (Xiang et al., 2002, 2011; Fan and Xiang 2003). However, relationships among these major lineages are uncertain and have yet to be resolved with strong support (Magallón et al., 1999; Albach et al., 2001; Xiang et al., 2002, 2011; Fan and Xiang, 2003;

Judd and Olmstead, 2004). Short branch lengths connecting the major clades, and

98 uncertainty at the root of the phylogeny in the results of analyses using only living species, are most likely due to an initial rapid radiation of Cornales (Xiang et al.,

2002, 2011; Fan and Xiang, 2003). Thus, molecular data from extant taxa alone may not be sufficient for inferring the early evolution of the order (Xiang et al.,

2002), and data from the fossil record will probably be required to resolve those deep internal branches. In this regard, morphological data from extinct Cretaceous representatives is already beginning to shed some light on the initial diversification of Cornales (Knobloch and Mai, 1986; Takahashi et al., 2002;

Manchester et al., 2015; Atkinson et al., 2016; Atkinson, 2016; Stockey et al.,

2016).

The oldest unequivocal record of Cornales are charcoalified fruits of

Hironoia fusiformis from the early Coniacian (~ 89 Ma) of Honshu Japan

(Takahashi et al., 2002). In addition, slightly younger Santonian deposits (~84

Ma) from Hokkaido, Japan have yielded permineralized fruits of Eydeia hokkaidoensis (Stockey et al., 2016). Both of these taxa have hypothesized plesiomorphic fruit vasculature patterns (see Eyde, 1963,1967, 1988; Atkinson,

2016). Endocarps of Hironoia have vascular bundles within the central axis

(Takahashi et al., 2002) and those of Eydeia have longitudinal rows of bundles throughout the septa (Stockey et al., 2016), vascular patterns which Eyde (1967) considered primitive within Cornales. These characters and stratigraphic ages of

Hironoia and Eydeia reveal that both types of vascularization were present within

Cornales in the mid-Late Cretaceous (Coniacian-Santonian 89-84 Ma).

99 By the Campanian (~80-73 Ma) cornalean fruits show great morphological diversity (Manchester et al., 2015; Atkinson et al., 2016; Atkinson

2016). Suciacarpa, in particular, displays a mosaic of hypothesized ancestral and derived fruit characters that are reminiscent of several families within the

Cornaceae/Alangiaceae and NMD clades such as: crescent-shaped locules

(Mastixiaceae and some Nyssaceae), short germination valves (Nyssaceae), secretory cavities (Cornaceae), and rows of vascular bundles within the septa

(Davidiaceae, some Mastixiaceae). Moreover, the endocarp of Suciacarpa xiangae contains interwoven fibers, which are characteristic of many mastixiaceous and davidiaceous taxa (Reid and Chandler, 1933; Eyde, 1963;

Manchester, 1994; 2002; Stockey et al., 1998). The mosaic of characters observed in the endocarp of S. xiangae and S. starrii further supports a close relationship between the Cornaceae/Alangiaceae and NMD clades (see Atkinson, 2016).

However, this hypothesis has yet to be tested by phylogenetic analyses.

The fruit of Sheltercarpa vancouverensis represents another Campanian cornalean with a mosaic of characters reminiscent of several families in two different major clades. These include an endocarp consisting of isodiametric sclereids and a lack of a central vascular strand (Cornaceae and Alangiaceae) and short germination valves (Nyssaceae) (Eyde, 1963, 1968, 1988; Noll, 2013).

Thus, Sheltercarpa shares characters with the Cornaceae/Alangiaceae and NMD clades.

100 Overall, cornalean fruits with apormorphic and plesiomorphic character mosaics from the Campanian are reminiscent of families within the

Cornaceae/Alangiaceae and NMD clades, which supports a close relationship between these two major groups. Moreover, a fruit representing the extant genus,

Cornus (Cornaceae) occurs in upper Campanian deposits of Shelter Point

(Atkinson et al., 2016). This striking diversity of fruits representing stem and crown group Cornales at a single locality suggests that the Campanian was a critical time for the evolutionary radiation of Cornales.

Conclusions

Due to unique combinations of characters, the Campanian fruits described in this study are recognized as two new cornalean taxa, Suciacarpa xiangae sp. nov. and Sheltercarpa vancouverensis gen. et sp. nov. Not only do these taxa increase the known diversity of Cretaceous Cornales, the newly discovered character mosaics also reflect the radiation of major cornalean lineages. These and other cornalean fossils demonstrate that the Campanian was a critical time interval for the evolution of the order (Manchester et al., 2015; Atkinson, 2016;

Atkinson et al., 2016). As the sampling density of Upper Cretaceous cornaleans increases, we are optimistic that fossils can significantly improve the resolution of the basal-most relationships within Cornales.

Acknowledgements

The authors would like to thank Graham Beard, Qualicum Beach

Museum, Qualicum Beach, British Columbia, for collecting and loaning the

101 concretion that contained the specimens for study. This work was supported in part by National Science Foundation grant DGE 1314109 to BAA.

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Figure 4.1. Suciacarpa xiangae sp. nov. Holotype SH 790. A, Specimen exposed in oblique longitudinal section on surface of cracked concretion. Scale bar = 2.0 mm. B, Counterpart of specimen in A. Note acuminate apex. Scale bar = 3.0 mm.

108

Figure 4.1

109

Figure 4.2. Suciacarpa xiangae sp. nov. Holotype SH 790 B1 Bot. A, Transverse section of endocarp towards apex showing valves, thick septum, central axis, and smooth exterior surface. Note high density of secretary cavities in valve tissue. #74, scale bar = 1.6 mm. B, Transverse section of endocarp showing four locules, two fertile, two abortive. Note crescent shaped locules. #139, scale bar = 1.9 mm.

110

Figure 4.2

111

Figure 4.3. Suciacarpa xiangae sp. nov. Holotype SH 790 B1 Bot. A, Transverse section of locule and germination valve (arrows). #121 scale bar = 1.0 mm. B, Secretory cavities surrounded by small isodiametric sclereids in valve. #92, scale bar = 336 µm. C, Transverse section of septum showing transversely elongated fibers and vascular bundles (arrowheads) with associated fiber bundles. #139, scale bar = 1.39 mm. D, Vascular bundle with fiber bundle. #72, scale bar = 36 µm. E, Septum displaying interwoven fibers and secretory cavities (arrows). #63, scale bar = 275 µm. F, Transverse section of ventral area of inner endocarp showing circum-locular narrow fibers tangential to locule. #121, scale bar = 120 µm. G, Transverse section of dorsal area of inner endocarp showing longitudinal fibers of inner endocarp tangential to locule. #121, scale bar = 42 µm. OE= outer endocarp, IE= inner endocarp, L= locule, Sd= seed.

112

Figure 4.3

113

Figure 4.4. Suciacarpa xiangae sp. nov. Holotype SH 790 B1 Bot. A, Transverse section of seed towards apex of locule showing membranous seed coat. #95, scale bar = 690 µm. B, Peridermal section of seed coat showing square to rectangular cells. #92, scale bar = 156 µm. C, Transverse section of seed showing endosperm (En) and embryo (Em). #95, scale bar = 322 µm.

114

Figure 4.5. Sheltercarpa vancouverensis gen. et sp. nov. Holotype SH790 B3. A, Transverse section of endocarp with four locules. Note locule at left showing germination valve (arrows). #10, scale bar = 690 µm. B, Transverse section of valve with isodiametric sclereids. #15, scale bar = 130 µm. C, Transverse section of septum showing isodiametric sclereids. Note arrow indicating possible locule lining of longitudinal fibers. #15, scale bar = 325 µm.

115

Figure 4.5

116

Table 4.1. Comparison of endocarp structure among similar cornalean taxa (modified from Atkinson 2016). Data from: Eyde 1963, 1968, 1988; Hammel and Zamora 1990; Manchester et al. 1999, 2010; Takahashi et al. 2002; Manchester and Hickey 2007; Noll 2013; Atkinson 2016; Stockey et al. 2016. *Note: the inner endocarp is a tissue layer, typically consisting of elongate sclereids or fibers, that surround the locule and markedly differs from bordering tissues by histology or cell orientation (see Morozowska et al., 2013; Morozowska and Wysakowska 2016). If present, septum and valve histology are characterized by the tissue surrounding the inner endocarp.

117

Table 4.1

No. of Locule Valve Septum Valve Endocarp Inner Endocarp Taxon Age locules shape length histology histology lacunae endocarp* vasculature Ellipsoidal ironoia to sub- Isodiametric Isodiametric Periphery of fusiformis Coniacian (3)-4 triangular Elongate sclereids sclereids Absent Fibers central axis Ellipsoidal Eydeia to sub- Isodiametric Isodiametric Rows in hokkaidoensis Santonian 4-(5) triangular Elongate sclereids sclereids Absent Absent septum Elongate Isodiametric Suciacarpa Crescent- sclereids, sclereids, Secretory Rows in starrii Campanian 4 shaped Short lacunae lacunae cavities Fibers septum Fibers, elongate Isodiametric Suciacarpa Crescent- sclereids, sclereids, Secretory Rows in xiangae Campanian 4 shaped Short lacunae lacunae cavities Fibers septum Ellipsoidal Sheltercarpa to sub- Isodiametric Isodiametric vancouverensis Campanian 4 triangular Short sclereids sclereids Absent Absent ? Septum Amersinia periphery, obtrullata Isodiametric Isodiametric periphery of (Nyssaceae) Paleocene 3 Ellipsoidal Short sclereids sclereids Absent Fibers central axis Browniea serrata (Nyssaceae) Paleocene 1 Ellipsoidal Elongate Fibers Fibers Absent ? ? Nyssa sylvatica Septum (Nyssaceae) Extant 1-(2) Ellipsoidal Short Fibers Fibers Absent Fibers periphery

118

Table 4.1 (Continued)

Nyssa talamancana Crescent- Septum (Nyssaceae) Extant 2-3 shaped Short Fibers Fibers Absent Fibers periphery Camptotheca acuminata Septum (Nyssaceae) Extant 1 Ellipsoidal Short Fibers Fibers Absent Absent periphery Davidia Varying involucrata Sub- sclereids, Rows in (Davidiaceae) Extant 6-9 triangular Elongate fibers Fibers Absent Fibers septum Mastixia philippinensis Crescent- Septum (Mastixiaceae) Extant 1 shaped Elongate Fibers Fibers Absent Fibers periphery Diplopanax Fibers, Scattered in stachyanthus Crescent- Fibers, parenchyma, Resin ventral (Mastixiaceae) Extant 1 shaped Elongate parenchyma lacuane canals Fibers endocarp wall Elongate, Cornus isodiametric Isodiametric mas sclereids, sclereids, Secretory Elongate Septum (Cornaceae) Extant 2-(3) Ellipsoidal Elongate lacunae lacunae cavities sclereids periphery Alangium chinense Isodiametric Isodiametric Septum (Alangiaceae) Extant 2 Ellipsoidal Elongate sclereids sclereids Absent Absent periphery

119

CHAPTER 5: TRACKING THE INITIAL DIVERSIFICATION OF ASTERIDS: ANATOMICALLY PRESERVED CORNALEAN FRUITS FROM THE EARLY CONIACIAN (LATE CRETACEOUS) OF WESTERN NORTH AMERICA.

Brian A. Atkinson, Ruth A. Stockey, and Gar W. Rothwell

Journal: Annals of Botany Oxford University Press, 198 Madison Avenue, New York, NY 10016, USA Submitted, in review.

120 Introduction

The fossil record indicates that the Late Cretaceous (100-66 Ma) was an important time for the initial radiation of core eudicots (>70% of extant angiosperm diversity;

Magallón et al., 1999; Friis et al., 2006a; 2011). The earliest evidence for this clade includes rosid flowers of Caliciflora (Friis et al., 2016) and an unnamed taxon (Basinger and Dilcher, 1984) from the early Cenomanian (~100 Ma) of North America. Slightly younger Cenomanian (~100-93.9 Ma) and Turonian (93.9-89.8 Ma) deposits have yielded a diversity of pollen representing additional rosids and other core eudicot lineages

(Doyle and Robbins, 1977; Jarzen, 1980; Pacltová, 1981; Wanntorp et al., 2004; Friis et al., 2006b; 2011). While these fossils strongly suggest that the radiation of core eudicots was well underway during the early Late Cretaceous, representatives of the largest core eudicot clade, the asterids, from Cenomanian-Turonian deposits are conspicuously absent.

The asterid clade consists of over 80,000 species (Magallón et al., 1999) and has an extensive post-Turonian fossil record (reviewed in Friis et al., 2011; Manchester et al.,

2015). While there have been several ambiguous reports of Cenomanian and Turonian asterids (see discussion), the most unequivocal, earliest evidence for this clade are charcoalified fruits of Hironoia fusiformis Takahashi, Crane & Manchester (2002) from the early Coniacian (~89 Ma) of Japan. More specifically, the morphology and anatomy of these fruits are highly diagnostic of the order Cornales (Takahashi et al., 2002), which holds an important phylogenetic position as the earliest diverging asterid lineage

(Magallón et al., 1999; Albach et al., 2001; Bremer et al., 2002; Judd and Olmstead,

121 2004; Angiosperm Phylogeny Group IV, 2016). Recent studies have documented a diversity of cornalean fossil fruits representing extinct and extant genera from the

Santonian (86.3-83.6 Ma) of Japan and Campanian (83.6-72.1 Ma) of North America

(Manchester et al., 2015; Atkinson, 2016; Atkinson et al., 2016; Stockey et al., 2016), which are beginning to shed light on the early evolution of the order.

In this study we characterize three new cornalean taxa, Eydeia vancouverensis sp. nov., Obamacarpa hokkaidoensis gen. et sp. nov., and Edencarpa grandis gen. et sp. nov., based on anatomically preserved (permineralized) fruits from the early Coniacian

(89 Ma) of western North America. These newly described cornaleans are the same age as Hironoia fusiformis. Together, these fruits represent the earliest evidence for Cornales and provide new insight into the timing of the initial diversification of the order, and the asterid clade as a whole.

