PHYLOGENETIC AND BIOGEOGRAPHIC RELATIONSHIPS OF

NUTT. EX. SEEMAN ()

A THESIS SUBMITTED TO THE GRADUATE DIVISON OF THE UNIVERSITY OF HAWAIʻI AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

BOTANY

AUGUST 2014

By

Chelsea Osaki

Thesis Committee:

Clifford Morden, Chairperson Donald Drake David Lorence

ACKNOWLEDGEMENTS

I would like to acknowledge the following people whom I am grateful for in helping me to complete this project: Dr. Clifford Morden for his patience and kindness with the project and throughout my time as a graduate student; Dr. Donald Drake for his insightful comments on my thesis; and Dr. David Lorence for his knowledge of the Marquesas .

I would also like to thank Joel Lau, whose ideas about the evolution and speciation of

Cheirodendron have served as the foundations of this project. To Mitsuko Yorkston, who has taught me every lab technique I know. Thank you to Dr. Anthony Mitchell for his comments and suggestions for primers and Dr. Ines Schönberger from Allan Herbarium (CHR) for providing outgroup samples.

There were many people who have been involved in field work, collecting specimen, sorting herbarium samples, performing DNA extractions, PCRs, and analyses, whose help I greatly appreciate: Dr. Gregory Plunkett, Dr. Pei-Luen Lu, Dr. Richard Pender, Nipuni

Sirimalwatta, Adam Williams, Seana Walsh, Dylan Morden, Wendy Kishida, Steve Perlman,

Dr. Timothy Gallaher, Vianca Cao, Jesse Adams, Peter Wiggin, Bao Ying Chen, April

Cascasan, Dylan Davis, Jacy Miyaki, Gavin Osaki, Erin Fujimoto, George Akau, Matthew

Campbell, Isaiah Smith, Dr. Daniel Rubinoff, Dr. Michael Thomas, and Robert Tamayo.

Finally, I would like to thank the Botany Department at the University of Hawaiʻi at

Mānoa for providing me with opportunities to do my research; National Tropical Botanical

Garden for excellent hospitality during my stay on Kauaʻi; and finally, the Kōkeʻe Resource

Conservation Program for providing me with the opportunity to learn about conservation, native Hawaiian and the beautiful of Kauaʻi.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... ii

LIST OF TABLES ...... v

LIST OF FIGURES ...... vi

CHAPTER 1. LITERATURE REVIEW AND THESIS PROPOSAL ...... 1

INTRODUCTION ...... 1

Family Araliaceae ...... 2

Taxonomic history of the Cheirodendron...... 3

Traditional uses ...... 5

PROPOSED RESEARCH AND HYPOTHESES ...... 6

MATERIALS AND METHODS ...... 7

Taxon sampling, outgroup sampling and DNA extraction ...... 7

DNA sequencing and analysis ...... 8

Preliminary data ...... 9

Future directions ...... 9

CHAPTER 2. PHYLOGENETIC AND BIOGEOGRPAHIC RELATIONSHIPS OF

CHEIRODENDRON NUTT. EX. SEEM. (ARALIACEAE) ...... 13

ABSTRACT ...... 13

INTRODUCTION ...... 14

MATERIALS AND METHODS ...... 19

iii

Sampling and DNA extraction ...... 19

DNA amplification and DNA sequencing ...... 20

Primer screening and analysis ...... 23

RESULTS...... 27

DISCUSSION ...... 33

Relationships among taxa ...... 34

Current vs. phylogenetic relationships ...... 37

Biogeographic relationships...... 39

Future directions ...... 40

CHAPTER 3. SYNTHESIS- HYPOTHESES REVISITED ...... 42

LITERATURE CITED ...... 44

iv

LIST OF TABLES

Table Page

1.1 Comparison of revisions of Cheirodendron………………………………………...10-11

2.1 Voucher information and locality of specimen used in this study………………….21-22

2.2 List of primers and references………………………………………………………24-26

v

LIST OF FIGURES

Figure Page

1.1 Infrafamilial phylogenetic relationships of Araliaceae………………………………12

2.1 ETS phylogeny of Cheirodendron…………………………………………………...29

2.2 ITS phylogeny of Cheirodendron………………………………………………….…30

2.3 ndhF-rpl32 phylogeny of Cheirodendron…………………………………………....31

2.4 Combined phylogeny comparing MP, ML, and BI methods………………………..32

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CHAPTER 1. LITERATURE REVIEW AND THESIS PROPOSAL

INTRODUCTION

Understanding infrageneneric relationships are crucial in providing a basis in analysis for other studies such as biogeography, ecology, macroevolution, systematics, and conservation biology (Sites & Marshall 2004). One way to determine these relationships is through . Understanding phylogeny, or how species are related to one another, provides a comprehensive interpretation of evolutionary processes, including speciation

(Harrison 1998). The study of and phylogenetic has provided a direct record of the speciation events that have led to the extant species we see today

(Barraclough & Nee 2001).

Phylogenetic analysis has been a useful tool in elucidating the microevolutionary and macroevolutionary relationships in a number of lineages, including the Araliaceae.

Progress has been made in resolving the placement of Araliaceae within the order

(Henwood & Hart 2001, Plunkett & Lowry 2001, Plunkett et al. 2004) as well as understanding relationships within and among closely related genera (e.g. Wen & Zimmer

1996, Mitchell & Wagstaff 1997, Costello & Motley 2001, Eibl, Plunkett & Lowry 2001).

Although studies have been done on some of the more horticulturally (Hedera L., ivy) or ethnobotanically (Panax L., ginseng) important genera, little attention has been given to other genera within Araliaceae, particularly Cheirodendron Nutt. ex Seem.

The phylogenetic relationships among species of Cheirodendron are currently unknown. To date, only a few taxonomical studies have assessed species relationships.

However, speciation is not always accompanied by clear morphological differentiation

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(Kenfack 2011). The importance of phylogenetics in discovering species relationships of

Cheirodendron will sort out the taxonomy of the genus. My proposed research aims to uncover species relationships by sequencing various regions of both chloroplast and nuclear

DNA. Using a molecular approach, genetic differences will be assessed, rather than morphological characters which can be influenced by the environment and highly plastic.

Family Araliaceae

The Araliaceae consists of about 50 genera (Liu et al. 2012) and 1500 species that are widely distributed in the tropics and subtropics of Asia, the Pacific Islands, and the

Neotropics (Wen et al. 2001, Yi et al. 2004). Most members of Araliaceae are woody with variable leaf morphologies (simple, palmately compound and pinnately compound), but maintain conserved floral characteristics (5-merous flowers with inferior ovaries in a compound umbel) (Wen et al. 2001, Yi et al. 2004, Liu et al. 2012). Some well-known species in Araliaceae include medicinal herbs such as Panax ginseng C. A. Meyer (ginseng), and ornamentals including Hedera helix L. (English ivy) and Schefflera actinophylla (Endl.)

Harms (umbrella ) (Liu et al. 2012).

Molecular studies of Araliaceae agree on a phylogeny with multiple polytomies and poorly resolved basal lineages (Figure 1.1) (Mitchell & Wagstaff 1997, Plunkett, Wen &

Lowry 2004). Poorly resolved genera that are basal in Araliaceae include Schefflera J. R.

Forster & G. Forster, Cheirodendron Nutt. ex. Seem., Seem., Cussonia Thunb.,

Osmoxylon Miq., and Hydrocotyle L. (Plunkett, Wen & Lowry 2004).

Implications about the origins of these taxa and explanations about the unresolved basal polytomies suggest that there was a rapid diversification and radiation in Gondwana

(Mitchell & Wagstaff 2000, Plunkett, Wen & Lowry 2004). These findings corroborate with

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Couper (1960) and Lee, Lee & Mortimer (2001) who place the origin of Araliaceae in New

Zealand during the Lower Eocene. These studies also suggest that the closest relative of

Cheirodendron is Raukaua, a genus of six species with a “Gondwanan distribution,” endemic to , Chile, , and Tasmania (Mitchell, Frodin & Heads 1997).

Taxonomic history of the genus Cheirodendron

The taxonomic history of Cheirodendron is convoluted. The first collections of

Cheirodendron were made by Gaudichaud, who named the Aralia trigyna in 1830

(Herat 1981). In his description, he noted the glabrous and opposite leaves of the plant and its triangular fruit (Herat 1981). De Candolle (1830) placed Gaudichaud’s Aralia trigyna into the genus Panax as P. gaudichaudii DC. Soon after, with collections made on Captain

Beechey’s voyage around the Pacific, Hooker & Arnott (1832) added two new species to De

Candolle’s Panax: P. ovatum and P. platyphyllum.

