“There is probably no magic bullet for species discovery and delimitation, but an

integrative and evolutionary framework provides taxonomists with a larger arsenal to face the realities of inventorying the actual — and woefully underestimated – biodiversity

of the planet.” (Padial et al. 2010, p.10)

A molecular systematic and taxonomic assessment of the Rhodymeniales

by

Gina V. Filloramo

B.Sc., Trinity College, 2010

A Thesis, Dissertation, or Report Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

in the Graduate Academic Unit of Biology

Supervisor: Gary W. Saunders, PhD, Biology

Examining Board: Aurora Nedelcu, PhD, Biology Janice Lawrence, PhD, Biology Scott Pavey, PhD, Biology (UNBSJ)

External Examiner: Brian Wysor, PhD, Biology, Roger Williams University

This thesis, dissertation or report is accepted by the Dean of Graduate Studies

THE UNIVERSITY OF NEW BRUNSWICK

July, 2016

©Gina Filloramo, 2016

Year of Graduation 2017 ABSTRACT

The assessment of biological diversity and understanding the evolutionary history of organisms is integral to understanding life on earth. The Rhodymeniales is a well-defined red algal order for which interfamilial relationships are incompletely understood and species identification is complicated by the inability of traditional morphology-based approaches to reconcile convergent features and phenotypic plasticity. In this thesis, I used an integrative taxonomic approach combining molecular and morphological techniques to address rhodymenialean phylogenetic relationships and species diversity. I implemented multi-gene phylogenetics and site-stripping analyses to uncover reasonable support for interfamilial relationships within the Rhodymeniales for the first time. As part of that study, I established the phylogenetic assignment of some taxa (Binghamiopsis,

Chamaebotrys, Minium) previously missing from molecular analyses, restored monophyly to notoriously polyphyletic genera by establishing Perbella gen. nov. and

Fushitsunagia gen. nov., and described three novel Australian species of Drouetia (D. aggregata sp. nov., D. scutellata sp. nov., D. viridescens sp. nov.) after clarifying tetrasporangial development for that genus. Identification of recently collected material from Australia as the generitype of Leptofauchea facilitated a re-examination of that genus and its constituent species. Multigene phylogenetics provided support for

Leptofauchea as a monophyletic genus for the first time. Additionally, inconsistencies with published accounts of some Leptofauchea species were clarified and two novel species (L.cocosana sp. nov., L. munseumica sp. nov.) assigned to that genus were recognized. A molecular-assisted alpha taxonomic approach employing the 5’ end of the mitochondrial cytochrome c oxidase subunit I (COI-5P) DNA barcode was implemented ii

to reassess species diversity and redefine inaccurate or inadequate species concepts for rhodymenialean taxa in British Columbia. I resolved 16 species in 10 genera where 13 species in 11 genera were previously reported, uncovering underestimated diversity for the genera Botryocladia, Faucheocolax, Fryeella, Gloiocladia and Rhodymenia. The previous necessitated a taxonomic transfer (Fryeella callophyllidoides comb. nov. for

Rhodymenia callophyllidoides), the resurrection of a previously synonymized species epithet (R. rhizoides) and the establishment of novel taxa (B. hawkesii sp. nov., R. bamfieldensis sp. nov.). That study also included reassessment of anatomical development for the monospecific genus Minium. Lastly, a comprehensive floristic survey of the genus Rhodymenia from Australia was performed using molecular-assisted alpha taxonomy with COI-5P and ITS sequence data as the genetic markers. Whereas five species were previously reported, I resolved 12 genetic groups. Four of those groups were attributed to previously recognized species, whereas some collections were attributed to a New Zealand species, R. novazelandica, expanding its biogeographical range. The remaining seven genetic groups were inconsistent with existing species of

Rhodymenia and established as novel taxa (R. compressa sp. nov., R. contortuplicata sp. nov., R. gladiata sp. nov., R. insularis sp. nov., R. lociperonica sp. nov., R. norfolkensis sp. nov., R. womersleyi sp. nov.).

iii

DEDICATION

To my parents, thank you for instilling in me a sense of curiosity and a desire to learn.

iv

ACKNOWLEDGEMENTS

When I arrived in Fredericton, New Brunswick, I never could have imagined the journey that would define the next six years. Words will never do justice for how grateful I am for having this experience, but this is a start.

First and foremost, this thesis would not have been possible without the support and encouragement of my advisor, Dr. Gary Saunders. Thank you for welcoming me into your lab and providing me with the incredible opportunity that was learning and studying alongside you. You patiently guided me in my transition from thinking like an undergraduate student to questioning like a researcher. From the early stages of conceptualizing this project through the ups and downs along the way, through countless drafts, biogeographical conundrums and marathon meetings, you never gave up on me and pushed me to explore my potential as a scientist. Thank you for the opportunities for travel, attending conferences and experiences that have enabled my integration into a larger scientific community.

To my incredible labmates in the Saunders lab— I am honoured to have met each of one of you. A special acknowledgement to Drs Bridgette Clarkston, Susan Clayden, Kyatt

Dixon, Sarah Hamsher, Katy Hind and Dan McDevitt — as the senior members of the lab when I began my degree, you served as my role models. I appreciate everything you did to help me over the years. To Tanya Moore, thank you for keeping the lab running smoothly and for assisting me with any lab crises as I generated my data. To Dr. Chris

Jackson — thank you for teaching me the basics of command line programs. I would v

have been very lost and confused without you! To Trevor Bringloe, Kirby Morrill and

Cody Brooks — thank you for helping me remember why I started this journey in the first place. I have so enjoyed getting to know each of you and have the opportunity to play a small role in your phycological education. To Dr. Caroline Longtin, from field adventures and conference road trips, thank you for being a constant form of support and keeping my spirits light. To Dr. Meghann Bruce, my officemate and later, roommate,

“thank you” just doesn’t do justice for how grateful I am for your generosity and love when I needed it the most. I am eternally grateful to call you my colleague and my friend.

To Amanda Savoie, thank you for always being a willing ear, for encouraging me, reminding me to find joy in little things and helping me keep sane especially during the writing process. To Josh Evans, thank you for your constant encouragement and for being the comedic relief of the office. To Rossella Calvaruso, your friendship over the past few years has been such a treasure…thank you for being a constant voice of reason! Lastly, to

Lesleigh Kraft, there are no words to describe how grateful I am that fate brought us to the lab at the same time. Thank you for being my friend, the best travel partner and for always helping me keep life in perspective.

To my friends near and far, thank you for your encouragement and support. A special thanks to A.B., L.M., D.F., B.C., H.S. and A.S. for their love and thoughtfulness, for never giving up on me and sharing memories that will last a lifetime. Each one of you has changed my life for the better.

vi

I would like to acknowledge the faculty at the University of New Brunswick, notably, my supervisory committee members, Dr. Linley Jesson and Dr. Adrian Reyes-Prieto, who provided support and toughtful feedback during the execution of my projects. Thank you to Margaret Blacquier, Rose Comeau and Melanie Lawson for your administrative assisstance. Marni Turbull, thank you for all of your help over the years especially when I co-organized the Biology Seminar Series and Let’s Talk Science.

Dr. Craig Schneider — you introduced me to red algae when I was an undergraduate and through your influence, I found an outlet for my curiosity and a passion that I could turn into a career. Your guidance and encouragement over the years have meant so much. To

Drs Michael Wynne, Louise Lewis and countless other phycological collegues — thank you for always expressing interest in my projects and serving as a source of inspiration. I would be remiss if I did not mention Dr. Frank Trainor — you taught me that no matter how tiny the alga, there is a wealth of knowledge waiting to be explored.

To my extended family who, despite the distance, have supported me every step of the way. Mom and dad — from a very early age, you taught me to explore my sense of curiosity and instilled in me an appreciation for education. Your love and support has never wavered. Thank you will never be enough. Laura — you are my rock, my inspiration and my role model. “There is no better friend than a sister and no better sister than you”.

vii

Last but certainly not least, to Tom — thank you. Your love, dedication and overwhelming support have seen me through the most challenging times. Through your influence, I have learned to look at the world differently. Thank you simply for being you.

viii

Table of Contents

ABSTRACT ...... ii DEDICATION ...... iv ACKNOWLEDGEMENTS ...... v Table of Contents ...... ix List of Tables ...... xiii List of Figures ...... xiv Chapter 1 General Introduction ...... 1 Statement of Publications and Intented Submissions ...... 16

Statement of Contribution to Research and Writing ...... 17

References ...... 18

Chapter 2 Application of multigene phylogenetics and site-stripping to resolve intraordinal relationships in the Rhodymeniales (Rhodophyta) ...... 31 Abstract ...... 32

Introduction ...... 33

Materials and Methods ...... 37

Phylogenetic analyses ...... 37

Site-Stripping ...... 39

Barcode analyses ...... 40

Morphological analyses ...... 41

Results ...... 41

Molecular results ...... 41

Taxonomic treatment ...... 45

ix

Discussion ...... 52

Interfamilial relationships ...... 52

Taxa included in molecular analyses for the first time ...... 53

Assessment of select polyphyletic genera ...... 55

Taxonomic reassessment of Drouetia...... 60

Conclusions ...... 61

Acknowledgements ...... 62

References ...... 63

Chapter 3 Assessment of the order Rhodymeniales (Rhodophyta) from British Columbia using an integrative taxonomic approach reveals overlooked and cryptic species diversity ...... 82 Abstract ...... 83

Introduction ...... 84

Materials and Methods ...... 86

Results ...... 88

Molecular results ...... 88

Morphological results ...... 94

Discussion ...... 104

Acknowledgements ...... 118

References ...... 119

Chapter 4 A re-examination of the genus Leptofauchea (Faucheaceae, Rhodymeniales) with clarification of species in Australia and the northwest Pacific ...... 146 x

Abstract ...... 147

Introduction ...... 147

Materials and Methods ...... 150

Results ...... 152

COI-5P Barcode results ...... 152

Phylogenetic results ...... 152

Morphological results ...... 154

Discussion ...... 157

Acknowledgements ...... 163

References ...... 163

Chapter 5 Molecular-assisted alpha taxonomy of the genus Rhodymenia (Rhodymeniaceae, Rhodymeniales) from Australia reveals overlooked species diversity ...... 175 Abstract ...... 176

Introduction ...... 176

Materials and Methods ...... 181

Results ...... 182

Molecular Results ...... 182

Taxonomic Results ...... 185

Discussion ...... 194

Acknowledgements ...... 201

xi

References ...... 202

Chapter 6 General Conclusion ...... 214 Future Research ...... 219

References ...... 225

Appendix A Supplementary Data for Chapter 2 ...... 228 Appendix B Supplementary Data for Chapter 3 ...... 243 Appendix C Supplementary Data for Chapter 4 ...... 298 Appendix D Supplementary Data for Chapter 5 ...... 303 Appendix E Multigene analyses resolve early diverging lineages in the Rhodymeniophycidae (Florideophyceae, Rhodophyta) ...... 332 Appendix F Key Kamchatkan collections provide new taxonomic and distributional insights for reportedly pan-North Pacific species of Rhodymeniophycidae (Rhodophyta) ...... 397 Curriculum Vitae

xii

List of Tables

Table 1.1 Comparison of the reproductive features currently emphasized for family-level distinction within the Rhodymeniales ...... 23

Table 2.1 Collection details and COI-5P GenBank accession numbers for Australian

Drouetia samples ...... 68

Table 2.2 Bayesian posterior probabilities, RAxML bootstrap values and likelihood scores for the full multigene alignment and the site-stripper subalignments...... 70

Table 2.3 Intraspecific COI-5P divergence and distance to nearest neighbour for

Australian species of Drouetia...... 71

Table 3.1 Intraspecific COI-5P divergence and distance to nearest neighbor for included rhodymenialean species ...... 122

Table 4.1 Collection details for samples included in molecular analyses ...... 166

Table 4.2 Intraspecific COI-5P divergence and distance to nearest neighbor for species of

Leptofauchea ...... 168

Table 5.1 Intraspecific COI-5P divergence and distance to nearest neighbor for

Australian Rhodymenia species ...... 205

xiii

List of Figures

Figure 1.1 Morphological diversity among members assigned to the Rhodymeniales ... 24

Figure 1.2 A generalized schematic of the triphasic life history of the Rhodymeniales.. 25

Figure 1.3 Example of rhodymenialean procarp ...... 26

Figure 1.4 Mature rhodymenialean cystocarps...... 27

Figure 1.5 Schematic of tetrasporangial division patterns ...... 28

Figure 1.6 Major modes of tetrasporangial development used for classifying families in the Rhodymeniales ...... 30

Figure 2.1 RAxML phylogeny for full multigene alignment partitioned by gene and then codon with subfamilial bootstrap support values appended...... 72

Figure 2.2 Morphology and anatomy of Drouetia aggregata Filloramo & G.W. Saunders sp. nov...... 73

Figure 2.3 Morphology and anatomy of Drouetia scutellata Filloramo & G.W. Saunders sp. nov...... 75

Figure 2.4 Morphology and anatomy of Drouetia viridescens Filloramo & G.W.

Saunders sp. nov...... 77

Figure 2.5 Morphology and anatomy of the tetrasporangial holotype of Drouetia coalescens (Farlow) G. De Toni ...... 79

Figure 2.6 Morphology and anatomy of Hymenocladia chondricola (Sonder) J. Lewis,

Hymenocladia conspersa (Harvey) J. Agardh and Perbella minuta (Kylin) Filloramo &

G.W. Saunders comb. nov...... 81

Figure 3.1 Simplified schematic of ITS1 for representatives of Faucheocolax attenuata and Gloiocladia species ...... 125 xiv

Figure 3.2 Phylogenetic tree resulting from RAxML analysis of the concatenated COI-5P and rbcL alignment for representatives of Botryocladia and Rhodymenia ...... 126

Figure 3.3 Phylogenetic tree resulting from RAxML analysis of the concatenated COI-5P and rbcL alignment for representatives of the Faucheaceae and Fryeellaceae ...... 129

Figure 3.4 Morphology and anatomy of Botryocladia hawkesii sp. nov...... 131

Figure 3.5 Morphology and anatomy of Fryeella callophyllidoides (Hollenberg & I.A.

Abbott) Filloramo & G.W. Saunders comb. nov ...... 133

Figure 3.6 Morphology and anatomy of Gloiocladia fryeana...... 135

Figure 3.7 Morphology and anatomy of Gloiocladia laciniata...... 137

Figure 3.8 Morphology and anatomy of Gloiocladia media (Kylin) Filloramo & G.W.

Saunders comb. nov...... 139

Figure 3.9 Morphology and anatomy of Gloiocladia vigneaultii sp. nov...... 141

Figure 3.10 Morphology and anatomy of Minium parvum...... 142

Figure 3.11 Morphology and anatomy of Northeast Pacific Rhodymenia spp...... 144

Figure 3.12 Morphology and anatomy of Northeast Pacific Rhodymenia spp...... 145

Figure 4.1 Phylogenetic tree resulting from RAxML analysis of the multigene alignment for Leptofauchea spp...... 169

Figure 4.2 Morphology and anatomy of Leptofauchea nitophylloides...... 170

Figure 4.3 Morphology and anatomy of the holotype for Leptofauchea cocosana sp. nov.

...... 171

Figure 4.4 Morphology and anatomy of Leptofauchea leptophylla from Jeju, South

Korea ...... 173

xv

Figure 4.5 Morphology and anatomy of the holotype for Leptofauchea munseomica sp. nov...... 174

Figure 5.1 Map of Australia and New Zealand showing Rhodymenia species distributions

...... 207

Figure 5.2 Phylogenetic tree resulting from RAxML analysis of COI-5P data for species of Rhodymenia included in this study ...... 208

Figure 5.3 Morphology and anatomy of select Australian and New Zealand Rhodymenia spp...... 209

Figure 5.4 Morphology and anatomy of select Australian Rhodymenia spp...... 211

Figure 5.5 Morphology and anatomy of select Australian Rhodymenia spp...... 213

xvi

Chapter 1 General Introduction

1

Humankind’s desire to discover, describe and categorize living things traces its roots to early hunter-gatherer societies and has gradually evolved from a means for survival to a widespread curiosity to understand life on earth (Godfray & Knapp 2004; Raven 2004).