Materials and methods

Three fossil fruit types have been identified from the Eden Main locality on

Vancouver Island, British Columbia, Canada. The fossils are anatomically preserved within calcareous shallow-marine concretions from two different exposures referred to as

“The Quarry” (49°49’52.04”N, 125°25’10.41”W) and “The Ditch” (49°49’24.02”N,

125°25’51.02”W). The exposures are part of the Upper Cretaceous Dunsmuir Member of the Comox Formation, Nanaimo Group (Mustard, 1994; Johnstone et al., 2006).

Biostratigraphy of bivalves, gastropods, and ammonites indicates an early Coniacian (~89

Ma) age for the locality (Haggart et al., 2003, 2005; Karafit and Stockey, 2008; Klymiuk et al., 2015).

122 A preliminary survey of the permineralized flora at Eden Main has documented a diverse assemblage of bryophytes, lycophytes, ferns, conifers, and numerous angiosperms (Karafit, 2008). So far, schizaeaceous fertile pinnules of Paralygodium meckertii (Karafit and Stockey, 2008), a cycadeoid stem of Saxicaulis meckertii (Jud et al., 2010), an arthropod-ridden cupressaceous seed cone of Acanthostrobus edenensis

(Klymiuk et al., 2015), termite infested lauraceous wood of Paraphyllanthoxylon vancouverense (Jud et al., 2017), and platanaceous inflorescences and flowers of

Ambiplatanus washingtonensis (Mindell et al., 2014) have been formally described from this locality.

Fossil containing concretions were cut into slabs using water and oil cooled saws with diamond-edged blades. Specimens were sectioned using the cellulose acetate peel technique (Joy et al., 1956). Eukitt mounting medium (O. Kindler, Freiberg, Germany) was used to mount peels onto microscope slides. Photographs of the specimens were made using a Better Light scanning camera (Precision Digital Imaging system,

Placerville, CA) mounted on a Leitz Aristophot stand. Summar lenses or a Zeiss WL compound microscope were used to focus the camera. Specimens are housed in the

University of Alberta Paleobotanical Collections (UAPC-ALTA) and at the Museum of

Natural and Cultural History, Condon Museum, University of Oregon, Eugene.

Systematic paleobotany

Order— Cornales Dumortier sensu Xiang et al. (2011)

Genus— Eydeia Stockey, Nishida et Atkinson

Species—Eydeia vancouverensis sp. nov. Atkinson, Stockey, et Rothwell

123 Specific diagnosis— Endocarps ovoid, at least 6.0 mm long, 3.0 mm in diameter; three- to five-loculate. Apex acuminate. One to two longitudinal ridges along external surface of germination valves. Outer periphery of septa forming longitudinally elongate ribs. Valves, septa, and central axis with elongate and isodiametric sclereids. Locule lining with one row of cells with slightly thickened walls and small lumens. Seed coat one cell layer thick.

Holotype hic designatus— F-55442 (Condon Museum) (Figs. 5.1E, 5.2C, 5.2D).

Paratypes— F-55443−F-55472 (Condon Museum); P13527 C bot, D bot, D top, G top, H top a, H top b; P14345 C bot, E top; P14460 E top, F bot, G top; P14462 B bot, C bot, C top, D top, E top; P14464 C bot, D top, E top; P14470 E top; P14489 B top;

P15037 A bot (UAPC-ALTA).

Locality. Eden Main— “The Quarry” (49°49’52.04”N, 125°25’10.41”W) and “The

Ditch” (49°49’39.40”N, 125°25;43.50”W), Vancouver Island, British Columbia, Canada.

Stratigraphic position and age— Comox Formation, early Coniacian, Late Cretaceous

(~89 Ma).

Etymology— The specific epithet refers to Vancouver Island, British Columbia,

Canada, where the fossils were recovered.

Description

Fifty-two fossil endocarp specimens of the same type have been recovered from the Eden Main sites of Vancouver Island, British Columbia, Canada. These fossil fruits consist of woody, ovoid endocarps, up to 6.0 mm long and 3.0 mm wide, with an acuminate apex (Figs. 5.1A and 5.1B) and three to five locules (only one specimen with

124 five) that are ellipsoidal to sub-triangular in transverse section (Figs. 5.1C-5.1G). Each locule is associated with a dorsal germination valve that opens apically and extends nearly the entire length of the locule. Each valve has one to two external ridges.

Furthermore, the ends of the septa form longitudinally elongate external ribs (Figs. 5.1C-

5.1F). In the center of the endocarp the septa intersect to form a distinct, thick central axis with three to five arms (Figs. 5.1C-5.1G, 5.2A).

Endocarps consist of at least two different types of sclerenchyma cells (Figs.

5.2A-C). Germination valves have small isodiametric sclereids and elongate sclereids located within the external ridges (Fig. 5.2B). The central axis and septa consist of a mixture of isodiametric and elongate sclereids (Figs. 5.2A, 5.2C, 5.2D). Each locule is lined with a uniseriate layer of sclerenchyma cells that have thick walls and small lumens

(Fig. 5.2E).

Endocarps lack a central vascular bundle (Fig. 5.2A). However, a small number of inconspicuous bundles can be found within the septa, adjacent to the locules (Figs. 5.2C,

5.2D, 5.2F; arrowheads).

There is one apically attached seed per locule (Figs. 5.1C-F, 5.2B, 5.2F). The seed coat is membranous and one cell layer thick (Fig. 5.2E). In one seed, there is a layer of well-preserved cells, one to a few cells thick (Fig. 5.2F), that we are interpreting as the outer zone of endosperm similar to that described for Eydeia hokkaidoensis (Stockey et al. 2016).

Order— Cornales Dumortier sensu Xiang et al. (2011)

Genus— Obamacarpa gen. nov. Atkinson, Stockey et Rothwell

125 Generic diagnosis— Fruits drupaceous; endocarps woody; mesocarps fleshy.

Germination valves short. Inner endocarp of circum-locular fibers. Outer endocarp of isodiametric sclereids. Endocarp vascular bundles in two rows within each septum, no central vascular strand. One seed per locule, apically attached. Ovular bundle ventral within locule.

Type species— Obamacarpa edenensis sp. nov. Atkinson, Stockey et Rothwell

Specific diagnosis— Fruits ovoid, 5.0 mm long 3.0 mm wide. Endocarp with elongate persistent style, smooth external surface, three locules. Transition zone of slightly lignified cells between endocarp and mesocarp, 1-4 cells thick. Mesocarp of radially elongate thin-walled parenchyma. Up to 10 vascular bundles surrounded by fibers within mesocarp near periphery of endocarp. Epicarp one cell layer thick.

Etymology— The generic name is designated in honor of the 44th president of the

United States, Barack Hussein Obama, due to his commitment to furthering scientific progress in the United States as well as understanding and preserving biodiversity. The specific epithet refers to the locality where the fossils were recovered.

Holotype hic designatus— P145604 B top University of Alberta Paleobotanical

Collections (UAPC-ALTA); Figures 5.3A, 5.4A, 5.4B.

Paratypes— F-55473−F-55477 (Condon Museum); P145604 A bot, P145345 D top,

P13527 H top (UAPC-ALTA).

Localities— Eden Main: “The Quarry” (49°49’52.04”N, 125°25’10.41”W) and “The

Ditch” (49°49’39.40”N, 125°25;43.50”W), Vancouver Island, British Columbia, Canada.

126 Stratigraphic position and age— Comox Formation, early Coniacian, Late Cretaceous

(~89 Ma).

Description

Eight fossil drupaceous fruit specimens were collected from the Eden Main locality of Vancouver Island British Columbia. Two fruits were found to have a fleshy mesocarp still attached (Figs. 5.3A, 5.3B, 5.4A). Fruits are ovoid, 5.0 mm long and 3.0 mm wide, with a smooth surface (Figs. 5.3A-5.3D). The endocarp has a long acuminate apex, which may represent a persistent style (Fig. 5.3E). Each fruit consistently has three locules that are ellipsoidal to sub-triangular in transverse section (Figs. 5.3A-5.3D).

Germination valves are associated with each locule and are restricted to the apical third to half of the fruit (excluding the acuminate apex) (Fig. 5.4C).

The mesocarp is up 2.5 mm thick and consists of large radially elongate thin- walled parenchyma (Figs. 5.3A, 5.3B, 5.4A). The epidermis of the fruit is one cell layer thick and composed of small parenchymatous cells that are rectangular in transverse section (Fig. 5.4A). There are up to ten vascular bundles within the mesocarp that form a ring around the periphery of the endocarp (Fig. 5.3A). Each bundle is associated with several longitudinally elongate, thick-walled fibers (Fig. 5.4B). There is a transition zone between the tissues of endocarp and mesocarp zone composed of slightly lignified cells, up to four cells thick (Fig. 5.4A).

The endocarp is completely sclerenchymatous with two tissue zones (Figs. 5.3A-

5.3E, 5.4D-5.4F). There is a multiseriate inner endocarp zone that of circum-locular fibers (Figs. 5.3C, 5.4D-5.4F), surrounded by an outer-endocarp zone composed of small

127 isodiametric sclereids (Figs. 5.3C, 5.3D-5.4F). There is no central vascular strand; however, in each septum there are two rows of vascular bundles (Figs. 5.3C, 5.4E, 5.4F).

There is one apically attached seed per locule (Figs. 5.3B, 5.3D, 5.3E, 5.4C,

5.4G). The seed coat is membranous and one cell layer thick (Fig. 5.4G). The raphe is located on the ventral side of the seed, adjacent to the central axis (Fig. 5.4C).

Systematics

Order— Cornales Dumortier sensu Xiang et al. (2011)

Genus— Edencarpa gen. nov. Atkinson, Stockey et Rothwell

Generic diagnosis—Endocarp wall thick, woody, no conspicuous external ridges or grooves. Germination valves elongate. Locules ellipsoidal to sub-triangular in cross section. Endocarp of isodiametric and slightly elongate sclereids. Rows of vascular bundles within septa, no central vascular strand. One apically attached seed per locule.

Seed coat membranous.

Type species— Edencarpa grandis sp. nov. Atkinson, Stockey et Rothwell

Specific diagnosis— Endocarps ovoid, up to 7.0 mm long, 6.0 mm wide. Endocarp with smooth external surface, three to four loculate.

Holotype hic designatus— F-55479 (Condon Museum); Figures 5.5A, 5.6.

Paratypes— F-55478, F-55480−F-55482 (Condon Museum), P14493 D bot (UAPC-

ALTA).

Localities— Eden Main: “The Quarry” (49°49’52.04”N, 125°25’10.41”W) and “The

Ditch” (49°49’39.40”N, 125°25;43.50”W), Vancouver Island, British Columbia, Canada.

128 Stratigraphic position and age— Comox Formation, early Coniacian, Late Cretaceous

(~89 Ma).

Etymology— The generic name refers to fruits (-carpa) recovered from the Eden

Main fossil locality (Eden-). The specific epithet, grandis (large), refers to the relatively large size of the fruits.

Description

Six endocarps were recovered from the Eden Main sites. These endocarps are relatively large 7.0 mm long and 6.0 mm wide with smooth external surfaces, lacking ridges and grooves (Figs. 5.5A-5.5C). There are three to four locules per fruit (Figs.

5.5A-5.5C), each with an elongate germination valve that extends nearly the entire length of the endocarp (Figs. 5.5A, 5.5C). The endocarps mostly consist of isodiametric sclereids; however, there are a small number of slightly elongate sclereids within the septa and central axis (Figs 5.5A-5.5C, 5.6A-5.6C). Endocarps lack central vascular bundles (Fig. 5.6B); however, there are two rows of bundles per septum (Fig. 5.5C).

There is one apically attached seed per locule with a membranous seed coat (Figs. 5.5A,

5.5B).

Discussion

Eydeia vancouverensis, Obamacarpa edenensis, and Edencarpa grandis differ from one another in endocarp size, sculpturing, and histology. Specifically, the endocarps of Eydeia vancouverensis are conspicuously ridged while those of Obamacarpa edenensis and Edencarpa grandis are smooth. Furthermore, the fruits of Obamacarpa have short germination valves and contain an inner endocarp composed of fibers, while

1 29 those of Eydeia and Edencarpa have elongate valves and lack an inner endocarp.

Edencarpa endocarps are significantly larger than those of Eydeia and Obamacarpa.

Despite the differences among Eydeia vancouverensis, Obamacarpa edenensis, and Edencarpa grandis, all three taxa are characterized by thick and woody endocarps with dorsal germination valves, one apically attached seed per locule, rows of vascular bundles within the septa, and no central vascular bundle. This suite of characters is clearly indicative of the order Cornales (Wilkinson, 1944; Eyde, 1963, 1967, 1968, 1988;

Tiffney and Haggard, 1996; Manchester, 2002; Manchester et al., 2010; Morozowska et al., 2013; Atkinson, 2016; Atkinson et al., 2016; Morozowska and Wysakowska, 2016;

Stockey et al., 2016).

Comparison of Eden Main taxa to extant cornaleans

There are currently ten recognized families within Cornales that are resolved within five major groups: 1) Cornaceae, Alangiaceae; 2) Nyssaceae, Mastixiaceae,

Davidiaceae; 3) Curtisiaceae, Grubbiaceae; 4) Hydrangeaceae, Loasaceae; and 5)

Hydrostachyaceae (Xiang et al., 2002, 2011). Unlike the Eden Main taxa, species within

Hydrangeaceae, Loasaceae, and Hydrostachyaceae typically have capsular fruits with many seeds per locule (Brown and Kaul, 1981; Leins and Erbar, 1988, 1990, Hufford,

1997, 2001; Roels et al., 1997; Weigend et al., 2004; Morozowska et al., 2012) and, therefore, need not be considered further.