Later, well-known botanist at the time Asa Gray (1854) changed the genus to Hedera.

However, when the revision of Hederaceae occurred, Berthold Seemann took up the name

Cheirodendron from Nuttall’s manuscripts and published it as a new genus in his Revision of the Natural Order Hederaceae (Seemann 1868). In his classification, he grouped five species with ranges around the Pacific: C. gaudichaudii (DC) Seem. and C. platyphyllum (Hook. &

Arnott) Seem. of the Hawaiian Islands; C. laetivirens (Gay) Seem. and C. valdiviense (Gay)

Seem. of Chile; and C. samoense (A. Gray) Seem. of the Samoan Islands (Seemann 1868).

However, since then, the Chilean and Samoan species were removed from Cheirodendron, leaving only two species (C. gaudichaudii and C. platyphyllum) in the genus.

Looking closer at Seeman’s Hawaiian species, Hillebrand (1888) was the first to describe different forms among the Hawaiian species. Hillebrand (1888) saw five forms in C.

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gaudichaudii, distinguished as: α, β, γ, δ, and ε. However, Heller (1897) recognized that C. gaudichaud was the same as Gaudichaud’s Aralia trigyna which had priority and created the new combination (Gaud.) Heller (Sherff 1954).

Cheirodendron fauriei Hochreutiner was described by Hochreutiner (1925) and C. dominii

Krajina, found only on , was described by Krajina (1931). Sherff (1954) later recognized 14 subspecies of C. trigynum. (Table 1.1).

A Marquesan species, C. marquense Brown, was described in 1935, but was later found to have been previously described in 1864 as Aralia bastardiana Decaisne (Frodin

1990). Frodin (1990) made a new combination: Cheirodendron bastardianum (Decne.)

Frodin.

Thus far, there have been three treatments classifying all species and subspecies of

Cheirodendron (Table 1.1). Sherff (1954) examined leaflet shape, carpel number, fruit size and number of flowers per umbellule. He recognized six species, along with associated variations and forms within the Hawaiian Islands. Although he takes note of a species in the

Marquesas, it was not included in his revision.

Herat examined the morphological and anatomical traits for many different characters. These include traits involving the petiole and petiolule, lamina, young stem, wood, pollen, and fruit characteristics (Herat 1981). In his taxonomic delineation, he recognized six species of Cheirodendron in and one species in the Marquesas (Herat

1981) (Table 1.1). His treatment greatly reduced the number of subspecific categories that

Sherff (1954) recognized.

The most recent revision of Cheirodendron was by Lowry (1990) as published in the

Manual of Flowering Plants of Hawaii. Lowry (1990) recognized five Hawaiian species and

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one Marquesan species, making C. keakuense Kraj. var. forbesii Sherff its own species, C. forbesii (Sherff) Lowry, and lumping C. helleri Sherff with C. trigynum (Gaud.) A. Heller.

This current taxonomy recognizes C. dominii, C. forbesii, C. fauriei, C. platyphyllum and C. trigynum as the Hawaiian taxa and C. bastardianum as the Marquesan taxon (Lowry 1990)

(Table 1.1).

In his revision, Lowry suggests that a number of taxa in Sherff’s (1954) classification may warrant proper species recognition. Although not noted in Table 1.1, Sherff (1954) recognized slight population differences in distinct island locations (e.g. Northwest Kauai,

Northernmost tip of Hawaii, etc.) where each subspecies was found. Lowry’s suspicions, as well as suspicions of others who have worked in the field, lead me to believe that these differences represent genetically distinct taxa. However, more evidence must be presented, in which this research intends to do.

Traditional uses

This leaf structure is indicative of the name, Cheirodendron, which is derived from the Greek word cheiros meaning “hand” and dendron meaning “tree,” referring to its five leaflets fixed at a common point (Rock 1913). In Hawaiian, Cheirodendron (spp.) is called

ʻōlapa or lapalapa which may have gotten its name from the similarities between the plant’s fluttering leaves in the slightest of wind and the movements of the skirt of a hula dancer of rank ʽōlapa (Lowry 1990). In the Marquesan language, Cheirodendron is called pimata

(Wagner & Lorence 2002).

Cheirodendron spp. was used traditionally in Hawaii for lei-making (leaves), dye- making (fruit), and weaponry such as spears (branches) (Abbott 1992). The leaves and bark of Cheirodendron spp. are recorded to have a carrot-like scent when crushed and, when

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extracted, provide a fragrance for traditional materials, such as kapa (Abbott 1992, Wagner

& Lorence 2002). Branches would also be slathered with gum from sticky Pisonia L. spp. seeds to ensnare birds (Abbott 1992). Traditional uses of C. bastardianum in the Marquesas

Islands included utilizing the leaves in lei-making (Wagner & Lorence 2002).

PROPOSED RESEARCH AND HYPOTHESES

The taxonomical work done by Sherff (1954), Herat (1981), and Lowry (1990) are helpful in discerning species relationships in Cheirodendron. However, genetic data were unavailable to clarify relationships among the populations and or species. My research will help to discern these relationships by comparing regions in the nuclear and chloroplast genomes. Phylogenies based on these regions will show if the current taxonomic classification corresponds or may show distinct genetic taxa that warrant species recognition.

Discovering the species relationships among Cheirodendron will also elucidate biogeographic implications regarding the source of the species in Hawaii and Marquesas, its closest relatives, and biogeographic patterns of dispersal within Hawaii. Three specific hypotheses have been established:

Hypothesis one: Phylogenetic analysis does not support the current taxonomic classification based on morphological data.

Based on herbaria specimen and personal field observations, vast morphological differentiation within the species, especially C. trigynum, causes me to believe that some taxa were left out of the classification.

Hypothesis two: Cheirodendron is monophyletic.

Mitchell et al. (2012) evaluated the phylogenetic relationships of Raukaua, a paraphyletic group whose New Zealand species share a sister relationship with

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Cheirodendron. However, this study only included a single species, C. trigynum, to represent the genus Cheirodendron (Mitchell et al. 2012). The proposed study will evaluate the monophyly of Cheirodendron by including all taxa in analyses.

Hypothesis three: The Pacific biogeography of Cheirodendron involves an Austral origin with long distance dispersal to Hawaii and then to the Marquesas.

Fosberg (1948) suggested the origin of Cheirodendron to be from a single introduction from an Austral origin. This claim was later supported with phylogenetic analyses evaluating generic relationships within Araliaceae (Wen et al. 2001, Plunkett, Wen

& Lowry 2004) and studies of Raukaua (Mitchell & Wagstaff 1997, Mitchell & Wagstaff

2000, Mitchell et al. 2012), the genus whose shares a sister relationship to Cheirodendron.

With precedence of other groups with Pacific distributions, Gillett (1972) suggested that Cheirodendron is likely to have spread from an Austral origin to Hawaii, and then to the

Marquesas. An in depth morphological assessment of the group suggested the same biogeographic pattern, as the leaf morphology of the Marquesan species, C. bastardianum, resembles an intermediate form between two Hawaiian species, C. platyphyllum and C. trigynum (Herat 1981). In addition, the fruit anatomy of the Marquesan species resembles C. platyphyllum (Herat 1981).

MATERIALS AND METHODS

Taxon sampling, outgroup sampling and DNA extraction

To ensure proper taxon sampling, collection will include all taxa mentioned in the current revision (Lowry 1990), including samples of the same taxon from different islands. If personal collection is not possible, collection by collaborators or herbarium specimen will supplement my personal collection. For all collections made, a voucher will be collected for

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reference, along with a tissue sample that will be extracted of DNA. Outgroup taxa chosen for this study will be (Hook.) A. D. Mitch., Frodin & Heads, R. edgerleyi

(Hook. f.) Seem., R. simplex (G. Forst.) A. D. Mitch., Frodin & Heads and Schefflera digitata

J. R. & G. Forst.; all shown to be monophyletic with Cheirodendron in a previous study

(Mitchell et al. 2012). Total genomic DNA will be extracted using a modified CTAB protocol (Doyle & Doyle 1987) and purified with phenol and chloroform.