During the 18th century, Linnaeus developed a system of binomial nomenclature that provided the first standardized common language for the naming of species, which laid the framework for a system of hierarchal classification based on common characteristics

(Paterlini 2007). Later, Darwin’s theory of evolution revolutionized the manner in which species were classified and prompted naturalists to consider how a species may have diversified over time when classifying an organism (Raven 2004).

The legacies of Linnaeus and Darwin persist as the scientific fields of taxonomy

(i.e., the practice of describing, classifying and naming living organisms) and phylogenetics (i.e., the practice of inferring evolutionary relationships among taxa), which are considered fundamental components of modern science (Hebert 2003). Indeed, an understanding of Earth’s biodiversity and the ability to accurately identify species provide a reliable foundation for population studies, ecological assessments and conservation efforts (Costello et al. 2013). Recent studies of global species diversity have predicted that there may be 5 +/-3 million species on Earth; however, only one-third of those species have been identified and there has been a substantial bias towards terrestrial vs. marine life (Mora et al. 2011; Appeltans et al. 2012; Costello et al. 2013). In an era of global changes that threaten marine environments [e.g., habitat loss, hunting, harvesting and climate-change induced acidification, stratification and deoxygenation (Brook et al.

2008; Hoegh-Guldberg & Bruno 2010)], there is increased pressure to rapidly advance

2

our understanding of marine biodiversity so that it can be managed properly and organismal prosperity may endure (Gamfeldt et al. 2015).

The Rhodophyta or red algae are common inhabitants of marine environments.

They are integral to the stability of marine ecosystems and support unique communities of marine organisms. Economically, some red algal species have been cultivated for use in the human food industry, to foster marine fisheries, support aquaculture and produce pharmaceuticals and fertilizers. Our ability to continue utilizing red algae as an important natural resource requires a thorough understanding of their diversity and evolution over time (Woelkerling 1990; Graham & Wilcox 2000).

Red algae are recognized as one of the three major lineages of primary photosynthetic organisms that evolved following primary endosymbiosis of a chloroplast

(Keeling 2004). Red algae are considered a highly diverse assemblage of taxa and are distinguished from other eukaryotes by the combination of the following biochemical and ultrastructural features: floridean starch stored in the cytoplasm, plastids with equidistant thylakoid membranes, accessory pigments phycoerythrin and phycocyanin occuring in stalked phycobilisomes on thylakoids, and, perhaps most uniquely, the absence of centrioles and flagella in all stages of their life history (Pueschel 1990; Hoek et al. 1995;

Ragan & Gutell 1995). Currently there are an estimated 7000 species in roughly 700 genera attributed to the red algae (Guiry & Guiry 2016).

Traditionally, red algal classification has relied on a morphology-based approach; however, that has presented a number of difficulties when applied to this group (Saunders

2005; Le Gall & Saunders 2007). Red algae are morphologically simple organisms providing few characters for taxonomic comparison (Saunders 2005). Although 3

reproductive criteria may be useful for distinguishing taxa, those features become problematic when only vegetative specimens are collected (Saunders 2005). Species of red algae may be phenotypically plastic (i.e., they have varied morphologies resulting from different environmental factors) or can have an alternation of heteromorphic generations, which may result in overestimated species diversity. In contrast, evolutionary convergence may lead to the misinterpretation of analogous features as homologous, while recent speciation and the retention of ancestral features can result in an underestimation of diversity (Saunders 2005). Additionally, morphological assessment is highly subjective and individual misinterpretations of anatomical and reproductive structures has likely served to exacerbate taxonomic confusion over time (Radulovici et al. 2010).

As a result of the numerous challenges associated with traditional morphology-based assessment, taxonomists and systematists have become increasingly reliant on molecular data, which provide a more objective and reliable alternative for both species diversity assessments and inferring evolutionary relationships (Saunders 2005; Cianciola et al.

2010). At the species level, “DNA barcoding” (i.e., using a short, standardized portion of

DNA to assign specimens to distinct genetic species groups according to the degree of nucleotide similarity) has been championed as an effective means for accurate species identification and discovery (Hebert 2003). The existence of a “barcode gap” between inter- and intra- specific genetic variation (i.e. interspecific variation should be greater than intraspecific variation) has resulted in a practical and standardized method of delineating genetic species groups (Hebert et al. 2003; Hebert et al. 2004; Barrett &

Hebert 2005). Although there is no universal barcode for eukaryotes, many lineages, 4

including the red algae, typically utilize a 664 bp region at the 5’ end of the mitochondrial cytochrome c oxidase subunit I (COI-5P) gene to assign collections to distinct genetic species groups (Saunders 2005, 2008). Mitochondrial markers are recognized as effective for species delineation as a result of their rapid rate of evolution, haploid state and uniparental mode of inheritance (Birky et al. 1983). Owing to the previous, the effective population size of mitochondrial genes is one quarter than that of nuclear genes (Palumbi & Cipriano 1998). As a result, genetic drift (i.e., fixation of mutations in a population) acts much faster at mitochondrial loci and closely related species are more likely to have fixed sequence differences at mitochondrial gene regions than they are at nuclear loci (Neigel & Avise 1986; Palumbi & Cipriano 1998). Analysis of intra- and inter- specific variation rates in COI-5P data for red algal species has indicated that the typical thresholds for intra- and inter- specific divergence are 0-0.5% and >4%, respectively (Saunders 2005). Other markers that are informative at the species level include the 3’ end of the plastid ribulose-1,5-biphosphate carboxylase large subunit

(rbcL-3P) and the nuclear internal transcribed spacer (ITS). Both rbcL and ITS may be used as complement to COI-5P data to confirm species identifications (Saunders &

Moore 2013). As ITS is biparentally inherited, that marker can provide insight into hybridization and introgression between populations and becomes particularly useful for assessing species boundaries when intra- or inter- specific divergence is resolved between

0.5-4% (e.g., McDevit & Saunders 2010; Hind & Saunders 2013; Savoie & Saunders

2015). It is imperative to emphasize that current red algal taxonomy is not exclusively inferred from molecular data, as it is standard practice to use morphological observation as a compliment to molecular findings to determine any defining characters for each 5

genetic species in an approach termed “molecular-assisted alpha taxonomy” [Saunders

(2005); popularized as “MAAT” in Cianciola et al. (2010)]. One of the benefits of

MAAT is that once taxonomists are equipped with morphological characters for each molecularly supported species, they may then refer to taxonomic guides and algal literature to assign each species group to an existing species name, or, if none exists, establish a new name (Saunders 2008). With accurate species concepts, it becomes possible to assess these groups beyond the species level to understand their phylogeny.

Over the past 10-15 years, the number of markers available for phylogenetic inference has increased substantially and phylogenetic studies have refined those nuclear, plastid and mitochondrial markers that are most useful at various taxonomic levels (Maggs et al.

2007; Verbruggen et al. 2010). Mitochondrial and some plastid markers [e.g., the mitochondrial COI-5P & plastid ribulose-1,5-biphosphate carboxylase large subunit

(rbcL)] have the highest resolution at lower taxonomic levels and are most useful for inferring recent evolutionary events (Maggs et al. 2007). Because those markers tend to saturate at higher taxonomic levels, resolving deeper evolutionary relationships requires use of other plastid [e.g., photosystem II thylakoid membrane protein D1 (psbA)] and nuclear markers [e.g., elongation factor 2 (EF2) & large subunit ribosomal DNA (LSU)]

(Maggs et al. 2007). While past red algal phylogenetics inferred evolutionary relationships from a single gene or multiple single gene phylogenies, those analyses typically lacked confidence in deeper nodes (e.g., Saunders et al. 1999). Recent phylogenetic approaches seldom rely on single gene phylogenies in preference for the concatenation of multiple markers from mitochondrial, nuclear and plastid genomes into a single comprehensive, robust dataset from which evolutionary relationships can be 6

inferred using improved model-based phylogenetic inference techniques (e.g., Bayesian

Inference and Maximum Likelihood) (e.g., Dixon et al. 2015; Lam et al. 2016). Although there are recommended divergence thresholds for delineation of red algae at the species level (see above), at this time there is a lack of standardization among taxonomic ranks and molecular thresholds for identifying and distinguishing between higher taxonomic categories are not recognized (beyond the requirement that those individual taxonomic units are monophyletic) (Groves 2014). As a result, the assignment and distinction of taxonomic ranks beyond the species level is somewhat arbitrary and reliant on the discretion of taxonomists who may use shared morphological features as a secondary tool to genetic data for establishing genera, families, etc. (Avise 2008; Avise & Liu 2011).

Overall, the application of molecular tools to red algal classification has revolutionized, and at times challenged, our present understanding of red algal taxonomy.

Genetic data have also resulted in the need for revised species concepts that are more consistent with evolutionary processes and molecular perspectives (Nixon & Wheeler

1990). Rather than recognizing species by exclusively employing a traditional morphological species concept (i.e. species were distinguished based on discontinuities in morphological variation) (Manhart & McCourt 1992; Leliaert et al 2014), contemporary red algal taxonomy delimits species based on the unification of diverse species definitions (De Queiroz 2007). The process of MAAT employs the genotypic clustering species concept (i.e., identification of species as distinct “genotypic clusters” in which members of the same species are genetically more similar to each other than to members of other species and lack intermediate forms; Mallet 1995), which may be reinforced secondarily by the morphological species concept. Additionally, the genotypic clusters 7

resolved via the molecular (DNA barcoding) component of MAAT are further characterized by the phylogenetic species concept (i.e. species are monophyletic groups of organisms diagnosable by qualitative, fixed genetic differences; Nixon & Wheeler

1990; Mayden 1997), which supports distinction of hierarchal groups and is consistent with various evolutionary explanations for the origin of species (i.e., sympatric speciation or allopatric speciation). Utilization of an integrative species concept has provided a practical, accurate and conclusive system of red algal identification and historical relationships while also indicating how greatly red algal biodiversity is underestimated

(Manhart & McCourt 1992; Davis 1996; Saunders 2005, 2008).

The red algal group of interest for this thesis was the Rhodymeniales, a well- defined order of red algae belonging to the subclass Rhodymeniophycidae in the most species-rich red algal class, Florideophyceae (Saunders & Hommersand 2004, Le Gall &

Saunders 2007). Established by Schmitz (1889) to accommodate florideophycean taxa united by a uniform female reproductive system (discussed below), the Rhodymeniales includes a variety of morphologies including large expansive plants, delicate diminutive thalli, anastomosing, peltate or stellate blades, solid thalli and mucilage filled sacs

(Figure 1.1). Owing to such vast morphological diversity, this group has been the subject of many systematic and taxonomic investigations (e.g., Dawson 1941; Sparling 1957;

Chapman & Dromgoole 1970; Lee 1978; Hawkes & Scagel 1986; Saunders & Kraft

1996; Saunders et al. 1999; Saunders et al. 2006; Dalen & Saunders 2007; Le Gall et al.

2008; Saunders & McDonald 2010). At present, there are six families, ~47 genera and over 300 species assigned to the Rhodymeniales (Guiry & Guiry 2016).

8

Members of the Rhodymeniales feature a triphasic red algal life history that is common among florideophyte lineages (Hommersand & Fredericq 1990). This specialized life cycle features an alternation of generations including a haploid gametophyte phase, a diploid sporophyte generation (carposporophyte) that develops directly on the surface of the female thallus, and a free-living diploid phase

(tetrasporophyte) (Figure 1.2) (Hommersand & Fredericq 1990). Understanding the details of this triphasic life history is essential for understanding traditional and current perspectives of rhodymenialean classification as reproductive features are commonly used for taxonomic distinction.

Occurring in subapical sori or scattered in patches on the thallus surface, the male gametangia (spermatangia) originate from the outermost cortical layer and produce male gametes (spermatia), which lack flagella and rely on water currents for dispersal to the female gamete (carpogonium) (Figure 1.2). The carpogonium is a flask-shaped cell, which is part of a “carpogonial branch” that includes a specific number of cells extending from a larger supporting (basal) cell (Figure 1.3). The carpogonium terminates with a long apical process called the trichogyne (Figure 1.3), which provides a receptive surface for the male gametes. A male nucleus travels down the trichogyne to the base of the carpogonium where it fuses with the female nucleus. After syngamy, the diploid nucleus is transferred via direct fusion or a connecting cell to a specialized cell called the auxiliary cell (Figure 1.3). The position of the carpogonium in relation to the auxiliary cell differs among red algal groups. “Procarpic” red algae are those with a carpogonial branch produced from the same supporting cell as the auxiliary cell branch, which is composed of two cells (the auxiliary cell and auxiliary mother cell in the Rhodymeniales) 9

(Figure 1.3). Once the diploid nucleus is transferred to the auxiliary cell, the cells of the carpogonial branch fuse and the auxiliary cell gives rise to diploid gonimoblast filaments that form the carposporophyte, which generates diploid carposporangia (Figure 1.4). An opening (ostiole) eventually forms in the pericarp (protective gametophytic tissue surrounding the carposporophyte) and serves as a portal for the release of the diploid carpospores (Figure 1.4b), which are produced by the carposporangia, carried passively in the water column and, after settling, give rise to the free-living tetrasporophyte generation (Figure 1.2). Tetrasporophytes produce tetrasporangia in sori or scattered over the thallus. Each tetrasporangium undergoes meiotic division (typically in one of four division patterns; Figure 1.5) to produce four haloid spores (tetraspores), which are released, carried in the water column and, after settling, complete the triphasic life cycle by giving rise to the haploid gametophyte generation (Figure 1.2) (Hommersand &

Fredericq 1990).

All members of this order are distinctively procarpic with 3- or 4- celled carpogonial branches and two-celled auxiliary branches (the auxiliary cell and auxiliary mother cell) (Figure 1.3) (Kylin 1931, 1956). In some groups, the layer of nutritive cells at the base of the cystocarp ruptures to form a hollow cavity surrounding the carposporophyte (Figure 1.4a) or becomes stretched around the mature carposporophyte to establish a stellate network of cells (tela arachnoidea) (Figure 1.4b) (Kylin 1931).

Tetrasporangial initials may be terminal or intercalary in position and mature tetrasporangia are typically cruciate (Figure 1.5a,b) or tetrahedral (Figure 1.5d) (Sparling

1957; Lee 1978; Saunders et al. 1999). Tetrasporangia are either located throughout the thallus or in discrete nemathecial sori [elevated, proliferous portions of the thallus that 10

also include sterile filaments (paraphyses) surrounding the developing tetrasporangia (Le

Gall et al. 2008)]. There are presently two major modes of tetrasporangial development attributed to the Rhodymeniales: (1) tetrasporangia that differentiate directly from the existing cortical cells (Figure 1.6a) or (2) tetrasporangia and paraphyses that develop from adventitious growth of the outer cortical cells (Figure 1.6b-e) (Dalen & Saunders

2007; Le Gall et al. 2008). In the latter case, during nemathecial development, the cells in the outer cortical layer differentiate, swell and produce sterile filaments (paraphyses) composed of smaller cells (the number of cells varies per species) (Figure 1.6b,c,d)

(Dalen & Saunders 2007). During this adventitious development those differentiated cells can produce tetrasporangia directly on the modified outer cortical cells, terminally or laterally on the paraphyses (Figure 1.6e) (Dalen & Saunders 2007; Le Gall et al. 2008).

Historically, classification of the Rhodymeniales has been complicated and the features emphasized for taxonomic distinction have been highly contested (Bliding 1928;

Kylin 1931, 1956; Sparling 1956; Lee 1978). Owing to the previous, this order was an ideal candidate for phylogenetic assessment to aid in establishing a more objective, natural system of classification. Molecular assessments of the Rhodymeniales have confirmed its monophyly and support six distinctive families (Champiaceae,

Faucheaceae, Fryeellaceae, Hymenocladiaceae, Lomentariaceae and Rhodymeniaceae), which are delineated according to a combination of reproductive features including ontogeny of tetrasporangia and paraphyses, tetrasporangial division patterns and the number of cells in the carpogonial branch (Table 1.1) (Saunders et al. 1999; Le Gall et al.