Many studies have emphasized the systematic importance of endocarp vasculature within Cornales (Horne, 1909, 1914; Wilkinson, 1944; Eyde, 1963, 1967, 1968, 1988;

Manchester et al., 2007; Yembaturova et al., 2009; Atkinson, 2016; Stockey et al., 2016),

130 and endocarp vascular patterns are useful for assessing systematic affinities of cornalean fruits. Curtisiaceous and grubbiaceous endocarps consistently have a central vascular bundle (Smith and Smith, 1942; Eyde, 1967; Manchester et al., 2007; Yembaturova et al.,

2009), while the Eden Main endocarps lack a central vascular bundle. Thus, Curtisiaceae or Grubbiaceae can also be removed from consideration.

Similar to the Eden Main taxa, fruits of Cornaceae, Alangiaceae, Nyssaceae,

Mastixiaceae, and Davidiaceae lack central vascular bundles. However, unlike the Eden

Main taxa, endocarps of Cornaceae and Alangiaceae consistently have vascular bundles located at the outer periphery of the septum for most of the endocarp length. This pattern is often called trans-septal (Horne, 1914; Wilkinson, 1944; Eyde, 1967, 1968, 1988;

Manchester et al., 2010; Table 5.1). Although, endocarps with trans-septal bundles occur in the Nyssaceae/Mastixiaceae/Davidiaceae clade, there are several taxa within this group that have rows of vascular bundles within the septa (Eyde, 1963, 1967, 1968, 1988; Eyde and Xiang, 1990; Stockey et al., 1998; Atkinson, 2016), a vascular pattern that is shared with the Eden Main taxa.

Nyssa ogeche Bartr. ex Marsh. is the only nyssaceous species with an endocarp vascular pattern similar to the Eden Main taxa. Eyde (1963) figured a uni-locular endocarp of N. ogeche showing several longitudinal bundles within the ventral wall, which is homologous to the septal position in multi-loculate fruits. However, all nyssaceous endocarps consist of only narrow fibers (Eyde, 1963; Noll, 2013), while the

Eden Main endocarps contain isodiametric sclereids. Moreover, endocarps of Nyssa are typically uni-locular, while those of the Eden Main fruits are multi-locular.

131 Within extant Mastixiaceae, Diplopanax Hand.-Mazz. is the only genus with endocarps that have several vascular bundles within the ventral wall (Eyde and Xiang,

1990; Stockey et al., 1998). However, all extant and extinct taxa within Mastixiaceae have crescent-shaped locules in cross section (Reid and Chandler, 1933; Kirchheimer,

1939; Knobloch and Mai, 1986; Mai, 1993; Manchester, 1994; Tiffney and Haggard,

1996; Stockey et al., 1998), which differ from the ellipsoidal to sub-triangular shaped locules in the Eden Main fruits (Table 5.1).

Similar to the Eden Main taxa, fruits within the monotypic family Davidiaceae

(Davidia Baill.) have several rows of bundles within the septa and the locules are sub- triangular in cross section (Horne, 1909; Eyde, 1963; Manchester, 2002; Atkinson, 2016,

Table 5.1). However, Davidia differs from the Eden Main cornaleans by having fruits with six to nine locules and fibrous endocarps with no distinct zones of isodiametric sclereids (Eyde, 1963; Manchester, 2002, Table 5.1).

Although the fruits of the Eden Main taxa have a vascular pattern seen only in the

Nyssaceae/Mastixiaceae/Davidiaceae clade, their endocarps contain a mosaic of characters not observed within the families of this major group (Table 5.1). Therefore, the taxa described in this study are not assignable to any extant family within Cornales.

Affinities of Eydeia vancouverensis

Eydeia vancouverensis is characterized by ridged endocarps with ellipsoidal to sub-triangular shaped locules in cross section, elongate germination valves, and rows of vascular bundles within the septa. Furthermore, the endocarp tissue of E. vancouverensis contains both isodiametric and elongate sclereids. This suite of characters is diagnostic of

132 the extinct genus Eydeia Stockey, Nishida & Atkinson (2016), previously known only from a single species, E. hokkaidoensis Stockey, Nishida & Atkinson, which has an uncertain phylogenetic position within Cornales. Although these two species have a similar general endocarp morphology, there are some distinct differences. Germination valves of E. hokkaidoensis have a single external ridge (Stockey et al., 2016), while those of E. vancouverensis have two external ridges. Furthermore, the valves of E. hokkaidoensis lack the elongate sclereids, that are present in those of E. vancouverensis

(Table 5.1). These differences indicate that the fruits of E. vancouverensis represent a new species of Eydeia.

Comparison of Obamacarpa and Edencarpa to extinct taxa

In addition to Eydeia, there are several other extinct cornalean genera, with uncertain phylogenetic relationships: Amersinia Manchester, Crane & Golovneva (1999),

Hironoia Takahashi, Crane & Manchester (2002), and Suciacarpa Atkinson (2016;

Atkinson et al., 2017). Similar to Obamacarpa and Edencarpa, the fruits of these extinct genera have combinations of characters not observed within any extant cornalean family.

Fruits, infructescences, and associated leaves of Amersinia obtrullata Manchester,

Crane & Golovneva (1999) have been described from the Paleocene of North America and Asia. Like those of Obamacarpa and Edencarpa, endocarps of Amersinia contain isodiametric sclereids (Table 5.1). Furthermore, Amersinia has endocarps with short germination valves and an inner endocarp layer, which also occur in Obamacarpa.

However, the endocarps of Amersinia have vascular bundles at the outer periphery of the septa and within the central axis (Manchester et al., 1999), while those of Obamacarpa

133 and Edencarpa have several bundles within the septa and lack bundles in the central axis

(Table 5.1). These differences in vasculature demonstrate that the Eden Main taxa are not representatives of the genus Amersinia.

Hironoia consists of one species, H. fusiformis Takahashi, Crane & Manchester

(2002) from the early Coniacian (89 Ma) of Japan. Fruits of Hironoia differ from those of

Obamacarpa and Edencarpa by having vascular bundles within the central axis

(Takahashi et al., 2002) rather than rows of bundles within the septa (Table 5.1).

Externally fruits of Hironoia have an external ridge on each valve, a character that is absent from Obamacarpa and Edencarpa (Table 5.1).

Suciacarpa contains one species, S. starrii Atkinson (2016) from the Campanian of western North America. Similar to endocarps of Obamacarpa and Edencarpa, those of

Suciacarpa have rows of vascular bundles within the septa (Atkinson, 2016). However,

Suciacarpa fruits have crescent-shaped locules in cross section (Atkinson, 2016), while those of Obamacarpa and Edencarpa are ellipsoidal to sub-triangular (Table 5.1).

Furthermore, the endocarps of Suciacarpa contain secretory cavities, which are lacking in those of Obamacarpa and Edencarpa (Table 5.1).

The fruits of Obamacarpa and Edencarpa have a combination of characters that do not conform to those of any previously described cornalean genus. Therefore, these fruit types represent two new genera and species, Obamacarpa edenensis and Edencarpa grandis.

Implications for the initial radiation of asterids

134 While there are no convincing reports of core eudicots from the Early Cretaceous

(145-100 Ma; see Friis et al., 2011, 2016; Manchester et al., 2015), the fossil record reveals that the Late Cretaceous (100-66 Ma) was a critical time for the initial radiation of the core eudicot clade (Friis et al., 2006a, 2011, 2016, Manchester et al. 2015). The earliest representatives of core eudicots are flowers of Caliciflora Friis, Pedersen &

Crane (2016) and an unnamed taxon referred to as the “Rose Creek Flower” (Basinger and Dilcher, 1984), from the early Cenomanian (~100 Ma) of North America. The floral morphologies of these taxa are strongly indicative of several lineages within the rosid clade (Friis et al., 2011, 2016). Additional rosids, represented by fagalean Normapolles pollen, have been reported from the Cenomanian (100-93.9 Ma) and Turonian (93.9-89.8

Ma) of Europe (Pacltová, 1981; Friis et al., 2006b; 2011). This evolutionary radiation is further highlighted by a diversity of late Cenomanian and Turonian pollen grains resembling Gunnerales and other core eudicot lineages (Doyle and Robbins, 1977;

Jarzen, 1980; Jarzen and Dettmann, 1989; Wanntorp et al., 2004; Friis et al., 2011).

However, there is a conspicuous absence of unambiguous Cenomanian-Turonian fossils of the most diverse core eudicot clade, the asterids (>80,000 extant species).

A number of fossils have been assigned to the two earliest diverging asterid orders, Cornales and Ericales, from putative Cenomanian and Turonian deposits; however, their affinities to the asterids are equivocal and/or their respective ages are uncertain (see Friis et al. 2016). A striking diversity of ericalean fossil flowers have been recovered from the Old Crossman Clay Pit locality of New Jersey, USA (e.g., Nixon and

Crepet, 1993; Martinez-Millan et al., 2009; Crepet et al., 2013; Martínez et al., 2016).

135 However, while the age of this locality has often been considered to be late Turonian

(~90 Ma) (Crepet et al., 2001), a Coniacian (89.8-86.3 Ma) and even a Santonian (86.3-

83.6 Ma) age have also been suggested (reviewed in Massoni et al., 2015; Friis et al.,

2016; Martínez et al., 2016). Thus, the precise stratigraphic age of this locality remains uncertain.

Fossil flowers of Tylerianthus crossmanensis Gandolfo, Nixon & Crepet (1998) have also been recovered from the Old Crossman Clay Pit locality and were tentatively assigned to Hydrangeaceae (Cornales). However, Tylerianthus displays a mosaic of floral characters also found in Saxifragaceae (Gandolfo et al., 1998). Therefore, the systematic affinities of Tylerianthus are unclear at this time.

Eoëpigynia burmensis Poinar, Chambers & Buckley (2007) was described from a single flower preserved in Burmese Amber that is possibly Cenomanian in age and cautiously assigned to Cornaceae. This taxonomic assignment was based on the flower displaying an epigynous tetra-merous morphology and in situ pollen that may be tricolporate (Poinar et al., 2007). However, this floral pattern also occurs in other core eudicot orders such as Saxifragales (rosids), Myrtales (rosids), and Asterales (asterids)

(Poinar et al., 2007). Thus, an affinity to Cornales or even the asterids is not strongly supported.

Pollen grains of the fossil form genus Nyssapollenites Thiergart have been extracted from the guts of fossil insects that were also preserved in Burmese Amber

(Huang et al., 2016). The taxonomy and systematics of Nyssapollenites, however, is highly problematic and many reports of this form genus may not even be representative

136 of Nyssaceae (Eyde, 1991). Therefore, an assignment to Nyssaceae or Cornales cannot be confirmed.

Conclusions

While there are no unambiguous reports of asterids from the Cenomanian or

Turonian, there is a considerable diversity of Cornales from slightly younger deposits represented by fruits of Hironoia fusiformis from the early Coniacian of Japan (Takahashi et al., 2002), and Eydeia vancouverensis, Obamacarpa edenensis, and Edencarpa grandis described here from the early Coniacian of North America. These fossils are the earliest unequivocal evidence for Cornales as well as the asterids. A striking aspect of this diversity is the disjunct geographic distribution of taxa (eastern Asia vs. western North

America), indicating that the order was already widespread at the time of its earliest appearance in the fossil record. The diversity, stratigraphic age, and geographic distribution of these early cornalean taxa clearly suggest that Cornales as well as the asterids diverged well before the Coniacian. Furthermore, these fossils indicate that

Turonian and possibly Cenomanian deposits are the most likely source of the most ancient asterids. We anticipate that a denser sampling of fossil fruits and flowers from these Late Cretaceous deposits will clarify the mode and tempo of early eudicot diversification.

Acknowledgements

We thank Dr. Dirk Meckert (Courtenay, BC), Rick Ross (Vancouver Island

Paleontological Society), Steven J. Karafit (University of Central Arkansas), Ludovic Le

Renard (University of British Columbia), Nathan A. Jud (Cornell University), for their

137 aid in collecting specimens in the field; and Ashley (AZ) Klymiuk (University of Kansas) for help in specimen preparation. This work was supported in part by National Science

Foundation grant DGE 1314109 to BAA and Natural Sciences and Engineering Research

Council of Canada grant A-6908 to RAS.

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Figure 5.1. Eydeia vancouverensis sp. nov. Atkinson, Stockey et Rothwell. A, Oblique longitudinal section of endocarp showing ovoid shape. F-55469, #2. Scale bar = 1.0 mm. B, Oblique longitudinal section of endocarp showing acuminate apex. F-55459, #2. Scale bar = 0.9 mm. C, cross section of fruit showing four locules associated with ridged germination valves. Note thick and woody septum. P14464 D top, #11. Scale bar = 1.0 mm. D, oblique cross section of fruit showing external ridges and one seed per locule. Bottom germination valve is abraded. F-55451, #9. Scale bar = 0.5 mm. E, Transverse section of fruit showing four locules and germination valve opening at left. F-55442, #1. Scale bar = 0.9 mm. F, Oblique transverse section of fruit showing three locules. F- 55453, #1. Scale bar = 245 µm. G, Oblique section of endocarp showing five locules. Note, level of locules within the endocarp vary showing one locule at its base (arrow) and one towards its apex (arrowhead). P13527 H top, #56. Scale bar = 637 µm.

146

Figure 5.1

147

Figure 5.2. Eydeia vancouverensis sp. nov. Atkinson, Stockey et Rothwell. A, transverse section of central axis displaying isodiametric and elongated sclereids. P14464 D top, #11. Scale bar = 325 µm. B, Transverse section of germination valve consisting of small sclereids. Note the elongate sclereids within the ridges as well planes of weakness of the valve at left and right. P 14464 D top, #10. Scale bar = 338 µm. C, Cross section of septum showing isodiametric to elongate sclereids and vasculature tissue (arrowhead). F- 55442, #1. Scale bar = 143 µm. D, Close up of vascular bundle in Fig. 2C. F-55442, #1. Scale bar = 52 µm. E, Transverse section of locule showing uniseriate lining. P14464 D top, #10. Scale bar = 195 µm. F, Cross section of seed showing outer layers of endosperm (arrow). Note vascular bundles (arrowheads) along the edge of the septum and central axis. P14460 F top, #161. Scale bar = 325 µm.