DNA sequencing and analysis

Primers will be chosen on the basis of having enough variability. Polymerase chain reaction will be performed to amplify DNA regions of interest. PCR products will be visualized on 1% agarose gel stained with EtBr to confirm amplification before being cleaned with ExoSAP –IT (USB, Cleveland, Ohio, USA). Cleaned PCR products will be sent to the Greenwood Molecular Biology Facility at the University of Hawaii at Manoa. PCR products will be sequenced in both the forward and reverse directions to confirm sequences, and completed sequences will be edited and assembled in Sequencher 3.0 (Gene Codes

Corporation, Ann Arbor, Michigan, USA). Sequences will be aligned using ClustalW in

MEGA version 6 (Tamura et al. 2013) and trimmed to be the same number of base pairs.

Molecular evolutionary analyses using maximum parsimony (MP) will be conducted using MEGA version 6 (Tamura et al. 2013). Maximum likelihood (ML) analyses will be carried out on a web-based server, molecularevoution.org, using GARLI version 2.0

(Bazinet, Zwickl & Cummings 2014). Bayesian inference will be carried out using MrBayes version 3 (Hueselenbeck & Ronquist 2001). If there is congruence among gene regions, a combined dataset will be analyzed under the phylogenetic methods mentioned above.

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Preliminary data

Thus far, primer screening for variable regions has been ongoing. The problem has been finding regions that have enough variability to resolve a . At present, both nuclear and chloroplast regions have been sequenced, including gene, spacer and intron regions of the genome. Nuclear regions sequenced include Phytochrome C (gene), Nitrate reductase (intron) and the internal transcribed spacer (ITS) (spacer). Chloroplast regions include atpB-rbcL (spacer), rbcL (gene), trnL-trnF (spacer), and ndhF-rpl32 (spacer). All regions tested to date have resulted in little to no variability

Future directions

Regions that show more genetic variation need to be found. Since regions used in many other phylogenetic studies (ie: trnL-trnF intergenic spacer) do not show variation in

Cheirodendron, faster-evolving regions need to be sequenced. Other chloroplast regions will be tested as described by Shaw et al. (2005, 2007). Nuclear regions described in Zimmer &

Wen (2012) can also be looked into. Good samples that are representative of the different taxa around Hawaii need to be analyzed. Thorough collection around all the main Hawaiian

Islands needs to be done.

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Table 1.1. Comparison of revisions of Cheirodendron. For location, K=Kauai, O=, Mo=Molokai, L=Lanai, M=Maui, H=Hawaii, Marq=Marquesas Islands

Sherff Herat Lowry (1954) (1981) (1990) Species Subspecies Form Location Species Subspecies Location Species Subspecies Location

platyphyllum O platyphyllum platyphyllum O platyphyllum platyphyllum O kauaiense keakuense K kauaiense K kauaiense K forbesii K keakuense keakuense K forbesii K dominii K forbesii K dominii K trigynum subcordatum H dominii K trigynum trigynum O, Mo, L, M, H

fosbergii O trigynum trigynum H helleri K 10

mauiense M acuminatum H fauriei K

oblongum latius M fosbergii O bastardianum Marq molokaiense angustius Mo, M hillebrandii O, L, Mo osteostigma Mo mauiense Mo, M halawanum O, M, L fauriei K hillebrandii O helleri K confertiflorum M marquesense Marq rockii L skottsbergii L, M ilicoides H acuminatum H degeneri pauciflorum H fauriei macdanielsii K

Table 1.1. (Continued) Comparison of revisions of Cheirodendron. For location, K=Kauai, O=Oahu, Mo=Molokai, L=Lanai, M=Maui, H=Hawaii, Marq=Marquesas Islands

Sherff Herat Lowry (1954) (1981) (1990) Species Subspecies Form Location Species Subspecies Location Species Subspecies Location

helleri microcarpum K multiflorum K

sodalium K

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Figure 1.1. Infrafamilial phylogenetic relationships of Araliaceae. Consensus from maximum parsimony analysis of combined ITS and trnL-trnF data. (from Plunkett, Wen & Lowry 2004). 12

CHAPTER 2. PHYLOGENETIC AND BIOGEOGRPAHIC RELATIONSHIPS OF

CHEIRODENDRON NUTT. EX. SEEM. (ARALIACEAE)

ABSTRACT

Cheirodendron is a genus of six arboreal species in the family Araliaceae, distributed in the Hawaiian and Marquesas Islands. Previous and current revisions were assessed using morphological characteristics, resulting in the present taxonomy that consists of five species and two subspecies in Hawaii and one species in the Marquesas. For the first time, molecular phylogenetic analyses were carried out to determine species and biogeographic relationships using sequences from the internal and external transcribed spacer regions of nuclear ribosomal DNA, and the ndhF-rpl32 chloroplast spacer region. The results suggest that

Cheirodendron is a monophyletic group with Marquesan species C. bastardianum sister to the Hawaiian Cheirodendron. Within the Hawaiian Cheirodendron, a of non-Kauai taxa was well-supported and a clade of Kauai taxa was weakly supported. However, species relationships within the Hawaiian were unresolved. Phylogenetic differences among subspecies suggest the recognition of two previously-recognized species, C. helleri Sherff and C. kauaiense Kraj. Results suggest two possible biogeographic patterns of

Cheirodendron in the Pacific: (1) a stepping stone pattern of dispersal from New Zealand to

Marquesas, and Marquesas to Hawaii or (2) a simultaneous colonization of both Hawaii and

Marquesas. Understanding species relationships and the biogeography of Cheirodendron adds to our knowledge of the evolution and speciation of Pacific island groups. Long distance dispersal, along with in situ speciation on island archipelagos provides interesting evolutionary and biogeographic patterns to be discussed.

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INTRODUCTION

Cheirodendron Nutt. ex. Seeman is a genus of six arboreal species in the family

Araliaceae. Araliaceae is composed of about 50 genera and 1500 species widely distributed in the tropics and subtropics of Asia, the Pacific Islands, and the Neotropics (Wen et al. 2001,

Yi et al. 2004). Most members of Araliaceae are woody with variable leaf morphologies, but maintain conserved floral characteristics (Wen et al. 2001, Yi et al. 2004, Liu et al. 2012).

Some well-known species in Araliaceae include medicinal herbs such as ginseng (Panax ginseng C. Meyer), and ornamentals including English ivy (Hedera helix L.) and the umbrella tree (Schefflera actinophylla (Endl. Harms) (Liu et al. 2012).

Cheirodendron has a tropical distribution in the Pacific Ocean with species occurring in the Hawaiian Islands and the Marquesas Islands. Mature individuals stand at 2-15 meters and are one of the dominant canopy constituents in mesic forests, wet forests and bogs from

600-1500 meters in elevation (Lowry 1990). All species have compound leaves, with 3-5(-7) leaflets with margins toothed to entire (Lowry 1990). Flowers are perfect and arranged in oppositely branched umbels, with carpel numbers varying from two to five (Frodin 1990,

Lowry 1990). When broken or crushed, plant parts emit a strong carrot-like odor (Frodin

1990, Lowry 1990). Fruits are small fleshy drupes that, when ripe, exude a deep purple liquid which traditional Hawaiians used for dyes.

The current taxonomy recognizes six species distinguishable by morphological characteristics of leaflet shape and carpel number (Lowry 1990). Cheirodendron trigynum

(Gaud.) A. Heller is the most common species and is known from all the high Hawaiian

Islands except Kahoolawe. It has been reported from Niihau (Hooker & Arnott 1832), but has since been extirpated (St. John 1959). This species is distinguished from the others by 14

having leaflets longer than wide, ovate to elliptic; margins entire or serrate-crenate and teeth curved upward and inward (Lowry 1990). The species maintains two morphological forms recognized as distinct subspecies, C. trigynum ssp. trigynum and ssp. helleri (Sherff) Lowry.

Subspecies helleri is only found on the island of Kauai and flowers have two (occasionally 3) styles and carpels, while ssp. trigynum is only present on the younger islands (Oahu, Maui,

Lanai, Molokai, and Hawaii) and has three to four (rarely 2) styles and carpels.