2008). These initial molecular phylogenetic studies were significant in providing a solid framework for numerous subsequent taxonomic assessments of rhodymenialean genera 11

and species (e.g., Saunders et al. 2006; Dalen & Saunders 2007; Saunders & McDonald

2010); however, those studies were unable to resolve relationships at the family level potentially owing to an inability to gain appropriate signal at the deeper nodes of interest

(Saunders et al. 1999; Le Gall et al. 2008). Recently, the phylogenetic reconstruction method of “site-stripping” (the systematic removal of quickly evolving sites, which as a result of substitution saturation introduce stochastic noise that overwhelms the signal of the historically informative, more slowly evolving sites) has addressed the challenge of retaining signal at deeper nodes and has provided means for more confidently resolving evolutionary relationships as higher taxonomic levels (Verbruggen et al. 2009; Cocquyt et al. 2010).

The primary objective of this thesis was to integrate molecular and morphological tools to reinvestigate evolutionary relationships and assess species diversity (notably from the biologically diverse areas of Australia and western Canada) among the

Rhodymeniales. In Chapter 2 I reassessed supraordinal relationships among families of the Rhodymeniales by increasing taxonomic representation (the most comprehensive phylogenetic assessment of the order to date), constructing a comprehensive multigene dataset of mitochondrial [cytochrome B (COB) & COI/COI-5P], nuclear [EF2 & large subunit ribosomal DNA (LSU)], and plastid (psbA & rbcL) markers, and implementing a fast-site removal reconstruction technique (i.e., SiteStripper; Verbruggen 2012) to prevent phylogenetic inference problems associated with substitution saturation. I resolved the Rhodymeniales as two major lineages: (i) the Fryeellaceae as sister to the

Faucheaceae and Lomentariaceae; and (ii) the Rhodymeniaceae allied to the

Champiaceae and Hymenocladiaceae and demonstrated that removal of 20% of variable 12

sites provided increased support at deeper nodes. This study was significant as it provided reasonable support for interfamilial relationships within the Rhodymeniales for the first time. In this study, I included three genera absent from previous phylogenetic investigations (Binghamiopsis I.K. Lee, J.A. West & Hommersand, Chamaebotrys

Huisman and Minium R.L. Moe) and provided solid phylogenetic support to confirm their respective family-level assignments (Lomentariaceae, Rhodymeniaceae and Fryeellaceae, respectively). In addition, I assessed two genera that were consistently polyphyletic in phylogenetic analyses, Erythrymenia and Lomentaria, and proposed the novel genera

Perbella gen. nov. and Fushitsunagia gen. nov. to restore monophyly. I reinvestigated the taxonomic position of the genus Drouetia G. De Toni by re-examining holotype material of the generitype species and clarifying tetrasporangial development in this genus. Lastly,

I recognized three novel Drouetia species in Australia: D. aggregata sp. nov., D. scutellata sp. nov., and D. viridescens sp. nov.

In Chapter 3 I used molecular-assisted alpha taxonomy to reassess species diversity, species limits and biogeography for rhodymenialean species in British

Columbia. Whereas 13 species in 11 genera were previously reported (Hawkes & Scagel

1986), COI-5P and rbcL-3P data resolved 16 genetic species groups in 10 genera. I confirmed the presence of species previously reported from this locale and also uncovered overlooked diversity for the genera Botryocladia (J. Agardh) Kylin, Fryeella

Kylin, Gloiocladia J. Agardh and Rhodymenia Greville, which warranted the description of three novel species (B. hawkesii sp. nov., G. vigneaultii sp. nov. and R. bamfieldensis sp. nov), the resurrection of R. rhizoides E.Y. Dawson and recognition of F. callophyllidoides (Hollenberg & I.A. Abbott) comb. nov. for the misplaced Rhodymenia 13

callophyllidoides Hollenberg & I.A. Abbott. I also uncovered genetic evidence suggesting that there may be more species of the parasitic genus Faucheocolax Setchell than currently recognized. Lastly, I reassessed anatomical development of the monospecific genus Minium R.L. Moe and suggest that M. parvum R.L. Moe represents a diminutive blade rather than a red algal crust.

In Chapter 4 I also used molecular-assisted alpha taxonomy (employing COI-5P) to assign recently collected specimens from Australia and South Korea to the genus

Leptofauchea Kylin. One of the Australian collections was identified as the generitype species L. nitophylloides (J. Agardh) Kylin and provided the necessary genetic data to assess monophyly of this genus for the first time. I used multigene phylogenetic analysis of COI-5P, LSU and rbcL data to demonstrate that species assigned to Leptofauchea in previous studies were allied with the generitype in a solidly supported monophyletic lineage. In this study, COI-5P data also revealed a second Australian species of

Leptofauchea from the Cocos (Keeling) Islands, Indian Ocean, which I described as L. cocosana sp. nov. In addition, COI-5P analyses resolved collections assignable to

Leptofauchea from South Korea as two genetic species groups, which were recognized as

L. leptophylla (Segawa) Suzuki, Nozaki, Terada, Kitayama, Hashimoto & Yoshizaki and the novel species L. munseomica sp. nov. Finally, I discovered that GenBank entries for two species of Leptofauchea previously reported in the northwest Pacific (L. leptophylla described from Japan and L. rhodymenioides described from the Caribbean) are conspecific (based on rbcL analyses) and morphologically assignable to L. leptophylla.

In Chapter 5 I conducted a comprehensive floristic survey for the genus

Rhodymenia Greville from Australia using a molecular-assisted alpha taxonomic 14

approach. Whereas five species of Rhodymenia were previously reported in Australia,

COI-5P data resolved 12 genetic species groups. Subsequent morphological assessment confirmed four of those groups as assignable to previously recognized species, viz. R. novahollandica G.W. Saunders, R. prolificans Zanardini, R. stenoglossa J. Agardh & R. wilsonis (Sonder) G.W. Saunders, while some collections from Lord Howe Island were attributed to the New Zealand species R. novazelandica E.Y. Dawson expanding its biogeographical range. I found that the remaining seven genetic groups were inconsistent with existing species of Rhodymenia and described those groups as novel taxa: R. compressa sp. nov., R. contortuplicata sp. nov., R. gladiata sp. nov., R. insularis sp. nov.,

R. lociperonica sp. nov., R. norfolkensis sp. nov. and R. womersleyi sp. nov. Although morphological features and biogeography were recognized as adequate for distinguishing some species of Rhodymenia, I suggest DNA sequencing in combination with morphology and biogeography as the most reliable means of identification.

Appendix E and Appendix F include additional research that I conducted and published while completing my thesis projects. For Appendix E, I used a multigene phylogenetic assessment that implemented site-stripping to resolve the earliest divergences in the red algal subclass Rhodymeniophycidae. The inclusion of key taxa

(new to science and/or previously lacking molecular data), sequence data from five molecular markers and phylogenetic analyses removing the most variable sites (site- stripping) have provided resolution for the first time at these deep nodes. The results of that study resulted in the establishment of a novel order, three new families, a new genus and a novel species all assigned to the Rhodymeniophycidae. For Appendix F, I used

DNA barcoding to compare Northwestern Pacific specimens of the red algal species 15

Callophyllis radula, Callophyllis rhynchocarpa, Pterosiphonia bipinnata and Sparlingia pertusa, all with type localities in Far East Russia, to reportedly conspecific populations in the Northeast Pacific. Analyses resolved genetically distinct species with a biogeographical split between the Northwest and Northeast Pacific for C. radula, C. rhynchocarpa and P. bipinnata. Comparison of Northwest to Northeast Pacific S. pertusa revealed COI-5P variation consistent with incipient speciation between these two regions; however, subsequent analysis of the ITS2 provided evidence for recognition of a single species with a pan-North Pacific distribution. Our results indicated greater floristic division between the Northwest and Northeast Pacific than is currently recognized.

Statement of Publications and Intented Submissions

Chapter 2 was published in the Journal of Phycology (Vol. 52, Issue 3; June 2016).

Chapter 3 is in preparation for submission to Botany.

Chapter 4 was published in Phycologia (Vol. 54, No. 4; July 2015).

Chapter 5 was published in the European Journal of Phycology (available online 26 May

2016).

Appendix E was published in the Journal of Phycology (available online 6 May 2016).

Appendix F was accepted (May 2016) for publication in Phycologia and is currently in revision.

16

Statement of Contribution to Research and Writing

I designed and implemented the objectives of this research with support and guidance from Dr. Gary Saunders.

I was involved with some of the fieldwork for this research; however, the majority of specimens included in this project were collected by former or current labmates. All collectors and collaborators have been recognized in the acknowledgements for each chapter.

I conducted the majority of the laboratory work for this project with some assistance from laboratory technicians (Tanya Moore, Dan McDevit, Kyatt Dixon and Ali Johnson) who processed much of the COI-5P data as part of the lab’s high through-put laboratory. Out of the ~700 COI-5P barcode sequences included in my research, I generated ~192 COI-

5P sequences. I generated all other sequence data excepting ~114 sequences of the 463 total sequences included in Chapter 2. Those 114 sequences were generated by Tanya

Moore as part of the Red Algal Tree of Life Project (RedToL). For Appendix E, I conducted the site-stripping molecular analyses, generated Table E.1, edited Figure E.1 and wrote the methods and results sections corresponding to the site-stripping analyses.

For Appendix F, I conducted all molecular and morphological analyses and wrote the manuscript for which all co-authors provided feedback.

I conducted all morphological assessments for this project.

17

I completed all of the data analyses for this project with guidance and assistance from Dr.

Gary Saunders.

I wrote all chapters of this thesis with guidance and editorial comments from Dr. Gary

Saunders.

References

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multi-locus time-calibrated phylogeny of the siphonous green algae. Molecular Phylogenetics and Evolution, 50: 642-653. Woelkerling, W.J. (1990). An introduction. In: Biology of the Red Algae. (Cole, K.M. & Sheath, R.G., editors), 305-346. Cambridge University Press, Cambridge.

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Table 1.1 Comparison of the reproductive features currently emphasized for family- level distinction within the Rhodymeniales. Modified from Le Gall et al. (2008;

Table 1).

Female Tetrasporangia Number of cells in Division Ontogeny the carpogonial pattern branch Champiaceae 4 Tetrahedral Directly from existing cortical cells Faucheaceae 3 Cruciate Adventitious growth of cortical cells Fryeellaceae 3 Cruciate or Adventitious growth of Tetrahedral cortical cells Hymenocladiaceae 4 Cruciate or Directly from existing Tetrahedral cortical cells Lomentariaceae 3 Tetrahedral1 Adventitious growth of cortical cells2 Rhodymeniaceae 3 or 4 Cruciate Directly from existing cortical cells

1 The genus Ceratodictyon is the only member of the Lomentariaceae to include cruciately divided tetrasporangia.

2 Based on Lee (1978), who reported a thickening of the cortex during tetrasporangial development as well as the conversion of the outermost cortical cells to tetrasporangia in species of Lomentaria.

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Figure 1.1 Members assigned to the Rhodymeniales are morphologically diverse including: a) large expansive blades, b) delicate, foliose thalli, c) anastomosing, peltate or stellate blades, and d), e) mucilage filled sacs of varying size and number.

All scale bars = 25 mm.

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Figure 1.2 A generalized schematic of the triphasic life history of the Rhodymeniales that includes an isomorphic alternation of generations including gametophyte (1N), carposporophyte (2N), and tetrasporophyte (2N) phases.

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Figure 1.3 The procarp for Sparlingia pertusa featuring a 3-celled carpogonial branch (carpogonium and trichogyne labelled) and 2-celled auxiliary branch

(auxiliary mother cell and auxiliary cell) borne on the same supporting cell.

Image adapted from Fritsch (1945).

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Figure 1.4 Mature cystocarps showing absence or presence of tela arachnoidea. a)

Mature cystocarp of Rhodymenia sp. with hollow cavity surrounding the carposporophyte (csp). Scale bar = 300 um. b) Mature cystocarp of Gloiocladia sp. showing the carposporophyte (csp) that is surrounded by well-defined tela arachnoidea (ta). The ostiole (o) is also visible. Scale bar = 400 um.

27

a b c d

Figure 1.5 Schematic showing tetrasporangial division patterns: a) cruciate, b) cruciate-decussate, c) zonate, and d) tetrahedral. For b and d, only three tetraspores are visible in one view and the dotted lines indicate the tetraspore that is not observable.

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Figure 1.6 Modes of tetrasporangial/paraphyseal development used for classifying rhodymenialean families. a) Tetrasporangial development for

Rhodymenia sp. showing tetrasporangial embedded in an unmodified cortex with maturing tetrasporangia (t) positioned on a cell of the inner cortex (icc). No adventitious growth of the cortex occurs. Scale bar = 40 um. b) Adventitious growth of the outer cortical cells during nemathecial development for Fryeella gardneri begins with swelling of the outer cortical cells (arrow), which produces paraphyseal filaments (double arrows) composed of smaller cells (the number of cells varies per species). Scale bar = 15 um. c) During early nemathecial development for Fryeella gardneri paraphyseal filaments retain inflated basal cells. Scale bar = 15 um. d) The basal paraphyseal cells eventually become compressed (double arrows) as the tetrasporangia mature for Fryeella gardneri.

Scale bar = 15 um. e) Mature tetrasporangial nemathecium for Leptofauchea sp. with tetrasporangia (t) borne on inner cortical cells (icc) and paraphyseal filaments (pf) borne from adventitious growth of the outer cortical cells. Scale bar = 35 um. Images b-d reproduced with permission from Dalen & Saunders

(2007).

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Chapter 2 Application of multigene phylogenetics and site-stripping to

resolve intraordinal relationships in the Rhodymeniales (Rhodophyta)

Citation for published manuscript: Filloramo, G.V. & Saunders, G.W. (2016).

Application of multigene phylogenetics and site-stripping to resolve intraordinal relationships in the Rhodymeniales (Rhodophyta). Journal of Phycology, 52: 339-355.

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Abstract

Previous molecular assessments of the red algal order Rhodymeniales have confirmed its monophyly and distinguished the six currently recognized families (viz.

Champiaceae, Faucheaceae, Fryeellaceae, Hymenocladiaceae, Lomentariaceae, and

Rhodymeniaceae); however, relationships among most of these families have remained unresolved possibly as a result of substitution saturation at deeper phylogenetic nodes. The objective of the current study was to improve rhodymenialean systematics by increasing taxonomic representation and using a more robust multigene dataset of mitochondrial (COB, COI/COI-5P), nuclear (LSU, EF2) and plastid markers (psbA, rbcL). Additionally, we aimed to prevent phylogenetic inference problems associated with substitution saturation (particularly at the interfamilial nodes) by removing fast-evolving sites and analyzing a series of progressively more conservative alignments. The Rhodymeniales was resolved as two major lineages: (i) the Fryeellaceae as sister to the Faucheaceae and Lomentariaceae; and (ii) the Rhodymeniaceae allied to the Champiaceae and Hymenocladiaceae.

Support at the interfamilial nodes was highest when 20% of variable sites were removed. Inclusion of Binghamiopsis, Chamaebotrys and Minium, which were absent in previous phylogenetic investigations, established their phylogenetic affinities while assessment of two genera consistently polyphyletic in phylogenetic analyses,

Erythrymenia and Lomentaria, resulted in the proposition of the novel genera

Perbella and Fushitsunagia. The taxonomic position of Drouetia was reinvestigated with re-examination of holotype material of D. coalescens to clarify tetrasporangial development in this genus. In addition, we added three novel Australian species to 32

Drouetia as a result of ongoing DNA barcoding assessments—D. aggregata sp. nov.,

D. scutellata sp. nov., and D. viridescens sp. nov.

Introduction

The Rhodymeniales is a well-studied, species-rich florideophycean order of extremely diverse frond morphologies united by a uniform procarpic female reproductive system

(i.e., the auxiliary cell is produced prior to fertilization and in close proximity to the carpogonium within a single branch system) and outward carposporophyte development.