148

Figure 5.2

149

Figure 5.3. Obamacarpa edenensis gen. et sp. nov. Atkinson, Stockey et Rothwell. A, Transverse section of fruit showing tri-locular woody endocarp and fleshy mesocarp. Note ten bundles (arrow heads) surrounding endocarp. P145604 B top, #2. Scale bar = 2.4 mm. B, Oblique transverse section of fruit showing tri-locular endocarp with fleshy mesocarp. Note single seed in bottom locule. P145604 A bot, #1. Scale bar = 0.5 mm. C, Transverse section of tri-locular endocarp displaying thick septa. P14345 D top, #18. Scale bar = 0.7 mm. D, Transverse section of endocarp. Note single seed at left. P13527 H top, #9. Scale bar = 0.4 mm. E, Tangential section of endocarp showing persistent style and apically attached seed at left. F-55474, #0. Scale bar = 0.8 mm.

150

Figure 5.3

151

Figure 5.4. Obamacarpa edenensis gen. et sp. nov. Atkinson, Stockey et Rothwell. A, Transverse section of fruit showing sclerenchymatous endocarp, transition zone of slightly less lignified cells, parenchymatous mesocarp with radially elongated parenchyma, and epicarp. P145604 B top, 2. Scale bar = 315 µm. B, Vascular bundle within mesocarp surrounded by fibers. P145604 A bot, #8. Scale bar = 182 µm. C, Cross section of endocarp towards apex showing germination valves. Note ventral ovular bundle (arrow) within locule. P14345 D top, #55. Scale bar = 312 µm. D, Transverse section of endocarp showing locule surrounded by fibers of inner endocarp which is surrounded by small sclereids of outer endocarp. P14345 D top, #31. Scale bar = 195 µm. E, transvers section of central axis showing inner endocarp layers and outer endocarp of small isodiametric sclereids. Note the vascular bundles (arrow heads) just outside of the inner endocarp. P14345 D top, #31. Scale bar = 195 µm. F, cross section of septum showing rows of vascular bundles (arrow heads). Note small isodiametric sclereids of outer-endocarp. P14345 D top, #8. Scale bar = 715 µm. G, Cross section of locules showing single seed. P14604 A bot, #2. Scale bar = 208 µm.

152

Figure 5.4

153

Figure 5.5 Edencarpa grandis gen. et sp. nov. Atkinson, Stockey, et Rothwell. A, transverse section of tri-locular endocarp showing smooth surface, germination valves, and on seed per locule. Note bottom germination valve is abraded. F-55479, #38. Scale bar = 1.5 mm. B, oblique cross section of tri-locular endocarp. F-55478, #41. Scale bar = 0.5 mm. C, Oblique section of endocarp showing four locules. P14493 D bot, #67. Scale bar = 2.1 mm.

154

Figure 5.5

155

Figure 5.6. Edencarpa grandis gen. et sp. nov. Atkinson, Stockey, et Rothwell. A, transverse section of valve showing small isodiametric sclereids. F-55479. Scale bar = 0.6 mm. B, cross section of central axis consisting of isodiametric sclereids. Note lack of central vascular bundle. F-55479, #41. Scale bar = 0.8 mm. C, transverse section of septum showing rows of vascular bundles more or less lining locule (at bottom). F- 55479, #12. Scale bar = 0.5 mm.

156

Figure 5.6

157

Table 5.1. Comparison of endocarp structure among similar fossil and extant cornalean species. Data from: Chandler, 1962; Eyde, 1963; 1968; 1988; Manchester, 1994, 2002; Manchester et al. 1999; 2007; 2010; 2015; Takahashi et al. 2002; Yembaturova et. al., 2009; Serbet, pers. comm. 2016; Atkinson, 2016.

158

Table 5.1

Number Locule Exterior Valve Septum Histology of Valve Inner Endocarp Taxon Age of shape in ridges length histology central axis histology endocarp* vasculature locules x.s. on valve Sub- Isodiametric Elongate & Eydeia triangular Elongate Longitudinal Coniacian 3-4-(5) Present Elongate & elongate isodiametric Absent vancouverensis to sclereids rows in septa sclereids sclereids ellipsoidal Sub- Obamacarpa triangular Isodiametric Isodiametric Isodiametric Longitudinal Coniacian 3 Absent Short Present edenensis to sclereids sclereids sclereids rows in septa ellipsoidal Sub- Edencarpa triangular Isodiametric Isodiametric Isodiametric Longitudinal Coniacian 3-4 Absent Elongate Absent grandis to sclereids sclereids sclereids rows in septa ellipsoidal Sub- Isodiametric Eydeia triangular Elongate Isodiametric Longitudinal Santonian 4-(5) Present Elongate & elongate Absent hokkaidoensis to sclereids sclereids rows in septa sclereids ellipsoidal Elongate & Elongate & Hironoia Sub- Isodiametric Periphery of Coniacian 3-(4) Present Elongate isodiametric isodiametric Present fusiformis triangular sclereids fruit axis sclereids sclereids Elongate Isodiametric Suciacarpa Crescent- sclereids & Elongate sclereids & Longitudinal Campanian 4 Absent Short Present starrii shaped secretory sclereids secretory rows in septa cavities cavities

159 Table 5.1 (Continued).

Isodiametric Fibers & Fibers & Suciacarpa sclereids & Longitudinal Campanian 4 Crescent Absent Shot secretory secretory Present xiangae secretory rows in septa cavities cavities cavities Sub- Septum Amersinia triangular Isodiametric Isodiametric Isodiametric periphery, Paleocene 3-(4) Absent Short Present obtrullata to sclereids sclereids sclereids periphery of ellipsoidal fruit axis Davidiaceae Sub- Longitudinal Paleocene 6-8 Absent Elongate Fibers Fibers Fibers ? Davidia antiqua triangular rows in septa Varying Davidia Sub- Longitudinal Extant 6+ Present Elongate sclereids, Fibers Fibers Present involucrata triangular rows in septa fibers

Cornaceae Isodiametric Isodiametric Isodiametric Septum Extant 3-(4) Ellipsoidal Present Elongate Absent Cornus sclereids sclereids sclereids periphery oblonga Cornus Isodiametric Isodiametric Isodiametric Septum Eocene 3 Ellipsoidal Absent Elongate Absent clarnensis sclereids sclereids sclereids periphery Cornus Sub- Elongate Elongate Septum Eocene 3-6 Absent Elongate ? Present multilocularis triangular sclereids sclereids periphery Vascular Curtisiaceae Sub- Isodiametric tissue & Isodiametric Central in Extant 4 Absent Elongate Present triangular sclereids isodiametric sclereids fruit axis Curtisia dentata sclereids

160

CHAPTER 6: EXPLORING THE INITIAL PHYLOGENETIC AND MORPHOLOGICAL DIVERSIFICATION OF CORNALES.

Brian A. Atkinson

Target Journal: American Journal of Botany

161 Introduction

The order Cornales (605 species) is the earliest diverging lineage within the most diverse core eudicot group, the asterids (>80,000 species; Magallón et al., 1999; Albach et al., 2001; Judd and Olmstead, 2004; Xiang, 2013; Angiosperm Phylogeny Group,

2016). However, deep node relationships within the order remain unstable due to an initial rapid radiation (Xiang et al., 2002; Fan and Xiang, 2003; Xiang et al., 2011).

Molecular divergence time analyses suggest that this initial diversification took place during the Late Cretaceous (100-66 Ma) and resulted in the divergence of all five major cornalean clades: 1) Cornaceae-Alangiaceae, 2) Nyssaceae-Mastixiaceae-Davidiaceae, 3)

Curtisiaceae-Grubbiaceae, 4) Hydrangeaceae-Loasaceae, 5) and Hydrostachyaceae

(Schenk and Hufford, 2010; Xiang et al., 2011). As reflected by short branch lengths, molecular data of extant taxa alone may not be sufficient for elucidating the early pattern of evolution for these major clades (Xiang et al., 2002).

The extensive fossil record of Cornales dates back to the Late Cretaceous

(Knobloch and Mai, 1986; Takahashi et al., 2002; Manchester et al., 2015; Stockey et al.,

2016). Recent paleobotanical studies have characterized a diversity of exceptionally well- preserved cornalean fossils, representing several extinct and extant lineages from Late

Cretaceous deposits (Atkinson, 2016; Atkinson et al., 2016; Stockey et al., 2016, Chapter

4; Chapter 5). Supporting molecular divergence time analyses, these fossils clearly indicate that the Late Cretaceous was an important time interval for the initial diversification of the order. Moreover, the majority of Cretaceous cornalean fossils are represented by fruits, which preserve well and display a number of systematically

162 informative characters (see Eyde, 1963, 1967, 1988; Eyde and Barghoorn, 1963;

Yembaturova et al., 2009; Atkinson, 2016; Stockey et al., 2016).

Conveniently, fruits within Cornales are useful for the systematic delineation of major clades, families, genera, and even sub-genera (Harms, 1898; Eyde, 1963, 1967,

1968, 1988; Carlquist, 1977; Leins and Erbar, 1988; Hufford, 2001; Weigend et al., 2004;

Yembaturova et al., 2009; Morozowska et al., 2012; Morozowska and Wysakowska,

2016). Therefore, fossil cornalean fruits, particularly from Cretaceous deposits, have great potential for elucidating early evolutionary patterns and relationships within the order.

In this study, I explore and elucidate deep-node relationships and early diversification patterns within Cornales using fruit morphology of extant and extinct taxa.

Fossils are directly incorporated into phylogenetic analyses to assess the effect of extinct taxa on inferring ancient evolutionary relationships. Morphological diversity (disparity) analyses are also conducted to explore phenotypic aspects of early cornalean diversification. Overall, cornalean fruits, particularly fossil species, prove to have exceptional utility for elucidating the initial radiation of Cornales.

Materials and methods

A morphological data matrix consisting of 77 discrete fruit characters (see appendix 1) scored for 58 taxa was constructed in Mesquite (Maddison and Maddison,

2017). All characters are equally weighted and unordered. Character scorings were based on observations of prepared microscope slides (R.H. Eyde collection in the Botany

Department, National Museum of Natural History, Smithsonian Institution, Washington

163 D.C.), unpublished photos from personal communications (S.R. Manchester, Florida

Museum of Natural History, University of Florida, personal communication, 2016; M.

Weigend, University of Bonn, personal communication, 2016; R. Serbet University of

Kansas, personal communication, 2016), living material from cultivated plants located on the Oregon State University campus, and published literature (see Appendix 1).

The ingroup consists of 55 cornalean taxa including all major groups and families within Cornales sensu Xiang et al. (2011). Of the 55 taxa, 23 represent extinct species.

Extinct taxa were chosen on the basis of available morphological and anatomical fruit data. Three taxa included in the analyses have not been formally named, Drumheller fruit

1 and 2, reported in Manchester et al. (2015) as Davidia and Nyssa, respectively, and

“Nyssa grayensis” described in the M.Sc. thesis of Noll (2013). Three representatives of the order Ericales were selected as outgroups because Ericales is the sister lineage to the remaining asterids (Magallón et al., 1999; Judd and Olmstead, 2004; Angiosperm

Phylogeny Group, 2016). The outgroup species represent three basal ericalean families:

Balsaminaceae, Marcgraviaceae of the balsaminoid clade, and Fouquieriaceae

(Schönenberger et al., 2005; Lens et al., 2007; Schönenberger, 2009; Schönenberger et al., 2010; von Balthazar and Schönenberger, 2013). The cladistic matrix will be made available through MorphoBank (O’Leary and Kaufman, 2012; Project 2537: Cornales fruit matrix).

Phylogenetic analyses

To infer phylogenetic relationships within Cornales, the data matrix was analyzed using maximum parsimony in TNT (Goloboff et al., 2003, 2008) spawned through

164 Winclada (Asado, version. 0.9.9 beta; Nixon, 1999). Seven out of the 77 morphological characters introduced high levels of homoplasy and were deactivated (see appendix 1). In addition, to assess the effects of fossils on phylogenetic inference, extinct species were excluded in a separate analysis that included only living taxa. Up to 10000 trees were kept and strict consensus trees were computed for both types of analyses. Utilizing the same criteria as described above, analyses were also conducted in PAUP* version

4.0a152 (Swofford, 2002) using the heuristic search option, which resulted in strict consensus trees of the same topologies as those obtained in the analyses employing TNT.

Tree statistics are reported from the TNT analyses. Support values for individual nodes of the strict consensus trees were assessed by bootstrap analyses using 1000 replicates in

PAUP*. Strict consensus trees were exported as .tre files in PAUP*.

To visualize the phylogenetic diversification of Cornales through time, a pruned version of the fossil-inclusive strict consensus tree was scaled to geologic time in the statistical software program R version 3.2.3 (R Core Team, 2015) using the Strap package (Bell and Lloyd, 2015). The duration or stratigraphic ranges of fossil taxa are relatively uncertain; therefore, the upper (first appearance datum, FAD) and lower (last appearance datum, LAD) bounds were set to the boundaries of the Cretaceous and

Paleogene stages of the deposits from which the fossils were recovered (Cohen et al.,

2013; updated). To avoid the appearance of non-existent polytomies, branches were forced to have positive lengths using the “equal” method, with minimal root lengths set to two million years (see Brusatte et al., 2008; Bell and Lloyd, 2015).

Morphospace and disparity analyses

165 To analyze the morphological diversity (disparity) of cornalean drupaceous fruits through time, the original data matrix was modified to include only taxa with drupaceous fruits and exclude non-informative characters (see appendix 1). The modified matrix was uploaded and analyzed in R using the cladistic disparity analysis package Claddis (Lloyd,

2016). The taxa were organized into three time bins, Late Cretaceous, Paleogene, and

Neogene-Recent. The modified matrix was transformed into a generalized Euclidean distance matrix, which was used to generate a principal coordinates ordination (PCO) resulting in an empirical morphospace with 47 dimensions (PCO axes). The two most informative PCO axes, coordinates 1 and 2, together explained 19.3% of the total variation within drupaceous fruit morphology, and were used to visualize patterns of morphospace occupation.