Cheirodendron platyphyllum (Hook. & Arnott) Seem. is characterized by broadly ovate to depressed ovate leaflets that are wider than they are long. This species is known from Kauai and Oahu, where two subspecies are present, differing in carpel number and leaflet shape. Subspecies kauaiense (Kraj) Lowry from Kauai has two carpels and rounded leaflet apices, while subspecies platyphyllum from Oahu maintains five (rarely 4) carpels and acuminate leaflet apices (Lowry 1990). Three other species, C. dominii Kraj., C. fauriei

Hochr. and C. forbesii (Sherff ) Lowry are restricted to Kauai each having differences in the degree of dentations on their leaflet margins, carpel numbers and locality. The characteristics of C. dominii include three broadly ovate leaflets longer than wide, margins caudate-dentate and spiny in appearance, and three to four (sometimes 2 or 5) styles and carpels (Lowry

1990). This taxon is also quite rare, occurring only in wet forest at 1525-1550 m in elevation near the slopes of Mount Waialeale (Lowry 1990). Similarly, C. fauriei has leaflets with the same spiny appearance as C. dominii; however they are more orbicular in shape. The ovary contains two (sometimes 3) carpels and is widely distributed in diverse mesic to wet forests at 650-1250 m in elevation (Lowry 1990). Cheirodendron forbesii is distinguished by its leaflets that are nearly twice as long as they are wide, margins entire, and five (sometimes 4) carpels (Lowry 1990). Cheirodendron bastardianum is the only species that occurs outside

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Hawaii and it shares similar morphological characteristics with the Hawaiian taxa (Frodin

1990). It is distinguished from Hawaiian Cheirodendron by morphological characteristics including leaves as long as or slightly longer than broad and rounded, truncate or slightly cordate at the base, longer pedicels, and smaller fruit (Brown 1935, Wagner & Lorence

2002).

Previous studies and revisions of Cheirodendron (Sherff 1954, Herat 1981, Lowry

1990) focus only on morphological distinctions of the group. However, these defining features often overlap. Speciation is not always accompanied by clear morphological differentiation (Kenfack 2011), and it is important to also look at genetic relationships in an evolutionary perspective. Studies that focus on infrageneric relationships and species delimitation are important because species are the basic fundamental unit in other disciplines, including biogeography, ecology, macroevolution, systematics and conservation (Sites &

Marshall 2004).

To date, no molecular work has been done to assess species relationships and the biogeographic relationships within Cheirodendron. However, several recent studies have focused on generic relationships within the family Araliaceae. Plunkett, Wen & Lowry

(2004) showed that there were three major clades within the family (Asian palmate group,

Polyscias J. R. Forst. & G. Forst.- K. Koch group, and Aralia L. group), although these clades along with numerous other taxa form an unresolved polytomy. These unresolved groups were attributed to rapid diversification during the Gondwana break up, which led to the widespread distribution of these groups (Plunkett, Wen & Lowry 2004).

Cheirodendron, along with a close relative, Raukaua Seem., are placed within this polytomy

(Plunkett, Wen & Lowry 2004). Much attention has been given to the biogeographic

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implications of Raukaua because of its “Gondwanan distribution” in Chile, Argentina, New

Zealand and Tasmania (Mitchell & Wagstaff 1997, Mitchell & Wagstaff 2000). Recently,

Mitchell et al. (2012) demonstrated that Raukaua is paraphyletic based on chloroplast and nuclear DNA sequence analysis because of the placement of cephalobotrys (F.

Muell.) Harms, Motherwellia haplosciadea F. Muell., Cheirodendron trigynum (Gaud.) A.

Heller and Schefflera digitata J. R. Forst. intermixed within the Raukaua clade.

Raukaua is a genus of six species with distribution of extant taxa in South America,

New Zealand and Tasmania. The Raukaua taxa closest to Cheirodendron are those from New

Zealand [R. anomalus (Hook.) A. D. Mitch., Frodin & Heads, R. simplex (G. Forst.) A. D.

Mitch., Frodin & Heads, R. edgerleyi (Hook. f.) Seem.], which form a monophyletic clade in previous analyses (Mitchell & Wagstaff 1997, Mitchell & Wagstaff 2000, Mitchell et al.

2012). Cheirodendron and New Zealand Raukaua share several synapomorphies including stipules or ligules reduced or absent, coriaceous adult leaf texture, pedicel articulating below the flower, wood intervessel pitting scalariform or opposite (Mitchell & Wagstaff 2000), fruit with laterally compressed endocarps, paniculate inflorescences with opposite umbellules, pentamorous flowers with 2-5 carpels, and palmately compound leaves (in Raukaua mostly in juvenile leaves) (Lowry, Plunkett & Wen 2004). This supports the idea that

Cheirodendron is a recent relative of an ancestor that probably originated in New Zealand

(Fosberg 1948) and that the current distribution of Cheirodendron is due to a long distance dispersal event (Mitchell & Wagstaff 2000).

The biogeographic patterns of Cheirodendron across the Pacific have not been well- studied. However, molecular phylogenetics has been a useful tool in discerning species relationships and the biogeographic patterns in many Pacific taxa, such as the Hawaiian

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Silverswords (Carlquist, Baldwin & Carr 2003), Cyrtandra J. R. Forst. & G. Forst.

(Gesneriaceae) (Cronk et al. 2005), Plantago L. (Plantaginaceae) (Dunbar-Co, Wieczorek &

Morden 2008), Astelia s.l. (Asteliaceae) (Birch & Keeley 2013), amonfelsenstieng many others. The evolution and speciation of these groups is especially interesting because the islands that they are native to lie miles from the nearest continental land mass and are resultant from a single, or sometimes, multiple events of long-distance dispersal (Wagner &

Funk 1995).

There has been one study assessing the biogeographic patterns of Cheirodendron.

Based on morphological analyses, Herat (1981) suggests an Austral origin for Cheirodendron with dispersal overseas to Hawaii, and subsequent dispersal to the Marquesas. This “stepping stone” dispersal pattern from island to island in the Pacific is well-known among other indigenous groups such as Metrosideros Banks ex Gaertn. (Myrtaceae) (Wright et al. 2001),

Astelia (Asteliaceae) (Birch & Keeley 2013), Coprosma J. R. Forst. & G. Forst. (Rubiaceae)

(Cantley et al. 2014) and many others. What is also interesting about Herat’s (1981) findings is that he concludes a Hawaiian species to be the progenitor of the single Marquesan species,

Cheirodendron bastardianum, a claim that Gillett (1972) also made, although his ideas were merely speculation on the basis that there were similarities between Hawaiian and Marquesan floras. Hawaii, being one of the Pacific archipelagos most distant from any continental landmasses, has often been assumed to be a sink for dispersal, rather than a source (Carlquist

1974). However, recent molecular analyses have found that Hawaiian groups such as

Melicope J. R. Forst. & G. Forst. (Rutaceae) (Harbaugh et al. 2009a) and Santalum L.

(Santalaceae) (Harbaugh & Baldwin 2007) have been the source of colonization on other

Pacific archipelagos.

18

The mode of seed dispersal in Cheirodendron across the Pacific is thought to be from ingestion by birds (Herat 1981) as with many other groups that have Pacific distributions and fleshy fruits (eg: Howarth & Baum 2005, Harbaugh et al. 2009a, Cantley et al. 2014). The importance of birds in carrying seeds over long distances is apparent, as a number of

Hawaiian taxa were brought to the islands by birds (Price & Wagner 2004).

The current study used molecular phylogenetics as a tool to determine species relationships and the biogeography of Cheirodendron by addressing the following questions:

(1) What are the species relationships in Cheirodendron? (2) Does the molecular phylogeny support the current taxonomy of five species distributed within the Hawaiian Islands? and (3)

Does the molecular data support a colonization of Cheirodendron to the Marquesas Islands via Hawaiian Islands as Gillett (1972) and Herat (1981) suggest or was there a stepping stone colonization across the Pacific from the Marquesas Islands to Hawaii? The following study includes molecular sequence data of two nuclear regions (ETS and ITS) and one chloroplast region (ndhF-rpl32) to construct a phylogenetic tree for Cheirodendron.

MATERIALS AND METHODS

Sampling and DNA extraction

Leaf tissue of all currently recognized taxa (Lowry 1990) was sampled. A total of 22 samples were analyzed, including at least two samples from each taxon in Cheirodendron, except C. bastardianum, in order to account for possible population differences (Table 2.1).

Although C. bastardianum persists on multiple islands in the Marquesas, only one sample was available for study from Hiva Oa. However, C. bastardianum has been found to be morphologically consistent among islands (Steve Perlman, National Tropical Botanical

19

Garden, personal communication). Outgroup taxa chosen for this study were Raukaua anomalus, R. edgerleyi, R. simplex and Schefflera digitata; all shown to be monophyletic with Cheirodendron (Mitchell et al. 2012).

Total genomic DNA was extracted from fresh or silica-preserved leaves or herbarium specimens using a modified CTAB protocol (Morden, Caraway & Motley 1996) and purified with phenol/chloroform. DNA from Hawaiian taxa were accessioned into the Hawaiian Plant

DNA library and stored at -20°C for future use (Morden, Caraway & Motley 1996).