Published phylogenetic analyses of florideophycean taxa regard the Rhodymeniales as monophyletic and closely related to the Halymeniales and Sebdeniales (e.g., Saunders &

Hommersand 2004, Withall & Saunders 2006, Le Gall & Saunders 2007, Verbruggen et al. 2010). Historically, subordinal classification of the Rhodymeniales has been complicated by conflicting perspectives regarding the most useful anatomical features for taxonomic distinction. A brief summary of rhodymenialean systematics is provided below; the reader is referred to Saunders et al. (1999) and Le Gall et al. (2008) for a more comprehensive review.

When Schmitz (1889) established the Rhodymeniales, he included six families:

Bonnemaisoniaceae, Ceramiaceae, Delesseriaceae, Rhodomelaceae, Rhodymeniaceae and Sphaerococcaceae. Soon after, all of those families were removed except for the

Rhodymeniaceae (Oltmanns 1904, Sjöstedt 1926). Bliding (1928) subsequently added the

Champiaceae, which was differentiated from the Rhodymeniaceae according to thallus construction (hollow thalli with longitudinal filaments bordering the cavity present vs.

33

solid thalli or hollow thalli but lacking longitudinal filaments), tetrasporangial division patterns (tetrahedral vs. cruciate), number of cells in the carpogonial branch (four vs. three) and the degree to which cells differentiated into carposporangia (only the terminal cells vs. most of the gonimoblast). Kylin (1931) maintained Bliding’s two families and further defined them by assigning subfamilies to each. Sparling (1957) rejected Kylin’s subclassifications and emphasized the presence (Champiaceae) or absence

(Rhodymeniaceae) of longitudinal filaments bordering the hollow parts of thalli for family-level distinction. Later, Lee (1978) used a combination of vegetative (thallus septation) and reproductive criteria (number of cells in the carpogonial branch and the position of the tetrasporangia) to define the Champiaceae, Rhodymeniaceae, and a third family, Lomentariaceae. Lee’s three-family classification system was widely accepted; however, some rhodymenialean genera featured diagnostic characters of more than one family and, therefore, could not be classified confidently (e.g., Dictyothamnion,

Ceratodictyon, Gelidiopsis, Hymenocladia, Hymenocladiopsis and Semnocarpa, see

Saunders et al. 1999).

The application of molecular techniques to systematic studies of the red algae has enabled a more objective means of assessing evolutionary relationships within the

Rhodymeniales. Although rhodymenialean taxa were represented in some of the earliest red algal molecular phylogenetic investigations (e.g., Freshwater et al. 1994, Ragan et al.

1994, Saunders & Kraft 1994, Saunders & Kraft 1996), these studies only included enough taxa to establish the order as monophyletic and allied to the Halymeniales.

Subordinal relationships remained largely unresolved until Saunders et al. (1999), using the nuclear small subunit ribosomal gene (SSU), embarked on the first extensive 34

molecular systematic study specific to the Rhodymeniales. That study included 56% of the recognized genera (Saunders et al. 1999). Challenging Lee’s three-family system,

Saunders et al. (1999) resolved the Rhodymeniales as six monophyletic assemblages distributed in two major lineages – the first included only a reduced Rhodymeniaceae while the second consisted of the Champiaceae, Lomentariaceae, their newly established

Faucheaceae, and two unassigned lineages. In light of their results, Saunders et al. (1999) emphasized reproductive features, including the number of cells in the carpogonial branch, tetrasporangial cleavage patterns and tetrasporangial position for family-level classification. Although that study was significant for providing a more natural system of classification for the Rhodymeniales, it was limited by its low representation of rhodymenialean taxa (both the absence of entire genera and many generitype species) and lack of resolution among interfamilial nodes (Saunders et al. 1999). Poor resolution was partially due to gene choice as SSU displayed highly variable rates in some families, which increased the potential for long-branch attraction artefacts (Saunders et al. 1999).

In a subsequent study Le Gall et al. (2008) sought to circumvent potential long-branch attraction artefacts by increasing taxonomic representation (by ~13%) and basing phylogenetic inference on two nuclear genes: elongation factor 2 (EF2) and the large- subunit nuclear ribosomal DNA (LSU). Le Gall et al. (2008) again resolved six fully supported family-level lineages. Although their newly described Fryeellaceae was strongly allied to the sister families Faucheaceae and Lomentariaceae, the relationships among the remaining families remained inconclusive (Le Gall et al. 2008).

The inability of the previous molecular phylogenetic analyses to generate meaningful interfamilial support was due at least in part to the general difficulty of 35

obtaining strong phylogenetic signal for lineages that have diverged deeper in evolutionary time (Verbruggen et al. 2009, Pisani et al. 2012, Zeng et al. 2014). Although a common approach for improving phylogenetic inference is to use larger datasets

(Holton & Pisani 2010), increasing the number of taxa and genes does not guarantee that signal at deeper epochs of interest will be enhanced and this approach may actually increase the potential for systematic errors (Jeffroy et al. 2006, Sperling et al. 2009, Pick et al. 2010). One of the major challenges for resolving deep evolutionary relationships is overcoming issues associated with substitution saturation at quickly evolving sites

(Cocquyt et al. 2010). Typically deep phylogenetic nodes are recorded in the historical signal of the more slowly evolving positions in an alignment; however, the stochastic noise introduced by quickly evolving sites can overwhelm this signal, which can lead to incorrect or poorly supported deep phylogenetic nodes (Ho & Jermiin 2004, Kostka et al.

2008). Fast site removal (site-stripping) is one technique that has been applied to reduce issues associated with systematic errors and substitution saturation to facilitate resolution of deeper relationships (Ruiz-Trillo et al. 1999, Morgan-Richards et al. 2008, Cocquyt et al. 2010). By removing quickly evolving sites, the signal to noise ratio is increased so that the signal of the historically informative more slowly evolving sites is maximized

(Brinkmann & Philippe 1999, Waddell et al. 1999, Delsuc et al. 2005, Rodríguez-

Ezpeleta et al. 2007, Kostka et al. 2008). The command line program SiteStripper

(Verbruggen 2012) was originally designed to better resolve deep nodes within the

Ulvophyceae (Chlorophyta). SiteStripper systematically removes variable sites from an alignment, 5% each time, to create a series of progressively more conservative sub- alignments that can be analyzed by phylogenetic inference techniques (Cocquyt et al. 36

2010). One of those sub-alignments should have enough of the quickly evolving sites removed while retaining sufficient signal to elucidate deeper relationships (Cocquyt et al.

2010).

The goals of the present study were: 1) to reassess and improve rhodymenialean systematics by expanding taxonomic representation (in conjunction with the Red Algal

Tree of Life project, http://dblab.rutgers.edu/redtol/home.php) and generating a more robust multigene dataset of mitochondrial (COB & COI/COI-5P), nuclear (EF2 & LSU) and plastid (psbA & rbcL) markers; 2) to minimize phylogenetic inference problems associated with substitution saturation and improve resolution at interfamilial nodes by assessing this dataset with a fast-site removal reconstruction technique (SiteStripper); 3) to revisit the classification system proposed by Le Gall et al. (2008) based on our generated topology; 4) to establish the phylogenetic affinities of some genera

(Binghamiopsis, Chamaebotrys, and Minium) absent from previous large-scale phylogenetic studies; 5) to assess genera consistently polyphyletic in published molecular analyses (notably Erythrymenia and Lomentaria); 6) to reinvestigate the taxonomic position of the problematic genus Drouetia; and, 7) to characterize novel taxa included in our analyses.

Materials and Methods

Phylogenetic analyses

Specimens used in phylogenetic analyses (Appendix A, Table S1) were pressed on herbarium paper or dried in silica to serve as vouchers with subsamples preserved in 37

silica for molecular analyses. Genomic DNA was extracted following Saunders &

McDevit (2012) with amplification of the following markers following Saunders &

Moore (2013): the mitochondrial cytochrome b gene (COB); the mitochondrial cytochrome c oxidase subunit 1 extended fragment or barcode region (COI and COI-5P, respectively); the nuclear elongation factor 2 gene (EF2); the nuclear large-subunit ribosomal RNA gene (LSU); the plastid photosystem II thylakoid membrane protein D1

(psbA); and the plastid ribulose-1, 5-biphosphate carboxylase large subunit gene (rbcL)

(Appendix A, Table S1). Amplicons were sequenced by the Génome Québec Innovation

Centre and raw data were edited in Sequencher™ 5.0 (Gene Codes Corporation, Ann

Arbor, MI, USA) and aligned in Geneious R7 version 7.1.7 (http://www.geneious.com,

Kearse et al. 2012) with additional sequences acquired from GenBank (Appendix A,

Table S1).

Six single-gene alignments were produced [COB (49% of ingroup taxa, 942 bp);

COI/COI-5P (90% of ingroup taxa, 1232 bp); EF2 (72% of ingroup taxa, 1641 bp); LSU

(100% of ingroup taxa, 2588 of 2841bp included in analyses,); psbA (62% of ingroup taxa, 953 bp) and rbcL (89% of ingroup taxa, 1358 bp); (Appendix A, Table S1)] and analyzed independently using Bayesian inference and Maximum likelihood under the

GTR+I+G model with partitioning by codon for protein-coding genes. Bayesian analyses were completed using MrBayes v.3.2.1 (Ronquist & Huelsenbeck 2003) in Geneious R7 version 7.1.7 with parameter settings (Tratio, Revmat, Statefreq, Pinvar, Shape,

Switchrates) unlinked, rate differentiation across the partitions enabled and branch lengths unconstrained with the branch length priors set to exponential (default exponential prior setting: 10.0). Analyses were run in parallel for 1,000,000 generations 38

with sampling performed every 100 generations. The burn-in was determined when both analyses converged into the stationary phase. Maximum likelihood analyses were performed using RAxML in Geneious R7 version 7.1.7 with a non-parametric bootstrap of 1,000 replicates.

All genes resolved congruent phylogenetic relationships and were combined to generate a multigene alignment (8,714 bp, 78% complete by site) for further phylogenetic analyses. Percent completeness for each taxon included in this study was recorded in

Table S1 (Appendix A). The multigene alignment was analyzed by Bayesian inference and Maximum likelihood under the GTR+I+G model with parameters settings as specified above with the exception of running Bayesian analyses for 3,500,000 generations with sampling performed every 3,500 generations. The impact of partitioning was evaluated by completing analyses first by partitioning data by gene and codon

(noPF) and then according to the evolutionary models and partitioning schemes determined by implementing PartitionFinder (PF) (Lanfear et al. 2012) under the BIC model selection criteria with linked branch length estimation. To assess the impact of missing data, Bayesian and Maximum likelihood analyses were repeated after first removing taxa less than 50% (n= 8) complete by site and then removing taxa less than

70% (n= 25) complete by site.

Site-Stripping

To assess the impact of substitution saturation on phylogenetic inference, site-specific rates were calculated for the multigene alignment using the program HyPhy (Pond et al.

39

2005) under the JC69 model with a Bayes phylogram as a guide tree. SiteStripper

(Verbruggen 2012) was used to order sites by rate and then remove quickly evolving sites, 5% at a time, to generate a series of progressively more conservative sub- alignments that were analyzed using RAxML version 7.3.5 (Stamatakis 2012) with a command line script available through SiteStripper. Analyses were partitioned by gene and then codon (noPF) under a GTR+I+G model and with the partitioning schemes and models of evolution as determined by PartitionFinder (PF). Branch support was estimated by 1,000 non-parametric bootstrap replicates.

To determine if site-stripping biases downstream analyses towards the phylogeny used as a guide tree, we recalculated site rates using an alternative starting tree topology

(Appendix A, Supplementary Fig. S1) that was generated using a neighbor-joining analysis under the HKY genetic distance model in Geneious R7. The resulting site rates were used by SiteStripper to generate a series of subalignments, which were analyzed by

RAxML. To assess the neighbor-joining tree against the original starting tree (Bayesian

Inference tree with partitioning by gene and codon), the Shimodaira-Hasegawa test (“-f h” option) was implemented using RAxML.

Barcode analyses

A total of 16 collections field identified to the genus Drouetia were collected from the subtidal in Coffs Harbour, New South Wales, Australia (Table 2.1) and dried in silica gel with voucher preservation, DNA extraction, and COI-5P amplification, sequencing,

40

editing and aligning as outlined above. The DNA barcode alignment (664 bp) was analyzed in BOLD to determine intraspecific variation and nearest neighbour distances.

Morphological analyses

Vegetative and reproductive features were analyzed by rehydrating algal tissue in 5% formalin and seawater and preparing sections using a freezing microtome (Leica

CM1850, Leica Microsystems, Wetzlar, Germany). Sections were stained with 1% analine blue solution in 6% 5N hydrochloric acid, rinsed with distilled water and permanently mounted in 50% corn syrup with 4% formalin. Photographs were taken using a Leica digital camera (DFC480) mounted to a Leica microscope (CTR5000) and plates were made using Adobe Photoshop Elements 11 (2012).

Results

Molecular results

To resolve subordinal relationships among the Rhodymeniales, a six gene concatenated alignment (80 species representing 42 genera) was assessed using Bayesian analysis and

Maximum likelihood (ML). Analyses were completed using the model GTR+I+G with partitioning by gene and codon. Then, analyses were performed again according to the model (GTR+I+G) and partitioning scheme (LSU) (COB1) (COB2, psbA1) (COB3,

COI3) (COI1, rbcL1) (COI2, psbA2, rbcL2) (EF21) (EF22) (EF23) (psbA3) (rbcL3) as determined by PartitionFinder. The phylogram inferred by ML analysis with partitioning

41

by gene and then codon is presented (Fig. 2.1) with bootstrap support values shown for subfamilial branches. Familial and interfamilial branch support are presented separately

(Table 2.2). The topology in Fig. 2.1 was resolved for all analyses of the full alignment except Bayesian inference with partitioning by gene and then codon, which failed to resolve branch C (lacked support in all full alignment analyses) (Table 2.2). Posterior probabilities and bootstrap support values were essentially not changed whether the alignment was fully partitioned or used the scheme selected by PartitionFinder. For both

Bayesian and ML analyses, tree scores were slightly better when data were partitioned by gene and codon rather than the partitioning schemes as defined by PartitionFinder (Table

2.2). Exclusion of incomplete taxa (both those < 50% and < 70% complete by site;

Appendix A, Table S1) did not change the tree topology (Fig. 2.1) and had little impact on posterior probabilities and bootstrap support values (data not shown). Maximum likelihood analyses of the progressively more conservative sub-alignments (partitioned by gene and codon only) produced the same tree topology (Fig. 2.1) as analyses of the full dataset; however, interfamilial branch support increased at three key branches (Table

2.1, branches B, C & I) with optimal signal gained when 20% of the quickly evolving sites were eliminated (i.e., 80% of the sites were retained). The results of the

Shimodaira-Hasegawa test indicated that the neighbor-joining guide tree topology

(likelihood -144156.83; Appendix A, Fig. S1) was significantly different (p<0.01) than the Bayes guide tree topology (likelihood -143596.69). Despite the previous, use of the neighbor-joining topology as the guide tree for calculating sites rates did not impact downstream site-stripping analyses as support for interfamilial and familial branches was consistent with SiteStripping analyses that used a Bayes guide tree. 42

All analyses (i.e., full alignment, site-stripped alignments, those for which incomplete taxa were removed) solidly resolved the Rhodymeniales (branch A, Fig. 2.1,

Table 2.2) as comprised of six monophyletic families distributed in two major lineages.