Two post-ordination morphological diversity (disparity) metrics, sum of variance and sum of range (Ciampaglio et al., 2001; Wills, 2001), were calculated on the first 46 axes, which explained over 95% of the overall variation. To normalize differences in the sample sizes between time bins, bootstrap re-sampling was conducted with 1000 replicates, and was used to calculate 95% confidence intervals for each time bin. To assess whether each group occupied significantly different regions of morphospace, a non-parametric multivariate analysis of variance (NPMANOVA; Anderson, 2001) was calculated in the software program, PAST (Hammer et al., 2001). Calculations using

NPMANOVA included the first 46 PCO axes that were transformed into a Euclidean distance matrix and 10,000 replications.

Results

166 Phylogenetic analyses

Phylogenetic analysis including fossils utilized 70 characters (seven were omitted, see appendix 1) in which 65 were parsimony-informative. The TNT analysis resulted in

118 most parsimonious trees (MPT) of 190 steps (consistency index [CI] = 37, retention index [RI] = 70). Seventeen nodes collapsed in the strict consensus tree (SCT), resulting in a monophyletic Cornales with well-resolved deep-node relationships (Fig. 6.1). Within the SCT there is a basal grade consisting of Loasaceae (Loasa sclareifolia + Mentzelia dispersa), Hydrangeaceae (Jamesia americana + (Deutzia corymbosa + (Philadelphus purpurascens + Hydrangea heteromalla))), Hydrostachyaceae, Grubbiaceae, a Hironoia-

Amersinia clade, and Curtisiaceae (Curtisia quadrilocularis + Curtisia dentata), respectively, leading to a ‘core’ cornalean clade (Fig. 6.1). The core group includes a clade consisting of a grade of extinct taxa leading to a clade of Nyssaceae (Nyssa and

Camptotheca), Mastixiaceae, and Davidiaceae (hereinafter designated NMD), which is sister to a Cornaceae-Alangiaceae clade.

The fossil grade leading to the NMD group (hereinafter referred to as stem NMD group) is composed of an early diverging lineage (Sheltercarpa, Edencarpa + (Eydeia hokkaidoensis + (Eydeia vancouverensis + Drumheller fruit 1))), Obamacarpa edenensis, and a Suciacarpa clade (Drumheller fruit 2 + (Suciacarpa xiangae + Suciacarpa starrii)), respectively. Familial/generic relationships within the crown NMD clade are not well resolved; however, Mastixiaceae and Davidiaceae (Davidia involucrata + Davidia antiqua) are monophyletic. Within the Cornaceae-Alangiaceae clade, Cornaceae is paraphyletic with Cornus oblonga sister to a monophyletic Alangiaceae (Alangium

167 villosum, A. javanicum + (A. vermontanum + (A. eydei + A. chinense))); the remaining cornaceous taxa form a clade with a polytomy at its base (Fig. 6.1).

The phylogenetic analysis of living taxa only employed the same 70 characters as the analysis with fossils included; however, only 53 characters were found to be systematically informative. Parsimony analysis yielded 116 most parsimonious trees of

132 steps (CI = 59, RI = 88). Thirteen nodes collapsed in the strict consensus tree. As in the analysis including fossils, Cornales is monophyletic with a basal grade consisting of

Loasaceae, Hydrangeaceae, Hydrostachyaceae, Grubbiaceae, and Curtisiaceae as well as a core cornalean clade (Fig 6.2). However, relationships within the core clade are not well-resolved (Fig. 6.2). More specifically, Cornus and Alangium form a polytomy with the NMD clade. Relationships within the NMD group are similar to those in the results of the fossil inclusive analysis.

Time-scaled phylogenetic tree

By using minimum clade-age dating using fossils at terminal nodes, the time- scaled tree (Fig. 6.3) reveals a major diversification of Cornales occurring during the Late

Cretaceous. More specifically, major divergence events occurred by the Coniacian (89.8

Ma). By the end of the Maastrichtian (66 Ma) at least some extant families within the core cornalean group had already diverged (Fig. 6.3).

Morphospace and disparity analyses of cornalean drupaceous fruits

As reflected by the morphospace and NPMANOVA analyses, Late Cretaceous taxa occupy a more restricted and significantly different area of morphospace than

Paleogene and Neogene-Recent taxa (Fig. 6.4, Table 6.1). Paleogene and Neogene-

168 Recent cornaleans occupy similar areas of morphospace (Fig. 6.4, Table 6.1) in which the occupied region of Paleogene taxa is almost entirely within the occupied region of

Neogene-Recent taxa (Fig. 6.4). Furthermore, taxa within the NMD clade are mostly concentrated to the opposite to taxa within the Cornaceae-Alangiaceae in the morphospace.

Post-ordination disparity analyses using sum of variance and range metrics obtained similar results (Fig. 6.5 A, B). The disparity of drupaceous fruits represented by

Late Cretaceous cornaleans is significantly lower than that of Paleogene and Neogene-

Recent cornaleans. No significant difference in disparity is observed between Paleogene and Neogene-Recent taxa (Fig. 6.5A, B).

Discussion

Inclusion vs. exclusion of fossil taxa in phylogenetic analyses

The wealth of Cretaceous cornalean fossils that are available for study provides an excellent opportunity to assess the relative contributions of extinct and extant species in resolving the overall pattern of phylogeny for Cornales. Whereas, the resolution for terminal nodes is excellent using nucleotide sequence characters for living species, the morphology of cornalean fruits is particularly useful for resolving deep internal nodes of the phylogeny. The results of analyses using living species only found higher resolution near the terminal nodes. The fossil inclusive and exclusive parsimony analyses resulted in trees with a basal grade consisting of Loasaceae, Hydrangeaceae, Hydrostachyaceae,

Grubbiaceae, and Curtisiaceae as well as a core clade comprising Cornaceae,

Alangiaceae, Nyssaceae, Mastixiaceae, and Davidiaceae. In the analysis excluding

169 extinct taxa, resolution among the core cornalean clades is markedly lower.

Relationships among the Cornaceae, Alangiaceae and NMD groups are not resolved (Fig.

6.2). However, when fossils are included in the analysis the relationships among the

Cornaceae, Alangiaceae and NMD clades are resolved. This increased resolution is most likely due to the unique character mosaics found within extinct taxa that reflect early cladogenic events that occurred in rapid succession (see Donoghue et al., 1989).

Phylogenetic relationships

Hydrangeaceae and Loasaceae, which compose the bulk of species diversity within Cornales, typically resolve as a major clade in molecular analyses (Xiang et al.,

2002; Fan and Xiang, 2003; Xiang et al., 2011). Although there is relatively strong support for a close relationship with Hydrangeaceae and Loasaceae, the phylogenetic position of Hydrostachyaceae has long been problematic (Albach et al., 2001; Xiang et al., 2011). In the current study Loasaceae, Hydrangeaceae, and Hydrostachyaceae were found to form a basal grade (here after referred to as LHH grade). Therefore, a close relationship between these families is supported.

Molecular analyses typically resolve Grubbiaceae and Curtisiaceae as a clade

(Xiang et al., 2002; Fan and Xiang, 2003; Xiang et al., 2011). However, there are no recognized morphological characters uniting this clade (Xiang et al., 2002; Yembaturova et al., 2009). In the current study, Grubbiaceae was found to be sister to the remaining

Cornales, all of which have drupaceous fruits. Furthermore, the monophyly of

Grubbiaceae and the remaining Cornales has strong bootstrap support in the analyses in the current study.

170 Hironoia Takahashi, Crane & Manchester and Amersinia Manchester, Crane &

Golovneva were suggested to have nyssaceous affinities due to their fibrous inner endocarps (Manchester et al., 1999; Takahashi et al., 2002). However, the phylogenetic analysis in this study indicates that these two extinct genera form a clade that is sister to

Curtisiaceae plus the core cornalean group (Fig. 6.1). Due to the presence of subtending bract scars, inflorescences of Amersinia most likely had large showy bracts that resemble those of Cornus and Davidia (Manchester et al., 1999). Therefore, petaloid bracts may be plesiomorphic within Cornales.

In the current study, Curtisiaceae was found to be sister to the core cornalean group, which contains Cornaceae, Alangiaceae, Nyssaceae, Mastixiaceae, Davidiaceae, and several extinct lineages. A close relationship between Curtisiaceae and these extant families was also found in a separate cladistic analysis using anatomical wood characters by Noshiro and Baas (1998). Furthermore, Curtisia (Curtisiaceae), Cornus (Cornaceae), and Mastixia (Mastixiaceae) have pollen with H-shaped endoaperatures, an additional character that suggests close affinities among these genera (Ferguson, 1977, Eyde 1988).

Within the core cornalean group, there are two major clades, one consisting of the

NMD clade with its associated stem group of extinct genera and the other consisting of

Cornaceae and Alangiaceae (Fig. 6.1). The monophyly of each clade is strongly supported by molecular data (Xiang et al., 2002; Fan and Xiang, 2003; Xiang et al.,

2011). The NMD and Cornaceae-Alangiaceae lineages have long been thought to form a monophyletic group (discussed in Eyde, 1988). In contrast, a recent phylogenetic analysis using cpDNA (Xiang et al., 2011) resolved the NMD clade as sister to a clade consisting

171 of Hydrostachyaceae plus the Hydrangeaceae-Loasaceae clade. However, this relationship was not supported in their total evidence analysis (Xiang et al., 2011).

Moreover, the cladistic analysis using wood anatomical data (Noshiro and Baas, 1998) suggested that Nyssaceae, Mastixiaceae, and Davidiaceae were more closely related to

Cornaceae, Alangiaceae, and Curtisiaceae than to Hydrangeaceae.

The NMD stem group consists entirely of Late Cretaceous taxa. As detailed below, the antiquity of this paraphyletic group has important implications for the timing and diversification of core-group Cornales. Moreover, within the NMD stem group, there are two fruit types from the Campanian of Canada, Drumheller fruits 1 and 2, that were preliminarily reported as Davidia and Nyssa, respectively (Manchester et al., 2015).

However, the analysis in the current study resolved Drumheller fruit 1 within an Eydeia clade and Drumheller fruit 2 as sister to Suciacarpa. Therefore, the Drumheller fruits may not be representative of Davidiaceae or Nyssaceae.

General trends in cornalean fruit evolution

Overall, species of Cornales are united by the presence of inferior (sometimes semi-inferior) ovaries and epigynous flowers (Horne, 1909; Smith and Smith, 1942;

Wilkinson, 1944; Eyde, 1963, 1968; Brown and Kaul, 1981; Eyde and Xiang, 1990;

Hufford, 1997; Roels et al., 1997; Hufford, 2001; Weigend, 2004b; Weigend et al., 2004;

Yembaturova et al., 2009). However, the fluvial aquatic Hydrostachys

(Hydrostachyaceae) is an exception due to the presence of highly specialized unisexual flowers that lack a perianth (Leins and Erbar, 1988, 1990). Taxa within Cornales can be roughly organized within two general groups based on fruit morphology:

172 dehiscent/capsular (LHH grade) and indehiscent/drupaceous (Grubbiaceae, Hironoia-

Amersinia clade, Curtisiaceae, and the core cornalean group).

Xiang et al. (2011) hypothesized that indehiscent drupaceous fruits with germination valves and no central vascular bundles are ancestral in Cornales. By contrast, the topology in the current study suggests that dehiscent capsular fruits are plesiomorphic within Cornales. The majority of species within the basal LHH grade have capsular fruits with many seeds per locule (Bensel and Palser, 1975; Leins and Erbar, 1988, 1990,

Hufford, 1997, 2001, Weigend, 2004a, b; Weigend et al., 2004). Moreover, many hydrangeacous and hydrostachyaceous gynoecia and fruits have a symplicate zone

(where the septa are discontinuous; Leins and Erbar, 1988, 1990; Roels et al., 1997;

Hufford, 2001) and have vascular bundles within the central axis of the synascidiate zone

(where the septa are continuous towards the base of the gynoecia). Furthermore, this fruit morphology is also present in the closely related basal ericalean outgroup taxa

(Schönenberger, 2009; Schönenberger et al., 2010; von Balthazar and Schönenberger,

2013).

Interestingly, Grubbiaceae was found to be phylogenetically situated between the

LHH grade and the remaining Cornales. This corresponds to the unique morphology of grubbiaceous fruits with woody endocarps containing symplicate zones and central vascular bundles within the synascidiate zones (Smith and Smith, 1942). Fruit morphology of Grubbiaceae resembles an intermediate between capsular fruits in the

LHH grade and the more ‘derived’ drupaceous fruits seen in the remaining Cornales, particularly in the core cornalean group (see Eyde, 1967, 1988).

173 Fruits within the Hironoia-Amersinia clade and the remaining Cornales are drupaceous with germination valves. Moreover, this clade and its successive sister lineage, Curtisiaceae, have fruits with multiple central vascular bundles (vestigial in

Amersinia; Manchester et al., 1999; Takahashi et al., 2002). It is worth noting that fruits of Amersinia also have transseptal bundles (see appendix 1 for definition, Manchester et al., 1999), a character that occurs primarily in core group taxa (Eyde, 1963, 1968, 1988).

All of the taxa within the core group have drupaceous fruits containing woody endocarps with germination valves, one apically attached seed per locule, and no central vascular bundle (Eyde, 1963, 1967, 1968; Eyde et al., 1969; Eyde, 1988; Manchester,

1994; Stockey et al., 1998, 2016; Atkinson, 2016; Atkinson et al., 2016). Eyde (1967,

1988) discussed the peculiarity of this fruit morphology, which appears to be relatively derived according to the topology recovered in the current study as opposed to the hypothesis of Xiang et al. (2011) that this fruit morphology is plesiomorphic.