Vouchers of each individual collected were deposited into the Joseph Rock Herbarium

(HAW).

DNA amplification and DNA sequencing

Each PCR reaction contained a total of 25 µL of 1X GoTaq Flexi Buffer (Promega,

Madison, Wisconsin, USA), 2 mM MgCl2 (Promega), 0.25 µg/µL bovine serum albumin

(Amresco, Solon, Ohio, USA), 0.1 µmol/L of each primer (forward and reverse, see Table

2.2), 0.2 mM each dNTP, 1U GoTaq Flexi DNA polymerase (Promega) and 1 µL of DNA diluted to 20 ng/µL. PCR was performed using a DNA Thermocycler (MJ Research, St.

Bruno, Quebec, Canada) programed for an initial denaturation at 95°C for 2 min, followed by

30 cycles of 93°C for 1 min, 45-55°C for 1 min, and 72°C for 2 min, followed by an extended elongation on the final step of 72°C for 3 min. When necessary, PCR products were cloned using the StrataClone PCR Cloning Kit with associated protocols (Agilent

Technologies, La Jolla, California, USA).

PCR products were visualized on 1% agarose gel stained with EtBr to confirm amplification before being cleaned with ExoSAP –IT (USB, Cleveland, Ohio, USA).

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Table 2.1. Voucher information and locality of specimen used in this study. Stars indicate samples used in primer screening.

Taxon Voucher number, associated Location collected herbaria

Cheirodendron trigynum K. R. Wood 14168 (PTBG, Laau, Kauai, HI, USA (Gaud.) A. Heller ssp. BISH, MBK) helleri (Sherff) Lowry Cheirodendron trigynum K. R. Wood 15220 (PTBG, Waiakoali, Kauai, HI, USA (Gaud.) A. Heller ssp. BISH, MO) helleri (Sherff) Lowry * Cheirodendron trigynum C. Osaki 52 (HAW, PTBG, Kokee, Kauai, HI, USA (Gaud.) A. Heller ssp. BISH) helleri (Sherff) Lowry * Cheirodendron trigynum C. Morden (HAW) Puulalaau, Hawaii, HI, USA (Gaud.) A. Heller ssp. trigynum Cheirodendron trigynum C. Osaki 72 (HAW) Manana trail, Oahu, HI, USA (Gaud.) A. Heller ssp. trigynum Cheirodendron trigynum S. Walsh (HAW) Munroe trail, Lanai, HI, USA (Gaud.) A. Heller ssp. trigynum Cheirodendron trigynum S. Walsh (HAW) Waihee trail, Maui, HI, USA (Gaud.) A. Heller ssp. trigynum * Cheirodendron C. Osaki 56 (HAW, PTBG, Circle Bog, Kauai, HI, USA platyphyllum (Hook. & BISH) Arnott) Seem. ssp. kauaiense (Kraj.) Lowry Cheirodendron K. R. Wood 14170 (PTBG, Laau, Kauai, HI, USA platyphyllum (Hook. & BISH, K, MBK) Arnott) Seem. ssp. kauaiense (Kraj.) Lowry Cheirodendron C. Osaki 32 (HAW) Konahuanui, Oahu, HI, USA platyphyllum (Hook. & Arnott) Seem. ssp. platyphyllum *Cheirodendron dominii K. R. Wood 12284 (PTBG, Waialeale, Kauai, HI, USA Kraj. BISH, UC) Cheirodendron dominii C. Osaki 70 (HAW, PTBG, Kilohana vista, Kauai, HI, Kraj. BISH) USA Cheirodendron fauriei K. R. Wood 15185 (PTBG, Kalalau rim, Kauai, HI, USA Hochr. BISH, UC, US)

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Table 2.1. (Continued) Voucher information and locality of specimen used in this study. Stars indicate samples used in primer screening.

Taxon Voucher number, associated Location collected herbaria

Cheirodendron fauriei K. R. Wood 14203 (PTBG, Kamooloa, Kauai, HI, USA Hochr. MBK, MO, UBC, US) Cheirodendron fauriei C. Osaki 42 (HAW, PTBG, Wahiawa bog, Kauai, HI, Hochr. BISH) USA Cheirodendron forbesii C. Osaki 61 (HAW, PTBG, Powerline trail, Kauai, HI, (Sherff) Lowry BISH) USA Cheirodendron forbesii C. Osaki 49 (HAW, PTGB) Makaleha, Kauai, HI, USA (Sherff) Lowry Cheirodendron Jon Price 205 (PTBG, US, Hiva Oa, Marquesas Islands, bastardianum (Decaisne) PAP, P, BISH) French Frodin Raukaua anomalus (Hook.) A. Mitchell CHR 529088 Banks Penninsula, Prices A. D. Mitch., Frodin & (CHR) Valley, New Zealand Heads Raukaua edgerleyi (Hook. f.) D. Grinstead, C. Jones CHR Eastern Nelson, Hira Forest, Seem. 553730 (CHR) New Zealand (G. Forst.) P.I. Knightbridge 70 Mount Wilberg, New A. D. Mitch., Frodin & (CHR) Zealand Heads Schefflera digitata J. R. & G. P. B. Heenan CHR 610132 B Cultivated, Christchurch, Forst. (CHR) New Zealand

22

Cleaned PCR products were sent to the Greenwood Molecular Biology Facility at the

University of Hawaii at Manoa. PCR products were sequenced in both the forward and reverse directions to confirm sequences, and completed sequences were edited and assembled in Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, Michigan, USA). Sequences were aligned using ClustalW in MEGA version 6 (Tamura et al. 2013) and trimmed to be the same number of base pairs.

Primer screening and analysis

Before choosing which regions to analyze, primers were screened based on their ability to amplify and the number of indels among individuals of the genus. To identify variable gene regions for analysis, four samples representing 3 species from different locations were used: C. trigynum ssp. helleri (Kauai), C. trigynum ssp. trigynum (Hawaii), C. dominii (Kauai) and C. platyphyllum ssp. kauaiense (Kauai) (See Table 2.1, taxa used for screening indicated with a star *). In total, 15 chloroplast and 9 nuclear regions were examined (Table 2.2). However, only three regions had enough variation where all samples amplified, and these were then sequenced for all individuals. ITS sequences for outgroup taxa (Schefflera digitata, Raukaua simplex, R. anomalus, and R. edgerleyi) were taken from

GenBank identified by the following respective accession numbers: JX106299.1, U63180.2,

U63164.1, U63171.1.

Before performing phylogenetic analysis, JModel Test 2 (Darriba et al. 2012,

Guindon & Gascuel 2003) was used to determine the best parameters for each region separately and combined according to the Akaike information criterion (AIC).

Molecular evolutionary analyses using maximum parsimony (MP) were conducted using MEGA version 6 (Tamura et al. 2013). All characters were equally weighted and gaps 23

Table 2.2. List of primers and references.

Locus Genome Primer names, sequences (5’-3’) and reference Approximate Number Percent origin, type of length of of indels variability DNA region (base (%) pairs) Internal Nuclear, ITS 5: GGAAGTAAAAGTCGTAACAAGG 600 9 1.5 transcribed spacer ITS 4: TCCTCCGCTTATTGATATGC spacer (ITS) (Baldwin 1992) Phytochrome C Nuclear, gene Phy C F: GAYTTRGARCCWGTDAAYC 600 1 0.2 (PHYC) Phy C R: GRATKGCATCCATYTCMAYRTC (Matthews & Donoghue 1999) Nitrate Nuclear, gene NIA F5: GCTGAACTTGCTAACGCTGA 600 5 0.8 reductase (NIA) NIA i2R: CCATGTCTCTCCTCCATCCA (Levin, Blanton & Miller 2009)

24 Nitrate Nuclear, NIA i3 F: 1300 22 1.7

reductase (NIA) intron AARTAYTGGTGYTGGTGYTTYTGGTC NIA i3 R: GAACCARCARTTGTTCATCATDCC (Howarth & Baum 2002) Alcohol Nuclear, gene Adhx2-1 F: CTTCACTGCTTTATGTCACACT Did not - - dehydrogenase Adhx8-1 R: GGACGCTCCCTGTACTCC amplify (Adh) (Small & Wendel 2000) Phytochrome A Nuclear, gene Phy A F: CCYTAYGARGRNCCYATGACWGC Did not - - (PHYA) Phy A R: GDATDGCRTCCATYTCRTAGTC amplify (Matthews & Donoghue 1999) 5S-Non- Nuclear, 5SFUL: TTAGTGCTGGTATGATCGCA Did not - - transcribed spacer 5SR: CACCGGATCCCATCAGAACT amplify spacer (Udovicic, McFadden & Ladiges 1995) External Nuclear, ETS 18S: GAGCCATTCGCAGTTTCACAG 500 4 0.8 transcribed spacer (Wright et al. 2001) spacer (ETS) jkETS 9: CGT WMA GGY GYA TGA GTG GT (Mitchell, Heenan & Patterson 2009)

Table 2.2. (Continued) List of primers and references.