The first lineage (branch B, Fig. 2.1) was strongly resolved in all analyses and included the Faucheaceae (branch G, Fig. 2.1), Fryeellaceae (branch D, Fig. 2.1) and

Lomentariaceae (branch F, Fig. 2.1). The second lineage (branch C, Fig. 2.1) included the Champiaceae (branch J, Fig. 2.1), Hymenocladiaceae (branch K, Fig. 2.1) and

Rhodymeniaceae (branch H, Fig. 2.1) and was variably supported in analyses of the full alignment; however, support at branch C increased (up to 75%, Table 2.2) as quickly evolving sites were removed. Within the first lineage the sister relationship of the

Faucheaceae to the Lomentariaceae (interfamilial branch E, Fig. 2.1) was fully supported across all analyses (Table 2.2). Within the second lineage, the Champiaceae was moderately resolved (Table 2.2) as sister to the Hymenocladiaceae (interfamilial branch I,

Fig. 2.1). Support at branch I (Fig. 2.1, Table 2.2) initially decreased (down to 71%,

Table 2.2) when 5% of the quickly evolving sites were removed (i.e., 95% of the sites were retained) and continued to fluctuate up and down with the generation of each sub- alignment; however, support was highest (81%, Table 2.2) when 20% of the sites were eliminated.

Analyses resolved phylogenetic relationships for genera included in a molecular context for the first time. The monospecific genus Binghamiopsis was positioned within the Lomentariaceae and strongly associated with Lomentaria hakodatensis (Fig. 2.1).

Within the Rhodymeniaceae, Chamaebotrys (represented by the generitype C. boergesenii and an unidentified species from Australia) was solidly allied to the sister 43

genera Halichrysis and Halopeltis (Fig. 2.1), while collections assigned to the genus

Drouetia were resolved as a fully supported monophyletic lineage distantly related to a lineage containing species of Chrysymenia and Maripelta (Fig. 2.1). The monospecific genus Minium was fully resolved within the Fryeellaceae as an unexpectedly close sister to Fryeella gardneri (Fig. 2.1).

Inclusion of select generitype species enabled assessment of monophyly for a few genera. Within the Hymenocladiaceae, species of Erythrymenia failed to form a monophyletic lineage with Erythrymenia minuta sister to Hymenocladia spp. rather than the type of the genus, Erythrymenia obovata (Fig. 2.1). The genus Perbella was established (below) to accommodate Erythrymenia minuta and render Erythrymenia monophyletic. Within the Lomentariaceae, species of Lomentaria were resolved as four independent lineages: (i) the generitype L. articulata joined L. clavellosa and L. orcadensis to form a fully supported monophyletic lineage; (ii) L. catenata and

Ceratodictyon spp. were allied with full support; (iii) L. divaricata resolved as sister to

Stirnia; and (iv) L. hakodatensis was fully resolved as sister to Binghamiopsis (Fig. 2.1).

The genus Fushitsunagia was established to accommodate L. catenata while the generic assignments of L. divaricata and L. hakodatensis could not formally be determined at this time (discussed below).

Analyses resolved some additional genera as polyphyletic (Fig. 2.1). Rather than resolving with other species of Botryocladia, the species B. leptopoda was more closely allied with Chrysymenia wrightii (which failed to resolve with other included

Chrysymenia spp. Fig. 2.1). The species R. delicatula was resolved as relatively distinct

44

from other species of Rhodymenia (including the generitype) and was most closely related to Cordylecladia erecta (Fig. 2.1).

Sixteen collections from Coffs Harbour, New South Wales, Australia were resolved as three genetic species groups and assigned to the genus Drouetia as novel taxa

(see below). Maximum COI-5P intraspecific divergence for these species was 0.31%

(Table 2.3). The nearest neighbour to D. aggregata was D. scutellata (7.19% divergent,

Table 2.3) while the closest species to D. scutellata was D. viridescens (2.5% divergent,

Table 2.3). The species D. scutellata and D. viridescens were included in multigene analyses where they were resolved in the Rhodymeniaceae and distantly related to an unidentified species of Drouetia from South Africa (Fig. 2.1).

Taxonomic treatment

Drouetia aggregata Filloramo & G.W. Saunders, sp. nov. (Fig. 2.2)

Description. Blades irregular in outline, peltate, typically anastomosing where they overlap (Fig. 2.2a), stipe 12-15 mm long x 2 mm wide (Fig. 2.2b); blades ca. 950 µm in transverse section at mid-thallus with 9-13 medullary layers of large axially elongated, non-pigmented cells (250-400 µm long x 80-100 µm wide) with frequent secondary pit connections (Fig. 2.2c) and distinct aggregations of smaller, round cells (50 µm in diameter) (Fig. 2.2d); medullary cells typically reducing in size toward the ~2 inner cortical layers of smaller cells subtending a dorsal cortex of 2-3 layers of densely packed, columnar-shaped, darkly staining cells (~10 µm in diameter) and a single-layered ventral

45

cortex of larger round cells (15-18 µm in diameter) (Fig. 2.2c); dorsal and ventral cuticle,

10 µm and 7.5 µm thick, respectively (Fig. 2.2c). Reproductive structures not observed.

Holotype. GWS032793 collected on December 12, 2012 at Mutton Bird Island (South),

Coffs Harbour, New South Wales, Australia (-30.30467, 153.14796), by G.W. Saunders and K. Dixon and deposited in the Connell Memorial Herbarium (UNB) of the University of New Brunswick, Fredericton, Canada. Images of holotype Figs 2.2a-c.

Holotype COI-5P Barcode. KU707873.

Isotype. GWS032792 (deposited in UNB, Table 2.1).

Etymology. Named for the aggregates of small cells interspersed throughout the larger cells of the medulla – this feature being pronounced when compared to other species of

Drouetia.

Geographical Distribution. Thus far only known from the type location.

Comments. Drouetia aggregata is distinguished from the other species of Drouetia in

Australia owing to its long stipe, thick thallus in cross section, larger medullary cells, and the aggregation of small round cells interspersed among the other medullary cells.

Drouetia scutellata Filloramo & G.W. Saunders, sp. nov. (Fig. 2.3)

Description. Peltate blades depressed at center and borne on short, narrow stipes 6-10 mm long x 0.5 mm wide (Fig. 2.3a); blades typically remaining saucer-shaped upon maturity, at times anastomosing at the margins (Fig. 2.3b); blades ca. 250-375 µm thick in transverse section at mid-thallus with (3-) 4-5 medullary layers of large elongated cells

(90-145 µm long x 72-87.5 µm wide) grading to 1-2 inner cortical layers of elongated cells subtending 2 dorsal cortical layers of darkly staining cells (12.5 µm in diameter) and 46

2 ventral layers of slightly larger rounded cells (17.5 µm in diameter) (Fig. 2.3c). Plants dioecious. Cystocarps clustered centrally on dorsal (upper) blade surface (Fig. 2.3b), rounded, ostiolate, 800-1000 µm in diameter, nutritive tissue present at base (Fig. 2.3d).

Spermatangial sori formed on the dorsal surface with spermatangia cut off from elongated spermatangial mother cells (Fig. 2.3e). Tetrasporophytes not observed.

Holotype. Female gametophyte GWS032729 collected on December 11, 2012 at Mutton

Bird Island (North), Coffs Harbour, New South Wales, Australia (-30.304706,

153.150959), by G.W. Saunders and K. Dixon and deposited in the Connell Memorial

Herbarium (UNB) at the University of New Brunswick, Fredericton, Canada. Images of holotype Figs 2.3b-d.

Holotype COI-5P Barcode. KU707880

Isotype. Male gametophyte GWS032752 (deposited in UNB, Table 2.1).

Etymology. Named for the plant’s typically saucer-shaped blades.

Representative specimen examined. GWS032664 (deposited in UNB, see Table 2.1 for collection details).

Geographical Distribution. Thus far known only from the type location and nearby South

Solitary I., Coffs Harbour, New South Wales, Australia.

Comments. Drouetia scutellata features a thin thallus in cross section with few medullary cell layers rendering it rather distinct when compared to other species of Drouetia from

Australia. Differences in internal anatomy are especially useful for distinguishing between D. scutellata and D. viridescens, which also has a single peltate blade with a depressed center and short stipe. Additionally, while some reproductive plants of D.

47

scutellata have been encountered as single blades, reproductive plants of D. viridescens have only been observed as anastomosed blades (below).

Drouetia viridescens Filloramo & G.W. Saunders, sp. nov. (Fig. 2.4)

Description. Juvenile blades peltate with shallow central depression (Fig. 2.4a), becoming stellate or lobed with age and typically anastomosing at margins and where blades overlap (Fig. 2.4b), borne on a short stipes 3-6 mm long x 1 mm wide. Transverse sections at mid-thallus 310-500 µm thick with 9-12 medullary layers of large, axially elongated cells (90-140 µm long x 50-80 (-120) µm wide) decreasing in size to 2 dorsal cortical layers of small, densely packed, darkly staining cells (10-15 µm in diameter) and

1-2 ventral cortical layers of round darkly staining cells (17.5-20 µm in diameter); outgrowths of the ventral cortical cells presumably involved in secondary attachments to substratum and other blades (Figs 2.4c-d); dorsal (~7.5 µm thick) and ventral cuticles (5 um thick) obvious (Fig. 2.4c). Tetrasporangial sori confined to the dorsal surface (Fig.

2.4d), tetrasporangia cruciately divided (30-40 µm long x 15-20 µm wide) (Fig. 2.4e), differentiating from existing cortical cells (Figs 2.4f-g); all outer cortical cells appear capable of differentiating into sporangia but do not convert simultaneously causing undifferentiated cortical cells to become elongated and compressed between developing tetrasporangia giving the appearance of paraphyses (Figs 2.4f-g). Cystocarps and spermatangia not observed.

Holotype. Tetrasporophyte GWS032612 collected on December 9, 2012 in Split Solitary

Island (Northwest) Coffs Harbour, New South Wales, Australia (-30.24210, 153.17921), by G.W. Saunders and K. Dixon and deposited in the Connell Memorial Herbarium 48

(UNB) at the University of New Brunswick, Fredericton, Canada. Images of holotype

Figs 2.4b, 2.4d-g.

Holotype COI-5P Barcode. KU707855

Isotype. GWS032624 (Table 2.1).

Etymology. The species epithet acknowledges the plant’s green iridescent glow underwater observed at the time of collection.

Representative specimens examined. GWS032599, GWS032600, GWS032642,

GWS032643, GWS032665, GWS032672, GWS032682, GWS032740, GWS032790

(deposited in UNB, see Table 2.1 for collection details).

Geographical Distribution. Thus far known only from the type locality and nearby sites in Coffs Harbour, New South Wales, Australia.

Comments. This species has the shortest stipe and the center of the blade typically

“glows” green when encountered underwater. Although D. viridescens has a similar number of medullary cell layers as D. aggregata, the former is much thinner in cross section with smaller medullary cells. The upper range of thallus thickness for D. scutellata overlaps with the lower range for D. viridescens; however, the fewer number of medullary cell layers for D. scutellata distinguishes it from the latter.

Generitype observations of Drouetia coalescens (Farlow) G. De Toni (Fig. 2.5)

Holotype material of D. coalescens was provided on loan from the Farlow Herbarium

(FH) (Fig. 2.5a). Blades were ca. 500 µm thick with 8-11 medullary cell layers of typically axially elongated cells of varying sizes (Fig. 2.5b). The dorsal blade surface was ca. 3 cortical layers thick while the ventral surface was composed of up to 6 layers of 49

small, round, tightly packed cells subtending a thick cuticle (Fig. 2.5b). Tetrasporangia were observed as deeply embedded in only the dorsal cortex (Figs 2.5b-c), cruciately divided (52-55 µm long x 15-18 µm wide) (Fig. 2.5d) with the tetrasporangial initials arising from the cells of the inner cortex (Fig. 2.5e). As the tetrasporangia matured, they lengthened and swelled causing neighboring cortical cells to become compressed and elongated between the developing tetrasporangia, taking on the appearance of paraphyses

(Figs 2.5c-e).

Comments. The generitype species shares a similar gross morphology with the novel species D. aggregata and D. viridescens in that all three species feature decumbent, peltate blades, which commonly anastomose at the margins. Compared to D. aggregata, the generitype is thinner in cross section and lacks aggregations of small cells within the medulla. The generitype species features longer tetrasporangia compared to those of D. viridescens and has never been reported to “glow” green underwater as is diagnostic of

D. viridescens. Although the habitat of the type specimen of D. coalescens is unknown,

Taylor (1945) collected material from the low-intertidal that was observed by Saunders et al. (2006) who recognized it as morphologically consistent with D. coalescens.

According to the previous, D. coalescens is distinct in its intertidal habitat relative to the three Australian Drouetia species, which have only been collected subtidally.

Fushitsunagia Filloramo & G.W. Saunders, gen. nov.

Diagnosis: Lomentariaceaen algae with hollow thalli divided by multi-rowed cellular septa. Spermatangial sori are borne on specialized fertile ramuli. The cells of the medulla become elongated and stretched appearing almost filamentous as they form a 50

conspicuous stellate network surrounding the developing tetrasporangia, which, when mature, are tetrahedrally divided.

Etymology: In recognition of the Japanese name “Fushitsunagi” (“fushi”= joint, knot, and node; “tsunagi”= connection) that was assigned to Lomentaria catenata by Okamura (Lee

1978).

Type and only species: Fushitsunagia catenata (Harvey) Filloramo & G.W. Saunders, comb. nov.

Basionym: Lomentaria catenata Harvey 1857 (Algae. In: Account of the Botanical specimens. Gray, A., [Eds.] Narrative of the expedition of an American squadron to the

China Seas and Japan, performed in the years 1852, 1853 and 1854, under the command of Commodore M.C. Perry, United States Navy. Volume II – with illustrations. Anon.

[Eds.]. Senate of the Thirty-third Congress, Second Session, Executive Document. House of Representatives, Washington, USA, pp. 331-332).

Perbella Filloramo & G.W. Saunders, gen. nov.

Diagnosis: Hymenocladiaceaen algae with smaller cells intermixed among the larger medullary cells from which they likely develop. Tetrasporangia are decussately divided.

Etymology: Latin for “very beautiful”, attributed to the breathtaking appearance of the type species when observed underwater or immediately after collection.

Type and only species: Perbella minuta (Kylin) Filloramo & G.W. Saunders, comb. nov.

Basionym: Erythrymenia minuta Kylin 1931 [Die Florideenordung Rhodymeniales. Acta

Universitatis Lundensis 27(11):1-48].

51

Discussion

Interfamilial relationships

The present study represents the most comprehensive molecular phylogenetic analysis of the Rhodymeniales based on the number of taxa included and markers used. Further, we have applied a technique to remove the most rapidly evolving sites from our multigene alignment to enhance resolution at the interfamilial nodes (Fig. 2.1). Results were largely congruent with previously published phylogenetic assessments of the order (Saunders et al. 1999, Le Gall et al. 2008) in that all of our analyses resolved the Rhodymeniales as comprised of six monophyletic families (Fig. 2.1); however, those previous studies consistently resolved only an alliance between the Faucheaceae and Lomentariaceae

(Saunders et al. 1999, Le Gall et al. 2008) while the current study resolved most interfamilial relationships with solid support (Fig. 2.1, Table 2.2). The Faucheaceae and

Lomentariaceae were resolved as sister groups and allied to the Fryeellaceae while the

Champiaceae and Hymenocladiaceae were resolved together and associated with the

Rhodymeniaceae (Fig. 2.1). Site-Stripping had the greatest impact on the short internal branch (branch C, Fig. 2.1) leading to the Champiaceae, Hymenocladiaceae and

Rhodymeniaceae. It is possible that this branch represents a lineage that radiated over a short period of evolutionary time. Generally, the limited signal associated with short branches is masked by more recent substitution events, which impacts accurate resolution of evolutionary relationships occurring over those short time intervals (Pisani et al. 2012).

Removing the quickly evolving sites (i.e., those prone to substitution saturation) from our original alignment reduced the stochastic noise and uncovered the historical signal. We

52

were able to provide meaningful resolution for previously unresolved relationships within the Rhodymeniales. The interfamilial relationships resolved herein were consistent with the classification system proposed by Le Gall et al. (2008) emphasizing ontogeny of the tetrasporangia.