Endocarps and ovules/seeds of the core cornalean group taxa are vascularized in two distinct ways. Rather than having central vascular bundles, core cornalean taxa have bundles that originate from the periphery of the septa either as: 1) transseptal bundles

(Cornaceae, Alangiaceae, most Nyssaceae, and some Mastixiaceae) or 2) several longitudinal rows of bundles within the septa (NMD stem group, some Nyssaceae, some

Mastixiaceae, and Davidiaceae; Eyde, 1967, 1988; Atkinson, 2016; Stockey et al., 2016).

Therefore, fruits lacking central vascular bundles unite the core cornalean group.

Timing of the initial diversification of Cornales

174 Using the stratigraphic ages of terminal fossil taxa as minimum clade-age dates provides a conservative and rigorous method for determining the timing of ancient phylogenetic divergences. Due to the nested arrangement of the earliest cornalean taxa

(Hironoia, Eydeia Stockey, Nishida & Atkinson, Edencarpa, and Obamacarpa) in the results of the time scale phylogeny (Fig. 6.3), it is clear that the primary diversification of

Cornales occurred prior to the Coniacian (89.8 Ma). Xiang et al. (2011) conducted the most conservative and comprehensive molecular divergence time analysis using inter- nodal fossil calibrations and suggested that the crown age of Cornales is ~96 Ma. Thus, the time-scaled phylogeny of the current study is congruent with the estimate of Xiang et al. (2011), supporting a pre-Coniacian origin for Cornales.

Morphological diversification of drupaceous fruits

The morphospace, NPMANOVA, and disparity analyses conducted in the current study provide concordant results. The disparity of drupaceous cornalean fruits during the

Late Cretaceous is relatively low (Figs. 6.4, 6.5). As revealed by the NPMANOVA analysis, Late Cretaceous taxa occupy a significantly different region of morphospace than Paleogene and Neogene-Recent taxa, indicating that fruits of Cretaceous cornaleans are significantly different from those of Cenozoic cornaleans. Moreover, by the

Paleogene, there was a significant shift in morphospace occupation, thus, indicating a significant change in fruit morphology. This shift coincides with a considerable expansion of morphospace occupation and increase in disparity. It appears that maximum levels of disparity were reached as early as the Paleogene and remained relatively constant throughout the rest of the Cenozoic.

175 Within a paleontological context, changes in morphospace occupation and disparity can be related to ecological responses to major environmental changes (e.g., mass extinction events; Foote, 1997; Erwin, 2007; Decombeix et al., 2011; Leslie, 2011;

Chartier et al., 2014; Xue et al., 2015; Foth et al., 2016). The morphological restriction of Late Cretaceous cornaleans may reflect the overall restricted ecological roles of angiosperms throughout the Late Cretaceous, at least in North America (Wing et al.,

1993; Wing and Boucher, 1998). The shift in morphospace occupation and increase in disparity of cornalean drupaceous fruits from the Late Cretaceous to the Paleogene may be attributed to an evolutionary exploration of different dispersal and seed germination mechanisms (see Eyde, 1988) that could have triggered an ‘ecological release’ as reflected by the expansion of morphospace occupation and increase in disparity.

Furthermore, this change in morphospace and disparity coincides with the major environmental changes that occurred throughout the Paleogene (Wilf and Johnson, 2004;

Wing et al., 2005), which suggests that cornaleans were exploiting new and available niches that appeared after the K/Pg event.

Another possible explanation for the changes of morphospace occupation and disparity may be due to developmental factors (see Boyce and Knoll, 2002; Erwin, 2007;

Chartier et al., 2014). Molecular analyses have indicated that the Cornaceae and

Alangiaceae clade experienced a whole genome duplication (WGD) event sometime between 85-66 Ma (Yu et al., 2017) followed by a subsequent increase in molecular evolution throughout the Paleogene (Xiang et al., 2008, 2011; Yu et al., 2017). Yu et al.

(2017) suggested that this WGD event played a critical role in the initial diversification of

176 Cornaceae, and probably Alangiaceae. Polyploidy and WGD are most likely to be important factors for morphological diversification and plant speciation (Soltis et al.,

2009; Schranz et al., 2012). Morphological diversification may be triggered by duplicated genes that could potentially enhance developmental toolkits, thus, encouraging the evolution of novel traits and character combinations.

The major shift in morphospace occupation revealed in the current study roughly coincides with the WGD event within the Cornaceae-Alangiaceae clade. The detection of similar synchronous WGD events in other cornalean clades, would help to explain the morphological diversification in drupaceous cornalean fruits through time.

Although the morphospace and disparity analyses in the current study utilize coarse time bins, they do provide a preliminary exploration into the earliest patterns of morphological diversification within Cornales. It appears that ecological and developmental factors may have played important roles in the early stages of the diversification of Cornales. This hypothesis, however, needs to be tested with the addition of more fossils particularly from Late Cretaceous and Paleocene deposits, which would allow for finer-scaled time bins.

Conclusions

Morphology of cornalean fruits, especially fossils, are crucial for elucidating the initial rapid radiation of Cornales. Cretaceous fruits contain data that reflect early patterns of divergence within the order and thereby resolve deep internal nodes of the tree. The nested arrangement of the oldest known cornaleans indicates that the initial diversification of the order occurred well before the Coniacian, supporting molecular

177 divergence time hypotheses (Xiang et al., 2011). Therefore, pre-Coniacian fossil fruits will be important for furthering our understanding of the pattern of initial cornalean diversification. The topology recovered in the current study suggests that capsular fruits are ancestral in Cornales and drupaceous fruits are derived. Among drupaceous fruits, those with central vascular bundles are ancestral, while those lacking central vascular bundles are derived. Thus, dehiscent fruits with central vascular bundles and many seeds per locule may be ancestral in asterids as a whole; however, this hypothesis needs to be tested with additional analyses.

A total evidence approach incorporating molecular data and morphological data of extinct and extant taxa would undoubtedly increase the resolution towards terminal nodes as well as serve as an ultimate test for inferring the overall pattern of phylogeny within Cornales. Exploration of the disparity of Cornales through time in the current study reveals that the morphological diversity of cornalean fruits has significantly changed and increased since the Late Cretaceous. This shows that Cretaceous cornalean fruits contain data important for reconstructing the early evolution of Cornales that otherwise is lacking in Cenozoic (including recent) fruits.

Acknowledgements

The author thanks Steven R. Manchester, Florida Museum of Natural History,

University of Florida, Maximillian Weigend, University of Bonn, and Rudy Serbet,

University of Kansas, for sending images of cornalean fruits. Appreciation also is expressed to Stan Yankowski Botany Department, National Museum of Natural History,

Smithsonian Institution for aid in obtaining microscope slides and images of living and

178 extinct cornalean fruits. Thanks to Tyler Schappe and Javier Tabima, Oregon State

University, for technical assistance in R. The author would also like to thank Gar W.

Rothwell and Ruth A. Stockey, Oregon State University, for their critical comments on the manuscript. This research was supported by NSF grant DGE-1314109.

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Figure 6.1. Strict consensus tree computed from most parsimonious trees obtained from fossil inclusive fruit morphological data set showing relationships in Cornales. Fossil species indicated with dagger (†). Numbers above branches indicate bootstrap support.

187

Figure 6.2. Strict consensus tree of most parsimonious trees obtained from fossil exclusive (i.e., living species only) fruit morphological data set.

188

Figure 6.3. Time-scaled phylogenetic tree of Cornales. Cladogram, pruned version of strict consensus tree in Figure 1. Thicker lines indicate upper and lower boundaries of approximate ages of terminal fossil taxa. Zero-length branches rescaled using ‘equal’ method and minimum branch length of 2 Ma.

189

Figure 6.4. Two-dimensional morphospace visualization of drupaceous cornalean fruits. Morphospace based on principal coordinate axes 1 and 2. Red = Late Cretaceous taxa, green = Paleogene taxa, blue = Neogene-Recent taxa.

190

Table 6.1. Non-parametric PERMANOVA test for statistical significance among time bins. Late Cretaceous (n = 12), Paleogene (n = 9), and Neogene-Recent (n = 27). Based on PCO axes (p<0.05**).

Neogene-Recent Late Cretaceous Paleogene

Neogene-Recent 0.0001** 0.6762

Late Cretaceous 0.0001** 0.0023**

Paleogene 0.6762 0.0023**

191

Figure 6.5. Drupaceous cornalean fruit disparity through time. A, Sum of variance metric. B, Sum of range metric. Plots display disparity value and associated 95% confidence intervals.

192

CHAPTER 7: CONCLUSIONS

193

Throughout this dissertation I have significantly increased the taxon sampling density of Late Cretaceous (100-66 Ma) cornaleans, developed new hypotheses on the early evolution of Cornales using fruit morphology, and reconstructed deep-node relationships and ancient patterns of morphological diversification within the order through phylogenetic and disparity analyses. Overall, these efforts have furthered our understanding of the initial radiation of Cornales and the asterid clade.

Prior to this dissertation there were six unambiguous cornaleans known from

Upper Cretaceous deposits (Knobloch and Mai, 1986; Takahashi et al., 2002; Stockey et al., 2016). Throughout this research endeavor seven taxa, including six new species and four new genera, were described from the Late Cretaceous of western North America:

Suciacarpa starrii gen. et sp. nov. (Chapter 2, Atkinson, 2016), Cornus subg. Cornus cf. piggae (Chapter 3, Atkinson et al., 2016), Suciacarpa xiangae sp. nov. (Chapter 4),

Sheltercarpa vancouverensis gen. et sp. nov. (Chapter 4), Eydeia vancouverensis sp. nov.

(Chapter 5), Obamacarpa edenensis gen. et sp. nov. (Chapter 5), Edencarpa grandis gen. et sp. nov. (Chapter 5). Characterizations of these taxa more than double the number of known Late Cretaceous cornaleans, clearly indicating that the Late Cretaceous was a critical time for the early evolution of Cornales. More specifically, the taxa described in

Chapters 2-4 are Campanian in age (83.6-72.1 Ma). The fruit character mosaics observed in the two species of Suciacarpa and Sheltercarpa vancouverensis are reminiscent of several cornalean families: Cornaceae, Alangiaceae, Nyssaceae, Mastixiaceae, and

Davidiaceae. Therefore, in Chapters 2 (Atkinson 2016) and 4, these five families are hypothesized to form a monophyletic group. This hypothesis is supported by the results

194 of the phylogenetic analysis presented in Chapter 6. The striking diversity of character mosaics displayed by Suciacarpa and Sheltercarpa indicate that the Campanian was an important time interval for the evolution of the Cornaceae-Alangiaceae and Nyssaceae-

Mastixiaceae-Davidiaceae (NMD) clades.

In Chapter 3, a permineralized fruit assignable to Cornus subg. Cornus cf. piggae was described from the Campanian of Vancouver Island, British Columbia, Canada. This fruit represents the oldest occurrence of Cornaceae to date, providing a minimum age of

73 Ma for the origin of Cornus. Furthermore, the occurrence of this cornaceous fossil also supports the hypothesis that the Campanian was an important time interval for the diversification of the Cornaceae-Alangiaceae and NMD clades.

As mentioned above, Cornales is the earliest diverging asterid lineage. Therefore, early cornalean fossils are important for understanding the timing and tempo of the initial diversification of asterids. Prior to this dissertation, the earliest cornalean and asterid fossils were represented by fruits Hironoia fusiformis from the early Coniacian (~89 Ma) of Japan. In Chapter 5, fruits of Eydeia vancouverensis, Obamacarpa edenensis, and

Edencarpa grandis were described from the early Coniacian of North America. Fruits of these taxa are synchronous with those of Hironoia, and together with Hironoia, are unequivocally the earliest evidence for Cornales. The disjunct geographic distribution of these early cornaleans (eastern Asia vs. western North America), clearly indicates that species of Cornales were already widespread by the Coniacian.

In Chapter 6, I conducted phylogenetic and morphological diversity (disparity) analyses that incorporated fruit morphological data from extinct and extant taxa. From

195 the results of those analyses, it is clear that the morphology of cornalean fruits, especially that of fossil fruits, is important for resolving the initial rapid radiation of Cornales. A time-scaled phylogeny indicates that the initial diversification of the order occurred well before the Coniacian, supporting previous molecular divergence time hypotheses (Xiang et al., 2011). Therefore, the discovery pre-Coniacian fossil fruits will be important for furthering our understanding of the origin and initial diversification of Cornales. The exploration of cornalean morphospace occupation and disparity through time reveals that the morphological diversity of cornalean fruits has significantly changed and increased since the Late Cretaceous. This change in disparity may be related to environmental and whole genome duplication events that occurred towards the end of the Cretaceous.

Additional fossils from Cenomanian (100-93.9 Ma), Turonian (93.9-89.8 Ma), and Paleocene (66-56 Ma) deposits are essential for testing the hypotheses presented in this study. The next step for inferring phylogenetic relationships within the order is to conduct a total evidence analysis incorporating molecular and morphological data, which would undoubtedly clarify relationships towards the terminal nodes. However, this dissertation is a solid step towards reconstructing the early evolution of Cornales.

The increase in Cretaceous taxon sampling density demonstrates that the recovery and characterization of fossil plants is essential for inferring evolutionary relationships.

As more fossils are collected, there is no doubt that cornalean fruits will continue to be an outstanding study system for analyzing macroevolutionary patterns and relationships among flowering plants.

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215

APPENDICES

216 Appendix 1: Cornalean fruit characters

General fruit morphology.

1. Ovary position (0, inferior/semi-inferior; 1, superior)

Hydrostachys spp. was scored as (-) because species of this genus are

imperfect, lacking a perianth (Leins and Erbar, 1988, 1990).

2. Fruit type (0, capsule; 1, drupaceous fruit)

Multi-carpelate fruits that dehisce to release their seeds are considered

capsular (character state, 0). Fruits with woody endocarps from which the

seed(s) directly germinate are considered drupaceous (character state, 1).

3. Capsule dehiscence (0, septicidal; 1, loculicidal; 2, apical interstylar region; 3,

apical valves)

Apical valve dehiscence should not be confused with germination valves of

drupaceous endocarps.

4. Capsule dehiscence procession (0, from basipetally from apex to base of fruit; 1,

acropetally from base to apex of fruits).