Locus Genome Primer names, sequences (5’-3’) and reference Approximate Number Percent origin, type of length of of indels variability DNA region (base (%) pairs) Xanthine Nuclear, gene X502F: TGTGATGTCGATGTATGC 1000 3 0.3 dehydrogenase X1599R: G(AT)GAGAGAAA(CT)TGGAGCAAC (Xdh) (Gorniak, Paun & Chase 2010) trnL-trnF Chloroplast, E: GGTTCAAGTCCCTCTATCCC 400 1 0.3 spacer F: ATTTGAACTGGTGACACGAG (Taberlet et al. 1991) trnL Chlroplast, C: CGAAATCGGTAGACGCTACG 535 2 0.4 intron D: GGGGATAGAGGGACTTGAAC (Taberlet et al. 1991) 25 psbA-trnH Chloroplast, psbA: GTTATGCATGAACGTAATGCTC 450 2 0.4 spacer trnH: CGCGCATGGTGGATTCACAATCC (Shaw et al. 2005) ndhA Chloroplast, ndhAx1: Did not - - intron GCYCAATCWATTAGTTATGAAATACC amplify ndhAx2: GGTTGACGCCAMARATTCCA (Shaw et al. 2007) atpB-rbcL Chloroplast, atpB: GAAGTAGTAGGATTGATTCTC 800 0 0 spacer rbcL: ATGTCAACAGGTACATGGTC (Manen, Natali & Ehrendorfer 1994) rbcL Chloroplast, ESRBCLF: 1200 0 0 gene ATGTCACCACAAACGGAGACTAAAGC ESRBCL1361R: TCAGGACTCCACTTACTAGCTTCACG (Schuettpelz & Pryer 2007) ndhF-rpl32 Chloroplast, ndhF: GAAAGGTATKATCCAYGMATATT 1200 8 0.7 spacer rpl32r: CCAATATCCCTTYYTTTTCCAA (Shaw et al. 2007)

Table 2.2. (Continued) List of primers and references.

Locus Genome Primer names, sequences (5’-3’) and reference Approximate Number Percent origin, type of length of of indels variability DNA region (base (%) pairs) trnQ-rps16 Chloroplast, trnQ: GCGTGGCCAAGYGGTAAGGC 2000 21 1 spacer rps16-1: GTTGCTTTYTACCACATCGTTT (Shaw et al. 2007) rpl16 Chloroplast, rpL16F71:GCTATGCTTAGTGTGTGACTCGTTG Did not - - gene rpl16R1516: CCCTTCATTCTTCCTCTATGTTG amplify (Shaw et al 2005) trnS-trnG Chloroplast, trnS: AACTCGTACAACGGATTAGCAATC 1000 4 0.4 spacer trnG: GAATCGAACCCGCATCGTTAG (Shaw et al. 2007)

26 ndhF Chloroplast, 536F: TTGTAACTAATCGTGTAGGGGA 800 1 0.1 portion of 1603R: gene GCATAGTATTGTCCGATTCAT(A/G)AGG (Olmstead & Sweere 1994) ndhC-trnV Chloroplast, ndhC: 1000 4 0.4 spacer TATTATTAGAAATGYCCARAAAATATCA TATTC trnVx2: GTCTACGGTTCGARTCCGTA (Shaw et al. 2007) trnT-trnL Chloroplast, trnT: CAAATGCGATGCTCTAACCT 800 4 0.5 spacer trnL: GGGGATAGAGGGACTTGAAC (Shaw et al. 2005) rpl32-trnL Chloroplast, rpl32F: CAGTTCCAAAAAAACGTACTTC 950 5 0.5 spacer trnL: CTGCTTCCTAAGAGCAGCGT (Shaw et al. 2007) petL-psbE Chloroplast, petL: AGTAGAAAACCGAAATAACTAGTTA Did not - - spacer psbE: TATCGAATACTGGTAATAATATCAGC amplify (Shaw et al. 2007)

or missing data were treated as complete deletion. Heuristic searches were conducted using the tree-bisection reconnection method. A consensus of equally parsimonious trees was constructed where branches of less than 50% consensus were collapsed into a polytomy. A total of 1000 bootstrap replicates were carried out to construct the tree. Maximum likelihood

(ML) analyses were carried out on a web-based server, molecularevoution.org, using GARLI version 2.0 (Bazinet, Zwickl & Cummings 2014). An optimal tree was inferred from 2000 non-parametric bootstrap replicates. If the best-fit model identified in JModel Test was not available, the general time-reversible (GTR) model was chosen. Bootstrap resampling in both

MP and ML (reported as percentages below) were used to estimate the robustness of the nodes (Felsenstein 1985). Bayesian inference (BI) was carried out using MrBayes version 3

(Hueselenbeck & Ronquist 2001). The GTR+I+G substitution model and default priors were used in analysis. Markov chain Monte Carlo (MCMC) sampling was done with two replicates of four chains (three cold, one hot). 10,000,000 generations were run with a subset sampling every 1000 generations. A burn-in period excluding the first 25% of trees was set.

Posterior probabilities (reported below as BI) for tree nodes give an overestimate of branch support (Suzuki, Glazco & Nei 2002) and were interpreted with caution. FigTree version 1

(Rambaut 2012) was used to view trees. Adobe Illustrator CS version 3 (Adobe Systems

Incorporated, San Jose, California, USA) was used to format and edit final trees.

RESULTS

In total, 15 chloroplast and 9 nuclear primer pairs were used to screen for possible regions for phylogenetic analysis (Table 2.2). Assessment of the percentage of indels within the region and the ability to amplify resulted in choosing two nuclear and one chloroplast

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region for phylogenetic analysis: a portion of the external transcribed spacer (ETS), the internal transcribed spacer (ITS) and ndhF-rpl32 (Table 2.2). There was no genetic variation among the Hawaiian Cheirodendron species for the other 21 regions examined. When aligned and trimmed, the total combined dataset included a total of 2365 characters including the nuclear regions ETS (510 bp), ITS (641 bp) and the chloroplast region ndhF-rpl32 intergenic spacer (1214 bp). JModel Test (Darriba et al. 2012, Guindon & Gascuel 2003) estimated the best fit model for each region to be HKY (ETS), TPM1uf+I (ITS), TVM+I

(ndhF-rpl32), and TPMuf+I+Γ (combined).

Comparison of each gene region under different phylogenetic methods (parsimony, maximum likelihood and Bayesian inference) produced similar topologies. Among trees constructed from the ETS region, maximum likelihood and Bayesian phylogenies showed weak support for C. bastardianum and C. forbesii as basal to the rest of Cheirodendron

(Figure 2.1) although the relationship among all Cheirodendron spp. is collapsed into a polytomy in the parsimony analysis. Among ITS trees (Figure 2.2), parsimony, maximum likelihood and Bayesian analyses agree on an unresolved, but well-supported monophyletic

Cheirodendron clade. Within this larger clade, three subclades were shared among all analyses: C. forbesii (from Kauai) and C. trigynum ssp. trigynum (from Hawaii) (MP: 75%,

ML: 57%, BI: 0.86), C. trigynum ssp. trigynum (from Maui) and C. trigynum ssp. trigynum

(from Lanai) (MP: 100%, ML: 55%, BI: 0.94), and two of the three collections of C. fauriei

(from Kauai) (MP: 100%, ML: 72%, BI: 0.99). Parsimony and Bayesian inference showed an additional relationship between two collections of C. trigynum ssp. helleri (MP: 100%, BI:

0.74) that was not inferred in maximum likelihood analysis. Phylogenetic analyses of the chloroplast region ndhF-rpl32 were congruent (Figure 2.3). In all analyses, C. bastardianum 28

Figure 2.1. ETS phylogeny of Cheirodendron. Bootstrap values and posterior probabilities are listed near each node as MP (%), ML (%), BI (probability), respectively. Collapsed branches are denoted as * for any particular analysis. If analysis does not support a particular relationship, a dash (-) is indicated. The location of where each taxon was collected is in parentheses next to each name. K=Kauai; O=Oahu; M=Maui; L=Lanai; H=Hawaii; Marq=Marquesas; NZ=New Zealand.