Taxa included in molecular analyses for the first time

Molecular data solidly resolved the monospecific genus Binghamiopsis within the

Lomentariaceae (Fig. 2.1), which was expected given that it has the diagnostic characteristics of this family (tetrahedrally divided tetrasporangia, three-celled carpogonial branches and characteristic lomentariaceous fusion cell in carposporophyte development, Lee et al. 1988, Saunders et al. 1999, Le Gall et al. 2008). The only species,

B. caespitosa, was closely allied to Lomentaria hakodatensis (Fig. 2.1). While

Binghamiopsis features thalli with a multilayered cortex and loosely interwoven medullary filaments in a central cavity (Lee et. al. 1988), L. hakodatensis is characterized by thalli with cortical and medullary layers surrounding a central cavity that is articulated at various intervals by multi-rowed cellular septa (Lee 1978). As L. hakodatensis was polyphyletic relative to Lomentaria sensu stricto, its proper generic-level assignment awaits future assessments of Lomentaria (see below); however, such substantial differences in thallus construction between B. caespitosa and L. hakodatensis warrant their recognition as belonging to distinct genera.

The generitype of Chamaebotrys, C. boergesenii, was resolved within the

Rhodymeniaceae (Fig. 2.1) where it was placed by Huisman (1996) when he established

53

this genus to accommodate some taxa originally assigned to another rhodymeniaceaen genus, Coelarthrum. Based on the species included here, his decision to recognize

Chamaebotrys was fully supported (Fig. 2.1). Unfortunately, our analyses did not include the generitype of Coelarthrum, C. cliftonii, and therefore, the distinction of Coelarthrum and Chamaebotrys remains untested by molecular analyses.

At the time that Moe (1979) described the only crustose rhodymenialean taxon,

Minium parvum, only two families (Champiaceae and Rhodymeniaceae) were recognized in the order. The criteria delimiting these families was greatly contested and poorly understood and Moe (1979) struggled to assign the monospecific genus Minium to either group especially after observing a variety of characteristics (tetrasporangial nemathecia, auxiliary cell branches with their associated sterile cells and the arrangement of the cells in the three-celled carpogonial branch) that did not ally Minium to either family. Moe

(1979) determined that the genus was best placed in the Rhodymeniaceae on account of a solid thallus construction, cruciately divided tetrasporangia and the overall appearance and development of the carposporophyte. Our molecular results solidly resolved Minium within the Fryeellaceae as an unexpected close sister to Fryeella gardneri (Fig. 2.1).

Analysis of tetrasporangial collections will confirm if one of the diagnostic features of the Fryeellaceae, the adventitious growth of the cortex in the formation of tetrasporangia and short, 3-4 celled paraphyses, is also characteristic of Minium. Those observations will be included in a future manuscript specific to rhodymenialean diversity in Canada.

54

Assessment of select polyphyletic genera

The present study was consistent with a previous molecular study that failed to resolve the type of the genus Erythrymenia, E. obovata from South Africa, and E. minuta from

Australia as monophyletic (Saunders et al. 2006). To accommodate E. minuta we established the novel monospecific genus Perbella, which was solidly resolved as sister to Hymenocladia (Fig. 2.1). The latter two genera are distinguished primarily by tetrasporangial division patterns, which are tetrahedral in Hymenocladia and cruciate in

Perbella, respectively. Additionally, Perbella is characterized by obovate blades while

Hymenocladia features pinnate blades (e.g., H. usnea) or complanate, broad, palmate, subdichotomously divided blades (e.g., H. chondricola, Womersley 1996). Nonetheless, we uncovered inconsistencies and taxonomic confusion in the literature related to the identification of E. minuta (= P. minuta) relative to species of Hymenocladia. Specimens of H. conspersa collected by Harvey from Garden Island, Western Australia (Trinity

College Dublin Herbarium, TCD) included foliose, irregularly divided plants with abundant laminar and marginal tooth-like proliferations [Harvey 132, TCD, annotated

“type” by Womersley, lectotypified by Lewis (1994)] and separately mounted smaller obovate plants (Harvey 300-A, TCD). Harvey (1862) considered the former to be mature

H. conspersa while the latter were juveniles. Additional juvenile specimens were collected from Port Fairy, Victoria (e.g., Harvey 300-D, TCD). Lewis (1994) and

Womersley (1996) concluded that Harvey’s “juveniles” were actually E. minuta (= P. minuta). Further, they speculated that the juvenile H. conspersa specimens (that they attributed to E. minuta), reportedly from Western Australia, were likely collected from

Port Fairy, Victoria (Lewis 1994). Owing to the previous, Lewis (1994) and Womersley 55

(1996) interpreted H. conspersa as limited to Western Australia with E. minuta (= P. minuta) restricted to Victoria.

As part of an ongoing barcode (COI-5P) survey of southern Australia (South

Australia, Victoria and Tasmania), three genetic species groups were resolved for

Hymenocladia including H. chondricola, H. conspersa and H. usnea. The first two were closely related species groups (1.24% maximum interspecific variation in COI-5P) and are in need of further molecular study. Our specimens identified as H. chondricola sensu stricto (Fig. 2.6a) were restricted to South Australia while collections attributed to H. conspersa were more widely distributed from South Australia, Tasmania and Victoria and included mature lanceolate morphs (Fig. 2.6b), intermediate morphs (Fig. 2.6c) and small, obovate juvenile morphs (Fig. 2.6d) similar to those collected by Harvey and superficially reminiscent of P. minuta specimens (Fig. 2.6e). As the type of H. conspersa is from Western Australia and our isolates from southern Australia lacked the laminar papillae considered by Harvey (1862) to be characteristic of this species (Fig. 2.6b), our assignment of H. conspersa remains tentative pending inclusion of topotype material. If our collections do represent H. conspersa, we provide genetic evidence that this species is indeed found in southern Australia and that small plants resembling P. minuta should be considered with caution as they are potentially juveniles of H. conspersa.

Previous and current phylogenetic assessments have consistently resolved the speciose genus Lomentaria as polyphyletic (e.g., Le Gall et al. 2008). Our molecular analyses resolved species assigned to Lomentaria as four independent lineages in the

Lomentariaceae (Fig. 2.1). The type of Lomentaria, L. articulata, was joined by L. clavellosa and L. orcadensis (Fig. 2.1). As L. articulata features branches with 56

multilayered septa while L. clavellosa and L. orcadensis have multilayered plugs only at the branch base, results herein do not support previously recommended revisions of this genus based on thallus construction and septation (Irvine & Guiry 1983). Thus,

Lomentaria sensu stricto remains a broadly defined genus characterized by various modes of thallus construction, tetrahedrally divided tetrasporangia in invaginated sori and conical cystocarps with prominent pores (Lee 1978).

The novel genus Fushitsunagia was established to accommodate L. catenata, which was resolved as sister to Ceratodictyon (Fig. 2.1). These genera are distinguished by tetrasporangial division patterns, which are tetrahedral in F. catenata (Lee 1978) and cruciate in Ceratodictyon (Price & Kraft 1991). The genus Fushitsunagia is unique from other lomentariaceaen taxa in that during tetrasporangial development, medullary cells become modified and form a conspicuous filamentous network that surrounds the developing tetrasporangia (Lee 1978).

The remaining Lomentaria species included in our multigene analyses, L. divaricata and L. hakodatensis, were resolved as sister to Stirnia and Binghamiopsis, respectively (Fig. 2.1). Lomentaria divaricata is distinguished from Stirnia according to thallus structure (hollow in the former and solid in the later) and the respective production of spermatangia and tetrasporangia on specialized fertile proliferations described only for the latter genus (Wynne 2001). Lomentaria hakodatensis is separated from Binghamiopsis based on overall thallus construction (see above). There are two available names to accommodate L. divaricata and L. hakodatensis: Hooperia J. Agardh

(1896), which would apply directly to its generitype taxon L. divaricata and,

Chondrosiphon Kützing (1843) for which the generitype species, L. firma, was not 57

available for inclusion in our molecular analyses. As L. firma shares numerous vegetative and reproductive characteristics with L. hakodatensis (flattened thalli, anastomosed intricate branches and well-developed cortication formed of a single layer of pigmented cells and 3-4 inner layers of subcortical cells, Curiel et al. 2006) it seems likely that these taxa will resolve together in the resurrected genus Chondrosiphon; however, it is possible that L. firma will resolve with L. divaricata, which would render Hooperia a synonym of

Chondrosiphon necessitating a new genus for L. hakodatensis. Until L. firma is molecularly assessed, L. divaricata and L. hakodatensis should remain incertae sedis in the Lomentariaceae. While we have established Lomentaria sensu stricto as a monophyletic lineage and clarified some of its taxonomic conflicts, there remains significant molecular and morphological work until this genus and the many species attributed to it are properly assigned.

Recent molecular assessment of Australian species assigned to Gloiosaccion [G. brownii Harvey (including G. brownii var. firmum and G. brownii var. coriaceum) and G. pumilum J. Agardh], in relation to species of Chrysymenia [including the generitype species C. ventricosa (J.V. Lamouroux) J. Agardh] warranted transfer of the former genus to the latter (Schmidt et al. 2016). In that study, “G.” brownii var. firmum

(collected from South Australia) was supported as distinct from G.” brownii var. coriaceum (collected from Western Australia) and the latter variety was raised to specific rank (Schmidt et al. 2016). Our routine barcoding studies in Australia have resolved collections (n= 31) field assigned to “G”. brownii in five genetically distinct morphologically cryptic species groups (data not shown). Two of those groups (one being our concept of “G.” brownii, which included specimens collected from near the type 58

locality, Tasmania) were included in the current study (Fig. 2.1; Appendix A, Table S1).

The rbcL data from our two “G.” brownii genetic groups did not match the GenBank rbcL data representative of “G.” brownii or “G.” coriacea from the Schmidt et al.

(2016) study and further study of “G.” brownii from Australia is needed.

Consistent with Le Gall et al. (2008), our analyses resolved the genus

Chrysymenia as polyphyletic with one of the species, C. wrightii most closely related to

Botryocladia leptopoda (rendering this genus polyphyletic as well). Assessment of that conundrum was beyond the scope of this study. Future molecular assessments including the generitype taxa for Botryocladia, Chrysymenia and Cryptarachne (previously recognized as a subgenus or a section of Chrysymenia, Guiry & Guiry 2015) are needed to clarify the species appropriately assigned to those genera.

Former morphological and molecular assessments of rhodymenialean taxa also identified the close alliance between Cordylecladia erecta and species of Rhodymenia, which are distinguished primarily according to habit (Brodie & Guiry 1988, Millar et al.

1996, Saunders et al. 1999). The addition of R. delicatula here goes further by resolving as sister to C. erecta and calling into question monophyly of Rhodymenia (Fig. 2.1).

While C. erecta features fully terete axes, species of Rhodymenia are typically compressed or foliose; however, the recent description of the novel Australian species R. compressa, which features compressed and sparsely branched thalli (Filloramo &

Saunders in press), challenges the utility of thallus habit for distinguishing C. erecta from

Rhodymenia spp. and more study is needed.

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Taxonomic reassessment of Drouetia

Prior to this study inclusion of the monotypic genus Drouetia in molecular analyses has been uncertain due to frequent misidentifications of numerous taxa currently assigned to

Asteromenia and Halichrysis as the reportedly cosmopolitan generitype of Drouetia, D. coalescens (Saunders et al. 2006). A recent study resolved material from South Africa that was vegetatively representative of Drouetia and identified as “D. coalescens” as an independent lineage in the Rhodymeniaceae (Saunders et al. 2006). In that study,

Saunders et al. (2006) viewed a prepared mica slide of holotype tetrasporangial material for the generitype, D. coalescens, and reported what they interpreted as adventitious growth of the cortex to form nemathecia of paraphyseal filaments and tetrasporangia on the dorsal surface only. Those observations were inconsistent with reports of tetrasporangial development for the South African samples (terminal tetrasporangia on both surfaces, Norris 1991) which, as a result, were not regarded as representative of D. coalescens (Saunders et al. 2006). The present study resolved three novel species from

Australia as morphologically assignable to the genus Drouetia (none were consistent with

D. coalescens) and two of these were included in multigene phylogenetic analyses where they were resolved in the Rhodymeniaceae sister to the previously discussed South

African collection (Fig. 2.1). Our observation of tetrasporangial development for the novel species D. viridescens did not suggest adventitious cortical growth in the formation of the tetrasporangial nemathecia (i.e., true paraphyses were absent) as reported by

Saunders et al. (2006) for the generitype, which prompted re-examination of holotype material of D. coalescens to clarify tetrasporangial development for this genus. That material was provided on loan from the Farlow Herbarium (FH) and freshly prepared 60

sections were examined. We observed cruciately divided tetrasporangia deeply embedded in the dorsal cortex only (Figs 2.5b-c). Tetrasporangial initials developed from the cells of the inner cortex and during maturation, became elongated and swollen, which resulted in neighbouring cortical cells becoming compressed and elongated between the developing tetrasporangia, taking on the appearance of paraphyses (Figs 2.5c-e). This mode of development was more consistent with that observed in D. viridescens and other rhodymeniaceaen taxa, which are typically characterized by tetrasporangia that differentiate from pre-existing cortical cells and lack true adventitious filaments

(paraphyses) (Le Gall et al. 2008, Saunders & McDonald 2010). More study of tetrasporangial development for the South African collections is needed to determine if previous reports of tetrasporangial sori occurring on both sides of the blade (Norris 1991) are incorrect and confounded by taxonomic misidentifications or if that species has an atypical mode of development. Either way, the South African species represents a fifth species best assigned to Drouetia, a conclusion that ultimately awaits inclusion of D. coalescens in phylogenetic analyses to provide molecular confirmation, as well as our decision to assign the three Australian species D. aggregata, D. scutellata and D. viridescens to Drouetia as distinct from the generitype.

Conclusions

The intention of this study was to enhance understanding of evolutionary relationships in the Rhodymeniales. Fast-site removal successfully improved resolution at a key node and provided clarification of interfamilial relationships. By expanding taxonomic

61

representation we have addressed the phylogenetic affinities of rhodymenialean genera absent from previous molecular assessments and have clarified two consistently polyphyletic genera. Additionally, re-examination of holotype material for Drouetia coalescens clarified rhodymeniacean tetrasporangial development for the genus and provided evidence that the three novel Australian species are correctly assigned to this genus, and that this genus is correctly assigned to the Rhodymeniaceae.

Acknowledgements

We thank all of the collectors for their efforts and contributions to this study

(http://www.boldsystems.org/index.php/MAS_Management_OpenDataSet?datasetcode=

DS-RHDYPHY). Thank you to Chris Lane and Thea Popolizio for supplying collections of L. divaricata from Rhode Island. Our sincerest gratitude to Genevieve Tocci at the

Farlow Herbarium for organizing a loan of D. coalescens type material. Many thanks to former and current members of the Saunders lab who generated some of the sequence data especially K. Dixon, L. Le Gall, D. McDevit and T. Moore. Thank you to C.W.

Schneider for his assistance with Latin nomenclature. Thank you to Hiroshi Kawai for his assistance translating Japanese names assigned to species of Lomentaria. Support for this research was provided by the Canada Research Chair Program, the Canada Foundation for Innovation, the New Brunswick Innovation Foundation, the Red Algal Tree of Life project (part of the National Science Foundation Assembling the Tree of life activity;

AToL), the Canadian Barcode of Life Network and Genome Canada through the Ontario

Genomics Institute and Natural Sciences and Engineering Research Council of Canada.