Modified from Hufford (1997) – Character 35

5. Fruit fusion (0, in fused heads; 1, not in fused heads)

6. Number of locules/ fruit (0, one; 1, two; 2, three; 3, four; 4, five or more)

While a number of studies have documented the plasticity of locule

numbers within endocarps (Eyde, 1963; Takahashi et al., 2002;

217 Manchester et al., 2010; Stockey et al., 2016), many taxa seem to have an

average number of locules per fruit (Eyde, 1963, 1988). Therefore, the

most common number of locules is coded for the appropriate taxon.

Note: due to high levels of homoplasy, this character was deactivated for

phylogenetic analyses.

7. Symplicate zone (0, present; 1, absent)

This is a zone typically found towards the apex of a multi-loculate ovary

where the septa do not fuse to form a central axis, thus the locules

coalesce to form a single cavity. See Hufford (2001).

8. General endocarp structure (0, thick and woody; 1, thin and not woody)

Drupaceous fruit endocarp morphology.

9. Endocarp shape (0, ovoid; 1, spheroid; 2, ob-ovoid; 3, spindle-shaped; 4,

fusiform; 5, conical; 6 funnel-shaped)

10. Apical depression on endocarp (0, present; 1, absent)

11. Acuminate apex (0, present; 1, absent)

Endocarps with an acuminate apex are considered to have a persistent style

(see Eyde 1988). These should not be confused with withered styles.

12. Germination valves (0, present; 1, absent)

13. Germination valve length (0, short; 1, elongate)

Length of valves is considered short when they only extend 1/3 or ½ of the

length of the endocarp (character state 0). Valves that extend beyond ½ of

218 the endocarp are considered elongate.

14. Germination valve apex (0, pointed; 1, rounded)

This character was deactivated due to high levels of homoplasy.

15. Pits/circular depressions on endocarp germination valve/dorsal exterior surface (0,

present; 1, absent)

Multiple rounded depressions on the endocarp surface, not including an

apical depression.

16. Grooves on endocarp valve/dorsal area exterior surface (0, present; 1, absent)

These groove(s) are located in the middle of the germination valve

exterior surface. The presence of this character is seen in many

mastixiaceous species (see Eyde 1963; Mai 1993; Tiffney and Haggard

1996). This should not be confused with a furrow between ridges.

Furthermore, these are not the fine grooves formed by vascular bundles.

17. Valve/dorsal area exterior groove morphology (0, shallow; 1, deep)

18. Groove(s) on endocarp valve/dorsal area interior surface (0, present; 1, absent)

These grooves extend the length of the inner surface of germination valve.

These should not be confused with furrows in between ridges.

19. Ridges on endocarp valve/dorsal area exterior surface (0, present; 1, absent)

These ridges are located in the middle of the germination valve’s exterior

surface and run longitudinally. This should not be confused with the area

between grooves.

219 20. Germination valve/dorsal exterior ridge morphology (0, broad/shallow; 1,

sharp/conspicuous)

21. Ridge(s) on endocarp ventral area interior surface (0, present; 1, absent)

These ridges are located within the locule running along the inner surface

of the central axis and/or septa.

22. Endocarp septal/ventral area exterior groove(s) (0, present; 1, absent)

These grooves are found on the exterior surfaces of the septa (or ventral

areas) and run longitudinally.

23. Endocarp septal/ventral area exterior ridge(s) (0, present; 1, absent)

These ridges are found on the exterior surfaces of the septa (or ventral

areas) and run longitudinally.

24. Locule shape in cross section (0, elliptical/sub-triangular; 1, crescent-shaped; 2,

~W-shaped; 3, D-shaped)

Ellipsoidal and sub-triangular locule shapes commonly overlap, so they

are coded as the same character state (0). Crescent-shaped locules are

often referred to as C- and U-shaped locules (see Tiffney and Haggard

1996).

25. Crescent-shaped locule morphology in cross section (0, U-shaped; 1, C-shaped; 2,

V-shaped)

26. Germination valve/dorsal area depression (0, present; 1, absent)

27. Fertile locules (0, equal-sized; 1, sizes differ).

220 Note, in some taxa, smaller locules can still be fertile, or crushed and

distorted (see Eyde 1968; Manchester 1994).

28. Abortive locules seen in mature fruits (0, present; 1, absent)

This pertains to locules that are aborted and distorted/closed or crushed by

growth of fruit and do not contain developed seeds.

Note: due to high levels of homoplasy, this character was deactivated for

phylogenetic analyses.

Drupaceous fruit endocarp linings and tissues.

29. Conspicuous locule lining (0, present; 1, absent)

This includes 1-2 layers of cells that line the locule that differ

histologically from neighboring cells of the septa and/or valves (Eyde,

1963; Stockey et al., 2016).

30. Conspicuous inner endocarp (0, present; 1, absent)

Multiseriate layer of cells surrounding the locule that form a distinct

tissue-zone (Morozowska et al., 2013; Morozowska and Wysakowska,

2016).

Drupaceous fruit endocarp histology.

31. Isodiametric sclereids in germination valve/dorsal area (0, present; 1, absent)

Sclereids with more of less equal dimensions. This character does not

include cells of the inner-endocarp, if present (see character 27).

32. Elongated sclereids in germination valve/dorsal area (0, present; 1, absent)

221 Sclereids that are more or less rice-shaped (Manchester et al., 2010). This

character does not include cells of inner-endocarp, if present (see character

27).

33. Fibers in germination valve/dorsal area (0, present; 1, absent)

In this study, fibers are considered as small diameter sclerenchyma cells in

which the length is over four times the width. This character does not

include cells of the inner-endocarp, if present (see character 27).

34. Parenchyma in germination valve/dorsal area (0, present; 1, absent)

35. Isodiametric sclereids in septa and central axis/ventral area (0, present; 1, absent)

This character does not include cells of inner-endocarp (if present, see

character 27).

36. Elongated sclereids in septa and central axis/ventral area (0, present; 1, absent)

This character does not include cells of inner-endocarp (if present, see

character 27).

37. Fibers in septa and central axis/ventral area (0, present; 1, absent)

This character does not include cells of inner-endocarp (if present, see

character 27).

38. Parenchyma in septa and central axis (0, present; 1, absent)

39. Sclerenchyma cells with large lumen diameter in central axis (0, present; 1,

absent)

40. Endocarp fibers in chaotic/interwoven multiseriate groups (0, present; 1, absent)

222 41. Secretory cavities (0, present; 1, absent)

Eyde (1988) used this to describe lacunae lacking an epithelium that are

filled with non-resinous substance. We have keyed this in fossil fruits that

lack an epithelium, although the contents may be unknown in some taxa.

42. Secretory cavities forming uniseriate cycle around locules (0, present; 1, absent)

43. Resin canals (0, present; 1, absent)

These are lacunae that are typically elongated with an epithelium and

contain resin.

44. Resin canals forming uniseriate cycle around locules (0, present; 1, absent)

45. Locule lining histology (0, fibers; 1, sclereids; 2, thin- walled cells)

Note: due to high levels of homoplasy, this character was deactivated for

phylogenetic analyses.

46. Inner endocarp histology (0, fibers; 1, elongated sclereids; 2, isodiametric

sclereids)

General fruit vasculature

47. Vascular bundles in central axis/ventral area (0, present; 1, absent)

48. Position of vascular bundles in central axis (0, center; 1, in margin of axis

opposite the locules).

Drupaceous fruit endocarp ventral vasculature.

Note: Ventral vasculature is used for vascular bundles that enter the ventral tissues of the endocarp. Eyde (1963, 1967, 1988) often called these bundles “ovular bundles”.

223 However, to avoid confusing the term ovular bundle with bundles that are directly attached to seeds within the locules, I refrain from using the term, ovular bundle, to refer to any ventral vasculature.

49. Vascular bundles towards outer periphery of septa or ventral endocarp tissue (0,

present; 1, absent).

This includes vascular bundles that are located towards the outer periphery

of the septa for most of the endocarp length. Near the apex these bundles

run across the septa to connect with the ovular bundles near the apex of

the locule. Eyde (1963, 1967) called this type of vasculature transseptal.

50. Transseptal (trans-septal) bundles ending blindly in septum (0, present; 1, absent)

51. Transseptal bundles entering endocarp (0, through grooves; 1, through pits)

52. Vascular bundles in longitudinal rows in septa (or ventral area of endocarp) for

most of the endocarp length (0, present; 1, absent).

53. Fibers associated with vascular bundles (0, present; 1, absent)

These fibers are markedly different from surrounding fibers (if present).

Drupaceous fruit endocarp dorsal vasculature.

54. Vascular bundles within exterior grooves on germination valve/dorsal area

surface (0, present; 1, absent)

55. Vascular bundles running parallel along exterior ridges on germination

valve/dorsal area surface (0, present; 1, absent)

224 56. Vascular bundles running parallel next to ridges on germination valve/dorsal area

surface (0, present; 1, absent).

57. Vascular bundles within germination valve/dorsal area tissue (0, present; 1,

absent)

Mesocarp and epicarp structure (at maturity).

Outgroup taxa with superior ovaries are not applicable for mesocarp and epicarp structure characters in this matrix (except for character 57). This is due to issues regarding homologies. For instance, the mesocarps and pericarps of in the in-group taxa (Cornales) with inferior ovaries are derived from the hypanthium while those of the out-group taxa with superior ovaries are directly derived from the carpel tissues.

58. Persistent calyx/tepal lobes (0, present; 1, absent)

59. Transition sclereids (0, present; 1, absent)

Transition sclereids are slightly lignified cells or cells with thicker walls

that are located between the mesocarp and endocarp. See Yembaturova et

al. (2009)

60. Sclereids in mesocarp (0, present; 1, absent)

Note: due to high levels of homoplasy, this character was deactivated for

phylogenetic analyses.

61. Radially elongated cells in mesocarp (0, present; 1, absent)

225 Note: due to high levels of homoplasy, this character was deactivated for

phylogenetic analyses.

62. Resin canals in mesocarp (0, present; 1, absent)

Lacunae with epithelia, containing resin.

63. Secretory cavities in mesocarp (0, present; 1, absent)

Cavities or canals without an epithelium that do not contain a resinous

substance.

64. Laticifers in mesocarp (0, present; 1, absent)

65. Mesocarp texture at maturity (0, leathery/dry; 1, fleshy)

66. Wings formed by pericarp (0, present; 1, absent)

67. Dense layer of large trichomes on exocarp at maturity (0, present; 1, absent)

Ovules/seeds/placenta.

68. Position of raphe within drupaceous fruits (0, ventral; 1, lateral/dorsal)

69. Placental morphology (0, intrusive, 1, non-intrusive)

This should be used for intrusive placentae take up the majority of the

locule space (Weigend, 2004).

70. Number of placentae per locule (0, one; 1, two or more)

71. Placentation (0, apical/sub-apical; 1, axile; 2, parietal l)

72. Number of ovules per locule (0, one; 1, two or more)

73. Number of cell layers in integument/seed coat (0, one to two; 1, three or more)

74. Cotyledon morphology (0, wide; 1, narrow)

226 Note: due to high levels of homoplasy, this character was deactivated for

phylogenetic analyses.

75. Chalazal wing (0, present; 1, absent)

76. Seed coat surface (0, sculptured; 1, smooth)

77. Endosperm copious (0, present, 1, absent)

Characters deactivated for phylogenetic analyses: 6, 14, 28, 45, 60, 61, 74

Data used for character scoring from: Kirchheimer, (1939); Smith and Smith (1942);

Wilkinson (1944); Eyde (1963, 1967, 1968, 1988, 1997); Eyde and Barghoorn (1963); Eyde et al. (1969); Leins and Winhard (1973); Bensel and Palser (1975); Carlquist (1977); Brown and

Kaul (1981); Knobloch and Mai (1986); Hufford (1988, 1997, 2001); Leins and Erbar (1988,

1990); Eyde and Xiang (1990); Hammel and Zamora (1990); Mai (1993); Wen and Stuessy

(1993); Manchester (1994, 2002); Tiffney and Haggard (1996); Roels et al. (1997); Stockey et al. (1998, 2016); Manchester et al. (1999, 2007, 2010); Takahashi et al. (2002); Xiang et al.

(2003); Weigend (2004); Weigend et al. (2004, 2005); Yembaturova et al. (2009);

Morozowska et al. (2012, 2013); Noll (2013); Woźnicka et al. (2015); Atkinson (2016);

Atkinson et al. (2016); Morozowska and Wysakowska (2016); S.R. Manchester, Florida

Museum of Natural History, University of Florida, personal communication, (2016); M.

Weigend, University of Bonn, personal communication, (2016); R. Serbet University of

Kansas, personal communication, (2016); B. Atkinson, personal observations (2016,

2017).