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Figure 2.2. ITS phylogeny of Cheirodendron. Bootstrap values and posterior probabilities are listed near each node as MP (%), ML (%), BI (probability), respectively. Collapsed branches are denoted as * for any particular analysis. If analysis does not support a particular relationship, a dash (-) is indicated. The location of where each taxon was collected is in parentheses next to each name. K=Kauai; O=Oahu; M=Maui; L=Lanai; H=Hawaii; Marq=Marquesas; NZ=New Zealand.

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Figure 2.3. ndhF-rpl32 phylogeny of Cheirodendron. Bootstrap values and posterior probabilities are listed near each node as MP (%), ML (%), BI (probability), respectively. Collapsed branches are denoted as * for any particular analysis. If analysis does not support a particular relationship, a dash (-) is indicated. The location of where each taxon was collected is in parentheses next to each name. K=Kauai; O=Oahu; M=Maui; L=Lanai; H=Hawaii; Marq=Marquesas; NZ=New Zealand.

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Figure 2.4. Combined phylogeny comparing MP, ML, and BI methods. Bootstrap values and posterior probabilities are listed near each node as MP (%), ML (%), BI (probability), respectively. Collapsed branches are denoted as * for any particular analysis. If analysis does not support a particular relationship, a dash (-) is indicated. The location of where each taxon was collected is in parentheses next to each name. K=Kauai; O=Oahu; M=Maui; L=Lanai; H=Hawaii; Marq=Marquesas; NZ=New Zealand.

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was closely associated with the outgroup species of Raukaua. The remaining species of

Cheirodendron form two clades, one consisting of the Kauai taxa and the other of non-Kauai taxa. The Kauai clade formed a polytomy that was strongly supported in MP analysis but weakly-supported in maximum likelihood and Bayesian inference (MP: 100%, ML: 65%, BI:

0.52). The non-Kauai clade was strongly supported in all three analyses (MP: 100%, ML:

100%, BI: 1).

Though individual genetic regions showed some inconsistencies, the combined analysis shows strong support for Cheirodendron as a monophyletic lineage, with C. bastardianum as sister to the Hawaiian species, and the Hawaiian species consisting of two clades representing Kauai and non-Kauai taxa. Bayesian analysis (Figure 2.4) shows branch support for C. bastardianum as sister to the rest of the Hawaiian species with a posterior probability of 1.0 and strong support for a Hawaiian clade (BI: 1). Within the Hawaiian group, the unresolved Kauai clade shows low support in Bayesian analysis (0.52), which is reflected in the ML and MP tree that shows all the Hawaiian taxa in a single large clade with the non-Kauai clade included as a strongly supported subclade within. The MP analysis was similar to the ML with the exception that the two C. forbesii collections group together and were sister to the remainder of the Hawaiian Cheirodendron (MP: 100%).

DISCUSSION

Results from this study indicate that Cheirodendron is a monophyletic group that shares a common ancestor with Schefflera digitata and a sister relationship to New Zealand

Raukaua. Though Raukaua is paraphyletic with the placement of Cheirodendron trigynum,

Schefflera digitata, Motherwellia haplosciadea and cephalobotrys intermixed

33

within the clade (Mitchell et al. 2012), New Zealand Raukaua and Cheirodendron share many similarities including laterally compressed endocarp fruits, paniculate inflorescences with opposite umbellules, pentamorous flowers with 2-5 carpels and an articulated pedicel, and palmately compound leaves (mostly in juvenile leaves in Raukaua) (Lowry, Plunkett &

Wen 2004). The current study also demonstrates a close relationship between Cheirodendron and Raukaua. In particular, the chloroplast phylogeny has C. bastardianum nested within

(BI) or sister to (MP and ML) Raukaua. Although analysis of the chloroplast region resulted in Cheirodendron being paraphyletic, combined analysis showed strong support for monophyly of Cheirodendron and for C. bastardianum as the to the Hawaiian clade. Consistent with this relationship, C. bastardianum shares many morphological characteristics with Hawaiian Cheirodendron, rather than Raukaua (Brown 1935) and looks almost identical to C. trigynum (Frodin 1990).

Relationships among taxa

Phylogenetic analysis revealed Cheirodendron as monophyletic, with C. bastardianum (from Marquesas) sister to the Hawaiian species. Within the Hawaiian species, two clades are supported, a Kauai taxa clade and a non-Kauai taxa clade. Both the Kauai clade and non-Kauai clade are unresolved, representing little or no gene sequence divergence among these taxa. Thus, fine-tuned phylogenetic relationships cannot be inferred at this time.

Further study at the population level might be of help in sorting out infraspecific relationships. However, within the scope of this project, this is not possible.

Short-branching polytomies within the Kauai clade and non-Kauai clade agree with a similar pattern of evolution found in other Pacific Island groups. Many Hawaiian radiations

34

exhibit great degrees of morphological diversity with little genetic variation (Wagner & Funk

1995). For example, the Hawaiian Cyrtandra (Gesneriaceae) is composed of 58 species, all resultant from a single ancestor and exhibits little infraspecific genetic variation (Cronk et al.

2005). Hawaiian Bidens L. (Asteraceae) is one of the most species-rich radiations within the genus, but has little differentiation in both nuclear and chloroplast regions (Knope et al.

2012). Many other examples, including the Hawaiian mints (Lindqvist et al. 2003),

Silverswords (Carlquist, Baldwin & Carr 2003), Lobeliads (Givnish et al. 2009) and

Coprosma (Cantley et al. 2014) have demonstrated great morphological diversity with little genetic differentiation.

The lack of genetic divergence within Kauai taxa and non-Kauai taxa, despite morphological differentiation, could be explained by a recent and rapid radiation of the group. Previous analyses have estimated the divergence rates of angiosperms to range from

0.078 to 0.091 net speciation events per million years (Magallon & Castillo 2009) which is considerably slower than other Hawaiian groups. Hawaiian Bidens was estimated to have a diversification rate of 0.3-2.3 species per million years (Knope et al. 2012). Baldwin &

Sanderson (1998) estimate the diversification rate of the Hawaiian Silverswords to be 0.56 ±

0.17 species per million years. Rapid speciation rates seen in other groups within Hawaii could be mirrored in Cheirodendron, although no analyses have estimated the age and rate of diversification of the genus. Further study using a molecular clock might give some insight as to the time frame and diversification rate of Cheirodendron.

Unresolved species relationships and poor branch support could suggest that

Cheirodendron is composed of only a few species with a great deal of morphological variation and that phenotypic plasticity might be the cause of variation in form. Phenotypic 35

plasticity is described as “the ability of an individual to express different features under different environmental conditions” (Travis 2009). To some extent, different morphologies might be the result of phenotypic plasticity in Cheirodendron, especially in C. trigynum, which exhibits local population differences (see Sherff 1954). However, phenotypic plasticity does not explain the morphological differences in taxa on Kauai that persist adjacent to one another. On Kauai, C. trigynum ssp. helleri can be found in lowland bogs to mesic forests

(440-1250 m) (Lowry 1990), overlapping with most Kauai species, including C. fauriei, which also occurs in low-elevation bogs (Lowry 1990). Although these two species can be found in the same ecological conditions, they still maintain separate morphologies. If plasticity relies heavily on environmental pressure to produce phenotypic differences, different morphological taxa would not be present next to each other.

The study by Knope et al. (2012) on Hawaiian Bidens revealed that these species consisted of closely related individuals with a great degree of morphological differences among them. Common garden experiments showed that these differences were not plastic traits, and forms held true to their phenotypes (Knope et al. 2012). Common garden experiments to rule out phenotypic plasticity have not been done with Cheirodendron before, thus phenotypic plasticity cannot be ruled out. However, it is more likely that the consistent differences found among individuals in similar or the same habitats are genetically based rather than a plastic response to ecological pressures, although genetic differences among them were not detected with the methods used in this study.

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Current taxonomy vs. phylogenetic relationships

Phylogenetic analysis suggests that two taxa of Cheirodendron presently regarded as subspecies (Lowry 1990) are to be recognized as distinct species. Cheirodendron trigynum ssp. helleri and C. platyphyllum ssp. kauaiense (both in the Kauai clade) are polyphyletic relative to their respective species (in the non-Kauai clade). This strongly supports the reclassification of subspecific taxa presently recognized in either C. trigynum or C. platyphyllum that they do not belong to the same species. As such, C. trigynum ssp. helleri

(Sherff) Lowry is now recognized as C. helleri Sherff and C. platyphyllum ssp. kauaiense

(Kraj.) Lowry is now recognized as C. kauaiense Kraj.