62

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resurrection of Halopeltis J. Agardh and description of Pseudohalopeltis gen. nov. Botany 88:639-67. Saunders, G. W. & Moore, T. E. 2013. Refinements for the amplification and sequencing of red algal DNA barcode and RedToL phylogenetic markers: a summary of current primers, profiles and strategies. Algae 28:31-43. Saunders, G. W., Lane, C. E., Schneider, C. W. & Kraft, G. T. 2006. Unravelling the Asteromenia peltata species complex with clarification of the genera Halichrysis and Drouetia (Rhodymeniaceae, Rhodophyta). Can. J. Bot. 84:1581-607. Saunders, G. W., Strachan, I. M. & Kraft, G. T. 1999. The families of the order Rhodymeniales (Rhodophyta): a molecular-systematic investigation with a description of Faucheaceae fam. nov. Phycologia 38:23-40. Schmidt, W.E., Gurgel, C.F.D. & Fredericq, S. 2016. Taxonomic transfer of the red algal genus Gloiosaccion to Chrysymenia (Rhodymeniaceae, Rhodymeniales), including the description of a new species, Chrysymenia pseudoventricosa, for the Gulf of Mexico. Phytotaxa 243: 54-70. Schmitz, F. 1889. Systematische ubersicht der bisher bekannten gattungen der Florideen. Flora 72:435-56. Sjöstedt, L. G. 1926. Floridean studies. Acta. Univ. Lundensis. 22:1-95. Stamatakis, A., Hoover, P. & Rougemont, J. 2008. A rapid bootstrapping algorithm for the RAxML web-servers. Syst. Biol. 57:758-71. Sparling, S. R. 1957. The structure and the reproduction of some members of the Rhodymeniaceae. Univ. Calif. Publ. Bot. 29:319-96. Sperling, E. A., Peterson, K. J. & Pisani, D. 2009. Phylogenetic-signal dissection of nuclear housekeeping genes supports the paraphyly of sponges and the monophyly of Eumetazoa. Mol. Biol. Evol. 26:2261-74. Taylor, W.R. 1945. Pacific marine algae of the Allan Hancock Expeditions to the Galapagos Islands. Allan Hancock Pac. Exped. 12:1-528. Verbruggen, H. 2012. SiteStripper v.1.01. GNU General Public License. http://www.phycoweb.net/software/SiteStripper/index.html Verbruggen, H., Ashworth, M., LoDuca, S. T., Vlaeminck, C., Cocquyt, E., Sauvage, T., Zechman, F. W., Littler, D. S., Littler, M. M., Leliaert, F. & De Clerck, O. 2009. A multi-locus time-calibrated phylogeny of the siphonous green algae. Mol. Phylogenet. Evol. 50:642-53. Verbruggen, H., Maggs, C. A., Saunders, G. W., Le Gall, L., Yoon, H. S. & De Clerck, O. 2010. Data mining approach identifies research priorities and data requirements for resolving the red algal tree of life. BMC Evol. Biol. 10:16. Waddell, P. J., Cao, Y., Hauf, J. & Hasegawa, M. 1999. Using novel phylogenetic methods to evaluate mammalian mtDNA, including amino acid-invariant sites- LogDet plus site stripping to detect internal conflicts in the data, with special reference to the positions of hedgehog, armadillo, and elephant. Syst. Biol. 48:31- 53. Withall, R. & Saunders, G. W. 2006. Combining small and large subunit ribosomal DNA genes to resolve relationships among orders of Rhodymeniophycidae (Rhodophyta): recognition of the Acrosymphytales ord. nov. Eur. J. Phycol. 41:379-94. 66

Womersley, H. B. S. 1996. The marine benthic flora of southern Australia – Part IIIB- Gracilariales, Rhodymeniales, Corallinales and Bonnemaisoniales. Vol. 5. Australian Biological Resources Study & the State Herbarium of South Australia, Canberra & Adelaide, 392 pp. Wynne, M. J. 2001. Stirnia prolifera gen. et sp. nov. (Rhodymeniales, Rhodophyta) from the Sultanate of Oman. Bot. Mar. 44:163-9. Zeng, L., Zhang, Q., Sun, R., Kong, H., Zhang, N. & Ma, H. 2014. Resolution of deep angiosperm phylogeny using conserved nuclear genes and estimates of early divergence times. Nat. Commun. 5:1-12.

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Table 2.1 Collection details and COI-5P GenBank accession numbers for Australian Drouetia samples used in molecular and morphological analyses. All samples were collected from Coffs Harbour, New South Wales, Australia. All data were generated for the present study.

Voucher COI-5P GenBank Species Number Habitat Geography Accession Drouetia aggregata GWS032792 4 m on rock Mutton Bird Island (South) KU707859 Filloramo & G.W. Saunders GWS032793 4 m on rock Mutton Bird Island (South) KU707873 Drouetia scutellata GWS032664 6 m on invert South Solitary Island (Southeast) KU707850 Filloramo & G.W. Saunders GWS032729 5 m on rock Mutton Bird Island (North) KU707846 GWS032752 5 m on invert Mutton Bird Island (North) KU707880 Drouetia viridescens GWS032599 5 m on rock Korora Beach KU707869 Filloramo & G.W. Saunders GWS032600 5 m on rock Korora Beach KU707868 GWS032612 8 m on rock Split Solitary Island (Northwest) KU707855 GWS032624 8 m on rock Split Solitary Island (Northwest) KU707881 GWS032642 10 m on invert Split Solitary Island (East) KU707878 GWS032643 10 m on coral rubble South Solitary Island (East) KU707874 GWS032665 6 m on invert South Solitary Island (Southeast) KU707863 GWS032672 10 m on invert Black Rock KU707870

68 GWS032682 6 m on invert Black Rock KU707847

Voucher COI-5P GenBank Species Number Habitat Geography Accession GWS032740 5 m on invert Mutton Bird Island (North) KU707852 GWS032790 4 m on rock Mutton Bird Island (South) KU707872

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Table 2.2 Bayesian posterior probabilities, RAxML bootstrap values and likelihood scores for the full multigene alignment with partitioning by gene and then codon (noPF) and with partitioning as determined by PartitionFinder (PF). RAxML bootstrap values and likelihood scores for the site-stripper subalignments with partitioning by gene and then codon (noPF).

Dashes (-) indicate branch support below 50% and n/a indicates a branch that was not resolved.

Bayes RAxML RAxML % of sites 100% 95% 90% 85% 80% 75% 70% retained ML noPF PF noPF PF noPF s cores -143267 -143395 -143595 -143787 -112089 -86686 -65296 -47116 -32139 -20852

A 1 1 100 100 100 100 100 100 100 100 B B 1 1 95 95 93 95 97 99 98 79 R C n/a - - 50 n/a 51 68 75 65 62 A D 1 1 100 100 100 100 100 100 100 100 N E 1 1 100 100 100 100 100 100 100 100 C F 1 1 100 100 100 100 100 100 100 100 H G 1 1 100 100 100 100 100 100 100 98 E H 1 1 100 100 100 100 100 100 100 100 S I 1 1 79 80 71 78 71 81 71 80 J 1 1 100 100 100 100 100 100 100 100 K 1 1 100 100 100 100 100 100 100 100

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Table 2.3 Intraspecific COI-5P divergence and distance to nearest neighbour for Australian species of Drouetia included in this study.

Max intraspecific Nearest Distance to nearest

Species divergence (%) species neighbour (%)

D. aggregata (n= 2) 0 D. scutellata 7.19

D. scutellata (n= 3) 0.15 D. viridescens 2.5

D. viridescens (n= 11) 0.31 D. scutellata 2.5

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Figure 2.1 RAxML phylogeny for full multigene alignment partitioned by gene and then codon with subfamilial bootstrap support values appended. Branch letters correspond to the support values in Figure 2.2. Taxa in bold represent generitype species. Asterisks denote fully supported branches. Species transferred to new genera have the previous genus name in parentheses. Outgroup species of the Halymeniales and Sebdeniales were 72 compressed to ordinal level to facilitate presentation.

Figure 2.2 Drouetia aggregata Filloramo & G.W. Saunders sp. nov. a. Top view of vegetative holotype (GWS032793). Scale bar = 8.75 mm. b. Side view of vegetative holotype with distinct stipes subtending anastomosed blades

(GWS032793). Scale bar = 8 mm. c. Transverse section at mid-thallus showing medulla and dorsiventral aspect (GWS032793). Scale bar = 200 µm. d.

Transverse section at mid-thallus showing ventral surface and aggregates of small cells interspersed among the larger cells of the medulla (GWS032792).

Scale bar = 100 µm.

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Figure 2.3 Drouetia scutellata Filloramo & G.W. Saunders sp. nov. a. Habit of single peltate blades showing distinct stipes and central depressions, lighter patches indicate spermatangial sori (arrows) (Isotype, GWS032752). Scale bar = 1 cm. b. Habit of cystocarpic holotype (GWS032729). Scale bar = 1 cm. c.

Transverse section at mid-thallus showing medulla and dorsiventral aspect

(GWS032729). Scale bar = 100 µm. d. Transverse section through mature cystocarp (GWS032729). Scale bar = 250 µm. e. Transverse section of male plant showing spermatangial development on the dorsal surface (GWS032752). Scale bar = 10 µm.

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Figure 2.4 Drouetia viridescens Filloramo & G.W. Saunders sp. nov. a. Habit of single peltate blades attached to substratum by single stipe (not pictured)

(GWS032740). Scale bar = 1 cm.b. Habit of tetrasporic holotype showing anastomosed blades that form an irregular outline (GWS032612). Scale bar = 1 cm. c. Transverse section at mid-thallus of vegetative plant (GWS032682). Scale bar =

93 µm. d. Transverse section at mid-thallus of tetrasporic holotype (GWS032612).

Scale bar = 108 µm. e. Mature cruciately divided tetrasporangium (GWS032612).

Scale bar = 33 µm. f. Transverse section at mid-thallus of tetrasporic holotype showing cortical cell (arrow) compressed between developing and mature tetrasporangia creating the appearance of paraphyses. Tetrasporangial initials pit connected (arrowheads) to inner cortical cell (GWS032612). Scale bar = 25 µm. g.

Intercalary tetrasporangial initial pit connected (arrowheads) to two neighboring cortical cells (GWS032612). Scale bar = 20 µm.

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Figure 2.5 Tetrasporangial holotype of Drouetia coalescens (Farlow) G. De Toni

(Harvard University Herbaria & Libraries, No. 00777108). a. Herbarium label and gross morphology of holotype. Scale bar = 21 mm. b. Transverse section of tetrasporangial thallus showing dorsal tetrasporangial sorus. Scale bar = 91 µm. c.

Developing tetrasporangia deeply embedded in the dorsal cortex. Scale bar = 47 µm. d. Cruciately divided tetrasporangia (arrows). Scale bar = 23 µm. e. Pit connection

(arrow) between two cortical cells modified into tetrasporangia surrounded by compressed cortical cells creating the appearance of paraphyses. Scale bar = 18 µm.

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Figure 2.6 Hymenocladia chondricola (Sonder) J. Lewis, Hymenocladia conspersa

(Harvey) J. Agardh and Perbella minuta (Kylin) Filloramo & G.W. Saunders comb. nov. a. Gross morphology of a vegetative specimen of H. chondricola from South

Australia (GWS029560). Scale bar = 25 mm. b. Mature morphology of a vegetative specimen of H. conspersa from Victoria (GWS017300). Scale bar = 25 mm. c.

Intermediate morphology of vegetative specimen of H. conspersa from Tasmania

(GWS015238). Scale bar = 19 mm. d. Juvenile morphology of vegetative specimen of

H. conspersa from Tasmania (GWS016539). Scale bar = 13 mm. e. Gross morphology of a vegetative specimen of Perbella minuta from Tasmania

(GWS015206). Scale bar = 25 mm.

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Chapter 3 Assessment of the order Rhodymeniales (Rhodophyta) from

British Columbia using an integrative taxonomic approach reveals

overlooked and cryptic species diversity

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Abstract

Molecular-assisted alpha taxonomy using COI-5P and rbcL-3P was employed to reassess species diversity for the Rhodymeniales (Rhodophyta) in British Columbia. A total of

563 collections from British Columbia were resolved as 16 genetic species groups, whereas 13 were previously reported. Collections attributed to B. pseudodichotoma

(Farlow) Kylin from British Columbia were resolved as distinct from collections of that species from California (type locality) and were assigned to B. hawkesii sp. nov.

Molecular data resolved an additional species of Fryeella for which the combination F. callophyllidoides (Hollenberg & I.A.Abbott) comb. nov. (for Rhodymenia callophyllidoides Hollenberg & I.A.Abbott) was established. Although two species of

Gloiocladia were recognized, genetic analyses resolved three: G. fryeana (Setchell)

N.Sánchez & Rodríguez-Prieto, G. laciniata (J.Agardh) N.Sánchez & Rodríguez-Prieto and G. vigneaultii sp. nov. Data also resolved G. media (Kylin) comb. nov. from

California. For the genus Rhodymenia, where two species were expected, molecular data resolved four. Both R. californica Kylin and R. pacifica Kylin were confirmed in British

Columbia, while some collections field identified as R. californica were genetically distinct and assigned to the novel species, R. bamfieldensis sp. nov. Some collections initially identified as R. pacifica were also resolved as a distinct group and assigned to the resurrected species R. rhizoides E.Y.Dawson. Anatomical development for the monospecific genus Minium was also reassessed in light of that taxon’s assignment to the Fryeellaceae. Our investigation clarified the number of rhodymenialean species in

British Columbia and resolved taxonomic and distributional uncertainties associated with some of these taxa. 83

Introduction

Bordered by the North Pacific, British Columbia is the westernmost province in Canada and is regarded as one of the most biologically diverse provinces in the country (Hebda

2007). Included in this province is Haida Gwaii, an archipelago situated west of the mainland and identified as a past glacial refugium (Warner et al. 1982; Clague & Ward

2011; Saunders 2014). The advance and retreat of glaciers during the Pleistocene in

British Columbia has made that province a particularly interesting locale for studying species diversification and evolution (Hebda 2007). An accurate understanding of species diversity in a given area requires the ability to accurately identify and distinguish taxa.

Traditional taxonomy and systematics for the marine organisms collectively termed seaweeds employed morphological features to identify and describe species; however, the subjective nature of this approach made it both frustrating and unreliable (Saunders

2005). Molecular-assisted alpha taxonomy (MAAT) aims to overcome the challenges of traditional morphology-based identification by using genetic similarity to assign algal specimens to distinct species groups, which are subsequently assessed morphologically and assigned to existing species or described as novel taxa (Saunders 2005, 2008;

Cianciola et al. 2010; Saunders & McDonald 2010).

A common molecular marker for assessing red algal species is the 5’ end of the mitochondrial cytochrome c oxidase subunit I gene (COI-5P), often referred to as the

“DNA barcode” (Saunders 2005). In cases where COI-5P amplification is unsuccessful, the 3’ end of the plastid ribulose-biphosphate carboxylase gene (rbcL-3P) has been

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recognized as an alternative barcode marker (Saunders & Moore 2013). Although not as variable as COI-5P, rbcL-3P is effective for distinguishing individuals of the same species as well as individuals of different species.

The Rhodymeniales is a red algal order whose members display extremely diverse habits and frond morphologies (expansive blades, small turf-like varieties, hollow sacs and mucilage-filled vesicles), which are united by a distinct pattern of procarpy and outward carposporophyte development (Saunders et al. 1999; Le Gall et al. 2008).

Currently, the Rhodymeniales includes six families, 49 genera and over 300 species distributed worldwide (Guiry & Guiry 2016). In 1986, Hawkes & Scagel published a monograph of the Rhodymeniales from British Columbia and northern Washington using traditional morphology-based methods and reported 12 species in 9 genera from British

Columbia: Botryocladia pseudodichotoma (Farlow) Kylin, Fauchea fryeana Setchell,

Fauchea laciniata J.G.Agardh, Faucheocolax attenuata Setchell, Fryeella gardneri

(Setchell) Kylin, Gastroclonium subarticulatum (Turner) Kützing, Lomentaria hakodatensis Yendo, Minium parvum Moe, Rhodymenia californica Kylin, Rhodymenia pacifica Kylin, Rhodymenia pertusa (Postels & Ruprecht) J.Agardh and

Rhodymeniocolax botryoidea Setchell. Subsequent to their monograph, F. fryeana and F. laciniata were transferred to the genus Gloiocladia (Rodriguez-Prieto et al. 2007), while the novel genera Neogastroclonium L.Le Gall, Dalen & G.W.Saunders and Sparlingia

G.W.Saunders, I.M.Strachan & Kraft were established to accommodate G. subarticulatum (Le Gall et al. 2008) and R. pertusa (Saunders et al. 1999). In addition,

Leptofauchea pacifica E.Y.Dawson was recognized in British Columbian waters (Dalen

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& Saunders 2007). Nevertheless, the monograph of Hawkes & Scagel (1986) remains the only large-scale assessment of the rhodymenialean flora in British Columbia.