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231 Appendix 2: R code for time scaling, morphospace, and disparity analyses

##########Time scaling############# library(strap) library(ape) library(geoscale)

# Uplaod data file ages<-read.csv("strapages.csv", row.names = 1, header = TRUE) ages

# Upload tree tree<-read.nexus("ConTree2.tre")

# Ladderize tree tree <- ladderize(tree, right = F)

# Scaling tree to geo-time scale tree.l<- DatePhylo(tree, ages, method="equal", rlen=2)

# Plotting tree and time scale geoscalePhylo(tree=tree.l, boxes="Age", ages = ages, cex.tip=.9, cex.ts=.9, cex.age = 1, quat.rm=TRUE, vers="ICS2012")

#######################Morphospace####################### # Load the Claddis package into R: library(Claddis)

# Upload morphological matrix nexus.data <- ReadMorphNexus("cornales disparity.nex.nex")

# Check names names(nexus.data)

# Look over matrix nexus.data$matrix

# Convert matrix into distance matrices dist.data <- MorphDistMatrix(nexus.data)

# Check at names names(dist.data)

# Principal coordinate ordination

232 cmdscale(dist.data$GED.dist.matrix)

# Checking distance matrix dist.data$GED.dist.matrix

# Ordinating all axes pco.data <- cmdscale(dist.data$GED.dist.matrix, k=nrow(dist.data$GED.dist.matrix) - 1, add=T)$points

# Scree plot library(ggplot2) scree.df <- data.frame(scree.data) scree.df$index <- 1:47 scree.df$percent <- scree.df$scree.data/sum(scree.df$scree.data)*100 qplot(index, percent, data=scree.df)

# Assigning axes to two-dimensional morphospace: PCOx <- 1 PCOy <- 2

# Plotting ordination: plot(pco.data[, PCOx], pco.data[, PCOy], xlab=paste("PCO ", PCOx, " (", round(scree.data[PCOx], 2), "% variance)", sep=""), ylab=paste("PCO ", PCOy, " (", round(scree.data[PCOy], 2), "% variance)", sep=""), pch=19)

# Adding names: text(pco.data[, PCOx], pco.data[, PCOy], rownames(pco.data))

# Making a better version with ggplot # Make pco.data into a dataframe pco.df <- data.frame(pco.data) # Add a column in pco.data for the taxa names pco.df$taxa <- as.vector(rownames(pco.df)) # Save as a csv so we can look at it and add Age data and save as seperate file write.csv(pco.df, "pco.df.csv") # Load the edited csv file with Age pco.ages <- read.csv("pco.df.age.csv", header=F) # Remove empty columms pco.ages <- pco.ages[,3] # Put this into the pco.df pco.df$Age <- pco.ages # Get rid of the underscore in taxa pco.df$taxa2 <- gsub('_', ' ', pco.df$taxa)

# Calculate convex hulls library(plyr) pco.hull <- pco.df[,1:2]

233 pco.hull$Age <- pco.df$Age hull <- function(pco.hull) pco.hull[chull(pco.hull$X1, pco.hull$X2), ] hulls <- ddply(pco.hull, "Age", hull) hulls.cretaceous <- hulls[hulls$Age == "Late Cretaceous" ,] hulls.neogene <- hulls[hulls$Age == "Neogene-Recent" ,] hulls.paleogene <- hulls[hulls$Age == "Paleogene" ,]

library(ggrepel) library(ggplot2) pco.df$Age <- as.factor(pco.df$Age) pco.df$Age <- factor(pco.df$Age, levels=c("Late Cretaceous", "Paleogene", "Neogene-Recent")) levels(pco.df$Age) (pco.plot <- ggplot(aes(x=X1, y=X2), data=pco.df)+ geom_polygon(data=hulls.cretaceous, fill="red", alpha=0.3)+ geom_polygon(data=hulls.neogene, fill="#3846d2", alpha=0.3)+ geom_polygon(data=hulls.paleogene, fill="green", alpha=0.3)+ geom_point(aes(x=X1, y=X2, color=Age), size=5, data=pco.df))+ geom_label_repel(aes(label=taxa2, color=Age), nudge_y = -0.1, show.legend = FALSE, data=pco.df)+ theme_bw()+ scale_color_discrete()+ xlab("Coordinate 1, 11.8% of variance")+ ylab("Coordinate 2, 7.5% of variance")

##################Disparity calculations#########################

# Organizing data into time bins.

# Load the CSV with the taxa and the age bin they each belong to taxa.age <- read.csv("pco.df.age.csv", header=F) # Gabbing the first 2 columns taxa.age <- taxa.age[,c(1,3)] # Naming the columns colnames(taxa.age) <- c("Taxa", "Age")

# Subset by Age late.cretaceous <- taxa.age[taxa.age$Age == "Late Cretaceous",] Paleogene <- taxa.age[taxa.age$Age == "Paleogene",] Neogene.recent <- taxa.age[taxa.age$Age == "Neogene-Recent" ,]

# Making vectors late.cretaceous.vec <- as.character(late.cretaceous$Taxa) Paleogene.vec <- as.character(Paleogene$Taxa) Neogene.recent.vec <- as.character(Neogene.recent$Taxa)

234 ######## Late cretaceous disparity

# Sum of variance late.cretaceous.sum <- sum(apply(pco.data[late.cretaceous.vec, 1:46], 2, var))

# Sum of range. late.cretaceous.sum.range <- sum(apply(apply(pco.data[late.cretaceous.vec, 1:46], 2, range), 2, diff))

######## Paleogene disparity

# Sum of variance: paleogene.sum <- sum(apply(pco.data[Paleogene.vec, 1:46], 2, var))

# Sum of ranges: paleogene.sum.range <- sum(apply(apply(pco.data[Paleogene.vec, 1:46], 2, range), 2, diff))

######## Neogene-recent

# Sum of variance: neogene.recent.sum <- sum(apply(pco.data[Neogene.recent.vec, 1:46], 2, var))

# Sum of ranges: neogene.recent.sum.range <- sum(apply(apply(pco.data[Neogene.recent.vec, 1:46], 2, range), 2, diff))

########################################## Bootstraps ##########################################

### Late cretaceous sum of variance

# Creating sum of variance object with 1000 random bootstraps sov.late.cretaceous<-vector(length = 1000)

# Start loop for (j in 1:1000) {

# Randomly samples 6 rows from recent_dataframe. df.temp is a new frame that the 6 rows will be dumped into. df.temp <- pco.data[late.cretaceous.vec, 1:46][sample(nrow(pco.data[late.cretaceous.vec, 1:46]), 6, replace=FALSE),]

235 # Creating a for_loop over each column (staring at column 2). var.temp is the vector that var(recent) dumps. i is the column. var.temp<-NULL for (i in 1:ncol(df.temp)){ var.temp[i]<-var(df.temp[,i]) } # Sum of variance for var.temp with sqrt. omitting na column. sov.late.cretaceous[j] <- sum(na.omit(var.temp)) }

# Gives 95% confidence interval (quantile.sov.late.cretaceous<-quantile(sov.late.cretaceous, probs = c(0.05, 0.95))) # Gives mean of bootstrap (mean.sov.late.cretaceous<-mean(sov.late.cretaceous)) qplot(sov.late.cretaceous, geom="histogram")

### Late cretaceous sum of range

# Creating sum of range object with 1000 random bootstraps sor.late.cretaceous<-vector(length = 1000)

# Start loop for (j in 1:1000) {

# Randomly samples 6 rows from recent_dataframe. df.temp is a new frame that the 6 rows will be dumped into. df.temp <- pco.data[late.cretaceous.vec, 1:46][sample(nrow(pco.data[late.cretaceous.vec, 1:46]), 6, replace=FALSE),] # Creating a for_loop over each column (staring at column 2). range.temp is the vector that range(recent) dumps. i is the column. range.temp<-NULL for (i in 1:ncol(df.temp)){ range.temp[i]<-diff(range(df.temp[,i])) } # Sum of range for range.temp with sqrt. omitting na column. sor.late.cretaceous[j] <- sum(na.omit(range.temp)) }

# Gives 95% confidence interval (quantile.sor.late.cretaceous<-quantile(sor.late.cretaceous, probs = c(0.05, 0.95))) # Gives mean of bootstrap (mean.sor.late.cretaceous<-mean(sor.late.cretaceous)) qplot(sor.late.cretaceous, geom="histogram")

###Paleogene sum of variance

# Creating sum of variance object with 1000 random bootstraps

236 sov.paleogene<-vector(length = 1000)

# Start loop for (j in 1:1000) {

# Randomly samples 6 rows from recent_dataframe. df.temp is a new frame that the 6 rows will be dumped into. df.temp <- pco.data[Paleogene.vec, 1:46][sample(nrow(pco.data[Paleogene.vec, 1:46]), 6, replace=FALSE),] # Creating a for_loop over each column (staring at column 2). var.temp is the vector that var(recent) dumps. i is the column. var.temp<-NULL for (i in 1:ncol(df.temp)){ var.temp[i]<-var(df.temp[,i]) } # Sum of variance for var.temp with sqrt. omitting na column. sov.paleogene[j] <- sum(na.omit(var.temp)) }

# Gives 95% confidence interval (quantile.sov.paleogene<-quantile(sov.paleogene, probs = c(0.05, 0.95))) # Gives mean of bootstrap (mean.sov.paleogene<-mean(sov.paleogene)) qplot(sov.paleogene, geom="histogram")

###Paleogene sum of range

# Creating sum of range object with 1000 random bootstraps sor.paleogene<-vector(length = 1000)

# Start loop for (j in 1:1000) {

# Randomly samples 6 rows from recent_dataframe. df.temp is a new frame that the 6 rows will be dumped into. df.temp <- pco.data[Paleogene.vec, 1:46][sample(nrow(pco.data[Paleogene.vec, 1:46]), 6, replace=FALSE),] # Creating a _loop for over each column (staring at column 2). range.temp is the vector that range(recent) dumps. i is the column. range.temp<-NULL for (i in 1:ncol(df.temp)){ range.temp[i]<-diff(range(df.temp[,i])) } # Sum of range for range.temp with sqrt. omitting na column. sor.paleogene[j] <- sum(na.omit(range.temp)) }

# Gives 95% confidence interval

237 (quantile.sor.paleogene<-quantile(sor.paleogene, probs = c(0.05, 0.95))) # Gives mean of bootstrap (mean.sor.paleogene<-mean(sor.paleogene)) qplot(sor.paleogene, geom="histogram")

###Neogene-recent sum of variance

# Creating sum of variance object with 1000 random bootstraps sov.neogene.recent<-vector(length = 1000)

# Start loop for (j in 1:1000) {

# Randomly samples 6 rows from recent_dataframe. df.temp is a new frame that the 6 rows will be dumped into. df.temp <- pco.data[Neogene.recent.vec, 1:46][sample(nrow(pco.data[Neogene.recent.vec, 1:46]), 6, replace=FALSE),] # Creating a for_loop over each column (staring at column 2). var.temp is the vector that var(recent) dumps. i is the column. var.temp<-NULL for (i in 1:ncol(df.temp)){ var.temp[i]<-var(df.temp[,i]) } # Sum of variance for var.temp with sqrt. omitting na column. sov.neogene.recent[j] <- sum(na.omit(var.temp)) }

# Gives 95% confidence interval (quantile.sov.neogene.recent<-quantile(sov.neogene.recent, probs = c(0.05, 0.95))) # Gives mean of bootstrap (mean.sov.neogene.recent<-mean(sov.neogene.recent)) qplot(sov.neogene.recent, geom="histogram")

###Neogene-recent sum of range

# Creating sum of range object with 1000 random bootstraps sor.neogene.recent<-vector(length = 1000)

# Start loop for (j in 1:1000) {

# Randomly samples 6 rows from recent_dataframe. df.temp is a new frame that the 5 rows will be dumped into. df.temp <- pco.data[Neogene.recent.vec, 1:46][sample(nrow(pco.data[Neogene.recent.vec, 1:46]), 6, replace=FALSE),]

238 # Creating a for_loop over each column (staring at column 2). range.temp is the vector that range(recent) dumps. i is the column. range.temp<-NULL for (i in 1:ncol(df.temp)){ range.temp[i]<-diff(range(df.temp[,i])) } # Sum of range for range.temp with sqrt. omitting na column. sor.neogene.recent[j] <- sum(na.omit(range.temp)) }

# Gives 95% confidence interval (quantile.sor.neogene.recent<-quantile(sor.neogene.recent, probs = c(0.05, 0.95))) # Gives mean of bootstrap (mean.sor.neogene.recent<-mean(sor.neogene.recent)) qplot(sor.neogene.recent, geom="histogram")

############################ #Make plots of SOV and SOR with 95% confidence intervals ############################

###Sum of variances # Get all sum of variance data into one dataframe sum.var.df <- data.frame(cbind(c(mean.sov.late.cretaceous, mean.sov.paleogene, mean.sov.neogene.recent), c(quantile.sov.late.cretaceous[1], quantile.sov.paleogene[1], quantile.sov.neogene.recent[1]), c(quantile.sov.late.cretaceous[2], quantile.sov.paleogene[2], quantile.sov.neogene.recent[2]) )) colnames(sum.var.df) <- c("Mean", "lower", "upper") rownames(sum.var.df) <- c("Late Cretaceous", "Paleogene", "Neogene- Recent") sum.var.df$Age <- factor(rownames(sum.var.df), levels=c("Late Cretaceous", "Paleogene", "Neogene-Recent")) ggplot(aes(x=Age, y=Mean), data=sum.var.df)+ geom_point(aes(x=Age, y=Mean, color=Age), size=5)+ geom_errorbar(aes(x=Age, ymin=lower, ymax=upper, color=Age), width=0.25, lwd=1.2)+ theme_bw()+ theme(legend.position = "none")+ theme( axis.title.x = element_text(size=20), axis.text.x = element_text(size=18), axis.title.y = element_text(size=20), axis.text.y = element_text(size=18) )+ ylab("Sum of variance")

239 ### Sum of ranges # Get all sum of range data into one dataframe sum.range.df <- data.frame(cbind(c(mean.sor.late.cretaceous, mean.sor.neogene.recent, mean.sor.paleogene), c(quantile.sor.late.cretaceous[1], quantile.sor.neogene.recent[1], quantile.sor.paleogene[1]), c(quantile.sor.late.cretaceous[2], quantile.sor.neogene.recent[2], quantile.sor.paleogene[2]) )) colnames(sum.range.df) <- c("Mean", "lower", "upper") rownames(sum.range.df) <- c("Late Cretaceous", "Neogene-Recent", "Paleogene") sum.range.df$Age <- factor(rownames(sum.range.df), levels = c("Late Cretaceous", "Paleogene", "Neogene-Recent")) ggplot(aes(x=Age, y=Mean), data=sum.range.df)+ geom_point(aes(x=Age, y=Mean, color=Age), size=5)+ geom_errorbar(aes(x=Age, ymin=lower, ymax=upper, color=Age), width=0.25, lwd=1.2)+ theme_bw()+ theme(legend.position = "none")+ theme( axis.title.x = element_text(size=20), axis.text.x = element_text(size=18), axis.title.y = element_text(size=20), axis.text.y = element_text(size=18) )+ ylab("Sum of range")