As far as the assessments of Sherff (1954), Herat (1981) and Lowry (1990), each hinted that the vast morphological diversity present within the Hawaiian Cheirodendron could represent more taxa. In his revision, Lowry (1990) takes note of the many different varieties seen by Sherff, stating that “some of these [populations] may represent genetically- based differences and could warrant taxonomic recognition.” This statement is supported for some taxa in the current study (see above), but is not supported for other species differences as Lowry (1990) suggests due to the lack of resolution within the Kauai and non-Kauai clades.

Disagreement between the molecular phylogeny and taxonomy could be caused by certain traits being convergent within Cheirodendron, the result masking the true evolutionary relationships. Convergent evolution is the process whereby similar characters arise independently in different lineages rather than as a result of common ancestry (Grande

& Rieppel 1994). These certain characters are said to be homoplasic, and can result from adaptive responses to similar selection pressures (Wake, Wake & Specht 2011). Homoplasy 37

and convergent evolution present a problem in phylogenetics because it gives a false sense of what the true relationships are. For example, thorns, spines and prickles are evolutionarily and ontogenetically different structures, though they arose from the same selective pressure to serve the same purpose—defense. If, then, this characteristic (having sharp protrusions for protection) was used to infer phylogentic relationships, Rosaceae (with prickles), Cactaceae

(with spines) and Rutaceae (with thorns), would infer a close relationship, however, this is definitely not the case (see Soltis et al. 2005).

Convergence in morphology could also be occurring in Cheirodendron. If the characters used to classify species were homoplasic due to convergent evolution, species delineations would not agree with phylogenetic relationships. Lowry’s (1990) classification of C. trigynum with two subspecies was based on indistinguishable characteristics in leaf morphology. Lowry’s (1990) ssp. helleri and ssp. trigynum are readily distinguished from each other based on carpel number, with ssp. helleri having two carpels and ssp. trigynum usually having three or occasionally four, although ssp. helleri is found to have three carpels in some individuals. This gives additional credibility to the separation of these as distinct species as noted above.

The question of why certain characters such as leaf morphology converged in different taxa is an interesting question that cannot be answered within the scope of this project. All C. trigynum share the same leaflet morphology (the most commonly recognized features being leaflets usually 3 to 5 and longer than wide, a large central leaflet, and margins thickened), and occur in similar habitats on their respective islands. Perhaps convergence on this particular leaf morphology has provided some ecological advantage to allow this form to occur in different lineages. 38

Biogeographic relationships

Fosberg (1948) was the first to suggest that the origins of Cheirodendron were from a single colonization event from the Austral region. Decades later, his ideas were confirmed with molecular data, supporting that Cheirodendron had an Austral origin and had moved northward via long-distance dispersal (Wen et al. 2001, Plunkett, Wen & Lowry 2004).

Recent studies have also shown that Raukaua, a paraphyletic group distributed in New

Zealand, Chile, Argentina and Tasmania, is Cheirodendron’s closest relative. The species sister to Cheirodendron are the New Zealand species of Raukaua: R. edgerleyi, R. anomalus, and R. simplex (Mitchell et al. 2012). Although there has been much recent information about the infrafamilial biogeographic relationships within Araliaceae, as well as the infrageneric biogeographic relationships of Raukaua, there has been no detailed phylogenetic work to assess these relationships within Cheirodendron until the present study.

Herat (1981) suggested a single colonization event from New Zealand to Hawaii with a subsequent dispersal event to the Marquesas (based on morphological similarities).

Although much less commonly seen, dispersal events originating from Hawaii have been confirmed through molecular work (see Harbaugh & Baldwin 2007, Harbaugh et al. 2009a).

However, this pattern of dispersal does not follow traditional ideas about biogeography in the

Pacific. Hennig (1966) proposed the concept of stepping stone biogeography patterns, where a given organism with a continental origin disperses to the next closest landmass (sometimes islands), evolutionarily “stepping” from landmass to landmass. This concept is especially embraced in Pacific biogeography when life on island archipelagos would have occurred via long distance dispersal along with in situ speciation. Evidence of this is well-supported by

39

many Pacific examples including Metrosideros (Wright et al. 2001), Astelia (Birch & Keeley

2013), and Coprosma (Cantley et al. 2014).

The biogeographic relationships of Cheirodendron inferred from this study suggest a stepping stone pattern of dispersal from New Zealand to Marquesas, and Marquesas to

Hawaii. As C. bastardianum and the Hawaiian clade have a sister relationship, an alternative could be that the most recent common ancestor could have colonized both the Marquesas and

Hawaiian Islands simultaneously. However, the previous hypothesis of Herat (1981) that C. bastardianum is descended from a colonist from the Hawaiian Islands can be discarded.

The biogeographic relationships implications of seven endemic species in the

Hawaiian Islands cannot be fully resolved at this time. Two Hawaiian clades, the Kauai clade and non-Kauai clade are sister to each other and could represent two simultaneous dispersal events from Marquesas to Hawaii. The biogeography of Cheirodendron within the Hawaiian

Islands cannot be inferred from this study, although Herat (1981) suggested that

Cheirodendron in Hawaii originated on Kauai, since Kauai appears to be the center of diversity and has the highest number of species. The lack of resolution within the two

Hawaiian clades suggests a recent and rapid radiation of these species.

Future directions

Although this study has shed light on the evolution and biogeography of

Cheirodendron, there are many questions that still remain. Are the forms of Cheirodendron a result of phenotypic plasticity? What are the phylogenetic relationships among Hawaiian species? What are the biogeographic implications of species within the Hawaiian Islands?

Can species relationships be resolved or are there underlying factors that limit our

40

understanding of the group? Perhaps Cheirodendron is part of a species complex, much like the unresolved species relationships of Metrosideros (see Harbaugh et al. 2009b).

The many challenges in sorting out species relationships have also been opportunities in deepening our understanding of the evolution of Cheirodendron. New techniques and methods to help answer these questions such as next generation sequencing, microsatellites, and restriction site associated DNA (RAD) sequencing may help in sorting out these relationships. Not only will these answers contribute to our understanding of Cheirodendron, but will advance our knowledge about the radiation and speciation of other Pacific groups.

Ultimately, this knowledge will provide valuable information for discerning relationships among all living things.

41

CHAPTER 3. SYNTHESIS- HYPOTHESES REVISITED

The conclusions of this study allow for the acceptance of two hypotheses and rejection of one hypothesis. The following discusses these conclusions as they relate to each individual hypothesis:

Hypothesis one: The molecular phylogeny does not support the current taxonomy of

Cheirodendron.

Conclusion: Accept hypothesis

Results of this study found disagreement between the molecular phylogeny and current taxonomy based on the placement of two Hawaiian subspecies. Based on these results, it is evident that species with subspecies recognized on different islands are not closely related and it is necessary to elevate the subspecies C. trigynum ssp. helleri to C. helleri Sherff and C. platyphyllum ssp. kauaiense to C. kauaiense Kraj. There are seven

Hawaiian species and one Marquesan species that should be recognized, as opposed to the current classification with five Hawaiian species and one Marquesan species.

Hypothesis two: Cheirodendron is monophyletic.

Conclusion: Accept hypothesis

This study shows evidence that Cheirodendron is a monophyletic lineage, consisting of C. bastardianum of the Marquesas along with Hawaiian species, C. trigynum, C. helleri,

C. platyphyllum, C. kauaiense, C. forbesii, C. fauriei and C. dominii. Cheirodendron has a sister relationship to New Zealand species of Raukaua, a paraphyletic genus with species in

New Zealand, Tasmania, Chile and Argentina. 42

Hypothesis three: The Pacific biogeography of Cheirodendron involves an Austral origin with long distance dispersal to Hawaii and then to the Marquesas.

Conclusion: Reject hypothesis

Phylogenetic analyses show Cheirodendron as a monophyletic clade of Austral origin. However, C. bastardianum has a sister relationship to the Hawaiian species rather than being derived from Hawaiian species. This infers that either two dispersal events simultaneously occurred from an Austral origin to Hawaii and the Marquesas, or dispersal occurred to Hawaii via Marquesas. There is no supporting data that suggests colonization of

Marquesas via Hawaii.

43

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