In light of the underestimated and cryptic diversity that MAAT has revealed for other red algal groups in British Columbia (e.g., Clarkston & Saunders 2013; Hind et al.

2014; Saunders & Millar 2014; Saunders et al. 2015), this study aimed to use an integrative taxonomic approach to reassess species richness, species limits and biogeography for rhodymenialean taxa in British Columbia. We confirmed for British

Columbia most of the species reported by Hawkes & Scagel (1986), while also identifying cryptic complexes for Botryocladia, Gloiocladia and Rhodymenia, an additional species of Fryeella, and potentially a second parasitic species of

Faucheocolax. We also analyzed recently collected specimens of Minium parvum to provide detailed anatomical observations in light of the recent phylogenetic assignment of this genus to the family Fryeellaceae (Filloramo & Saunders 2016a).

Materials and Methods

British Columbian specimens (n= 563) that were field identified to various rhodymenialean genera were collected from either the intertidal as attached or drift material or in the subtidal by SCUBA (Appendix B, Table S1). Additional rhodymenialean collections from California (n= 41), Oregon (n= 2) and Washington (n=

7) were also included in this study to explore species distributions along the west coast of

North America (Appendix B, Table S1). All specimens were either pressed on herbarium paper or dried in silica gel to serve as vouchers (housed at the Univeristy of New

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Brunswick Connell Memorial Herbarium; UNB) with a subsample preserved in silica for molecular analyses (Saunders & McDevit 2012).

Total Genomic DNA was extracted following Saunders & McDevit (2012). The

5’ end of the mitochondrial cytochrome c oxidase I gene (COI-5P; DNA barcode; 664 bp) was amplified (Saunders & Moore 2013) with primer combinations recorded on the

Barcode of Life Database (BOLD; www.boldsystems.org) and GenBank. Full-length rbcL (1358 bp) sequences were amplified (Saunders & Moore 2013) for at least one representative of each genetically verified COI-5P species group. Full-length rbcL was used as a reference for amplicons of the 3’ end of the plastid ribulose-1, 5-bisphosphate carboxylase oxygenase region (rbcL-3P, 786 bp), which were amplified as a secondary barcode marker when amplification of COI-5P was unsuccessful. To validate some genetic species groups resolved with COI-5P and rbcL-3P, the nuclear internal transcribed spacer of the ribosomal cistron (ITS) was amplified (Saunders & Moore

2013). All amplicons were sequenced by the Génome Québec Innovation Centre and raw data were edited and aligned in Geneious R7 version 7.1.7 (http://www.geneious.com;

Kearse et al. 2012). Additional COI-5P, rbcL and ITS data were acquired from GenBank for comparative purposes (Appendix B, Table S1).

To determine intraspecific variation and nearest neighbor distances, the COI-5P alignment was subjected to a barcode gap analysis that was implemented in BOLD. The rbcL-3P sequences were compared to full-length rbcL and both were included in distance analyses to determine within and between species variation. Total differences in ITS were assessed by analyzing the alignments in Geneious R7.

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A multigene alignment was constructed using COI-5P and full-length rbcL data for one representative of each genetic species group that was resolved for Botryocladia and Rhodymenia. To place the British Columbian species in a broader context, all relevant GenBank data (Appendix B, Table S1) for Botryocladia and Rhodymenia were added to the previous alignment. Other rhodymeniacean taxa were selected for the outgroup based on Filloramo & Saunders (2016a). A second multigene alignment of

COI-5P and rbcL data was created for representatives of the Faucheaceae and

Fryeellaceae and was combined with pertinent data from GenBank (Appendix B, Table

S1). The previous multigene alignments were analyzed separately in RAxML under a

GTR+I+G model (determined by PartitionFinder) with partitioning by gene and then codon and support was determined by 1000 rounds of bootstrap resampling. The

Faucheaceae and Fryeellaceae were rooted on one another.

Anatomical observations were made by rehydrating algal tissue in 5% formalin and seawater and then sectioning it by hand or with a freezing microtome (CM18250,

Leica, Heidelberg, Germany). All sections were stained with 1% aniline blue solution in

6% 5N hydrochloric acid and permanently mounted in 50% corn syrup with 4%

Formalin. Features were photographed with a Leica digital camera (DFC480) and Leica microscope (CTR5000).

Results

Molecular results

COI-5P 88

The DNA barcode was successfully amplified for 577 specimens. These collections were resolved in 18 genetic species groups (Table 3.1). Maximum intraspecific variation for

COI-5P was 0.77% with the exception of Fryeella callophyllidoides (1.53%; Table 3.1) and Neogastroclonium subarticulatum (0.93%; Table 3.1). In both cases the greatest divergence was between collections from British Columbia and California. The within species variation for British Columbian specimens of Fryeella callophyllidoides and

Neogastroclonium subarticulatum was 0% and 0-0.75%, respectively. In general, interspecific variation for COI-5P ranged from 2.67% to 20.46% (Table 3.1). The collections morphologically assigned to the following six species resolved in single genetic species groups: Fryeella garderni, Leptofauchea pacifica, Lomentaria hakodatensis, Minium parvum, Neogastroclonium subarticulatum and Sparlingia pertusa

(Table 3.1), while other morphospecies were resolved as part of cryptic complexes

(discussed below).

Californian samples (n= 7) identified as Botryocladia pseudodichotoma were resolved in a distinct genetic species group (5.13%; Table 3.1) from British Columbian and northern Washington collections, which were described herein as B. hawkesii sp. nov. A British Columbian collection and two Californian specimens were morphologically identified as Rhodymenia callophyllidoides, but genetically most similar

(6.50 %; Table 3.1) to Fryeella gardneri necessitating transfer of this species to Fryeella.

Collections assigned to Gloiocladia resolved as four genetic species groups, three found in British Columbia, where only two were expected. Gloiocladia fryeana (British

Columbia n= 20) was distantly (13.14 %; Table 3.1) related to a single specimen from

California attributed to G. media. Additional collections from British Columbia were 89

assigned to G. vigneaultii sp. nov. (n= 14) and the closely allied (3.73%; Table 3.1) G. laciniata (British Columbia n= 73), which also included a collection from Washington.

Some collections (n= 3) of the parasitic Faucheocolax attenuata were genetically identical to their host species (Table 3.1), G. laciniata, while two other collections were distinct and most closely related to G. media (2.67%; Table 3.1). Specimens that were field identified as R. californica were resolved as two distinct genetic groups (8.81% interspecific variation) assigned to R. californica (British Columbia n= 128, California n=

8, Oregon n= 2) and R. bamfieldensis sp. nov. (British Columbia n= 5, Washington n= 2)

(Table 3.1). Similarly, collections morphologically assigned to R. pacifica formed two distantly related groups (8.27% interspecific variation) assigned to R. pacifica (British

Columbia n= 9, California n=7) and the resurrected R. rhizoides (British Columbia n= 25,

California n= 2) (Table 3.1).

rbcL-3P

Consistent with COI-5P, rbcL fully resolved 18 genetic species groups. Using rbcL-3P data, we were able to assign 36 collections for which COI-5P amplification failed to genetic species groups (Appendix B, Table S1). A single Californian collection was resolved as B. pseudodichotoma while a specimen from British Columbia that was field identified as the former species was resolved as B. hawkesii. Specimens field identified as

Gloiocladia spp. were resolved as G. fryeana (n= 4), G. laciniata (n= 22) and G. vigneaultii (n= 2) and five collections attributed to Rhodymenia were resolved as R. bamfieldensis (n= 1), R. californica (n= 1), and R. pacifica (n= 3), respectively, while a

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single collection (GWS012983; Appendix B, Table S1) field identified as Rhodymenia sp. was in fact a dichotomously branched Sparlingia pertusa.

Comparison to rbcL in GenBank

A GenBank entry attributed to G. fryeana (FJ713148) from Washington was resolved with our collections of G. fryeana from British Columbia (0-3 bp different across 650 bp available for comparison). One entry for G. laciniata (FJ713149, California) had a single base pair difference (across 1363 bp) from our Californian specimen assigned to G. media, while a second (FJ713150, British Columbia) joined (0-3 bp differences across

1363 bp) our G. vigneaultii. Four additional GenBank entries for G. laciniata

(AY294355, Alaska; KM253823, KM253724, KM253834, California) matched (0-3 bp differences across 1363 bp) our collections assigned to that species.

ITS

Our ITS data for B. hawkesii and B. pseudodichotoma were consistent with COI-5P in that two distinct genetic species groups were resolved (21-29 bp differences across 616 bp; Appendix B, Table S1). As with the COI-5P results, the ITS for three collections of

Faucheocolax (GWS004768A, GWS014315 and GWS020832; all parasitic on G. laciniata), were resolved as genetically identical to their host collections. Also consistent with COI-5P, the ITS for one of the Faucheocolax collections (GWS001423) was distinct

(352 bp differences across 1475 bp) from those of the previous three parasite collections, as well as from its host species, G. laciniata (352 bp differences across 1462) (Appendix

B, Table S1). The ITS1 for G. fryeana was virtually unalignable to that of G. laciniata, 91

G. media and G. vigneaultii; however, although highly divergent, the ITS2 was more comparable. A comparison of ITS data for G. laciniata to that of G. vigneaultii suggested that there were two distinct genetic species (330-333 bp differences across 1494 bp), which corroborated with the COI-5P results. Further examination of Faucheocolax and

Gloiocladia data revealed a ~160 bp repetitive sequence in the ITS1 for G. laciniata, G. media and G. vigneaultii as well as both genetic groups assigned to F. attenuata. In those species, that repetitive sequence occurred in variable numbers as complete entities, truncated units or as a modified sequence that was shared with some but not all species

(Fig. 3.1). The previous differences resulted in variable sequence lengths and contributed to increased interspecific variation.

Comparison of our ITS data to relevant data in GenBank indicated 2 bp differences (across 1255 bp) between G. vigneaultii and a GenBank representative

(U30363; Washington) that was assigned to G. laciniata with those differences possibly attributed to errors in the GenBank sequence at otherwise conservative sites. Similarly, a

GenBank entry of G. fryeana (U30364; Washington) was most similar (4 bp differences across 1494 bp) to our data for G. laciniata and, again, those differences were potentially a result of errors in the GenBank sequence. Both U30363 and U30364 were host to parasitic F. attenuata (represented by the GenBank entries U30366 and U30365, respectively). The GenBank data for U30366 and U30365 were identical to each other but distinct from the ITS data generated for British Columbian collections of F. attenuata included in the present study (Fig. 3.1) raising the distinct possibility of a third species included in this morphospecies.

92

Phylogenetic Analyses

RAxML analysis included eight representatives of Botryocladia, which were resolved in two groups (Fig. 3.2). Botryocladia species from the Northeast Pacific were resolved as sister to each other in the first group, which also included two species from the Atlantic

Islands/tropical & subtropical Northwestern Atlantic (Fig. 3.2). The second Botryocladia clade was comprised of B. neushulii from Pacific Mexico, two species from Australia, the

South Pacific species B. ebriosa and the Northwest Pacific species Chrysymenia wrightii

(Fig. 3.2).

The 24 species of Rhodymenia that were included were resolved in three groups and an unresolved R. pseudopalmata (Fig. 3.2). The first group included representatives predominantly from Australia in addition to species from the Central Pacific, New

Zealand, Northeast and Northwest Atlantic (Fig. 3.2). The second clade included our four

Northeast Pacific Rhodymenia species, among which, a specimen of R. intricata from the

Northwest Pacific was resolved. Also, weakly associated as sister to that clade was the generitype species R. pseudopalmata from the Northeast Atlantic. Lastly, the third clade was comprised exclusively of species from the Southern Hemisphere (i.e., Australia,

South Africa and South America; Fig. 3.2).

Phylogenetic analyses strongly placed Fryeella callophyllidoides in the

Fryeellaceae as sister to F. gardneri (Fig. 3.3). Minium parvum was fully resolved in a monophyletic lineage with the previous species of Fryeella (Fig. 3.3). In fact, it was more closely related to the two Fryeella spp. than were species of Leptofauchea,

Gloiocladia and Webervanbossea relative to one another (Fig. 3.3).

93

Fifteen specimens of Gloiocladia and Gloioderma spp. and two representatives of

Faucheocolax were included in our phylogenetic analyses and were resolved in four clades (Fig. 3.3). The first clade included G. halymenioides and an unidentified species from Western Australia, which were distantly related to G. spinulosa from the Northwest

Pacific (Fig. 3.3). The second clade was comprised of closely resolved Northwest Pacific

G. japonica and the Gulf of Mexico species G. tenuissima, which were weakly allied to the Northwest Pacific species G. iyoensis (Fig. 3.3). The third clade included three

Northeast Atlantic species, which were closely resolved, in addition to the more distantly related species G. pelicana from the Gulf of Mexico (Fig. 3.3). Lastly, the fourth clade was comprised of taxa exclusively from the Northeast Pacific and relationships among those taxa were resolved with strong to full support (Fig. 3.3). Included were two representatives of parasitic F. attenuata — one, which was resolved alongside its host G. laciniata, and the other, which was fully supported as sister to G. media (Fig. 3.3).

Morphological results

Botryocladia hawkesii Filloramo & G.W.Saunders sp. nov. Fig. 3.4a-g

DIAGNOSIS: Thalli typically 26-65 mm tall with a discoid holdfast and solid, terete stalk either bearing a single mucilage-filled, saccate vesicle (11-33 mm long x 7-22 mm wide) or irregularly branched and bearing numerous vesicles (Fig. 3.4a). Vesicle outer cortex 2

(-3) layers of small cells 5 μm in diameter grading to an inner cortex of 1 (-2) layers of oblong cells (15-26 μm long x 5-12 μm wide); medulla of 1 (-2) large axially elongated

(96-178 μm long x 54-81 μm wide) cells surrounding the mucilage filled cavity (Fig.

94

3.4b). Gland cells scattered on medullary cells, rarely single and typically in groups of 7-

15, subspherical, 12-50 μm in diameter (Fig. 3.4c,d). Spermatangia (2.5-3 μm long) in sori cut off from cortical cells (Fig. 3.4d). Carpogonial branches and auxiliary cell branches not seen. Cystocarps scattered (Fig. 3.4e), 600-900 μm in diameter, protruding both inwards and outwards, ostiolate (Fig. 3.4f). Tetrasporangia in scattered cortical sori, modified from existing cortical cells, cruciately divided, 38-43 μm long x 22.5-38 μm wide (Fig. 3.4g).

HOLOTYPE: G.W. Saunders, B. Clarkston, K. Hind & D. McDevit, 16 May 2008 (UNB,

GWS009487) (Fig. 3.4a); isotype GWS009486 (Appendix B, Table S1).

TYPE LOCALITY: Pipers Point (49.54668, -123.79256), Sechelt, British Columbia,

Canada; subtidal (10 m) on rock.

HOLOTYPE COI-5P BARCODE: HM917100.

ETYMOLOGY: Named for Dr. Michael Hawkes in recognition of his significant contributions to our understanding of rhodymenialean diversity in British Columbia and northern Washington.

DISTRIBUTION: Genetically verified collections from San Juan County, Washington,

United States to Prince Rupert, British Columbia, Canada.

REPRESENTATIVE COLLECTIONS: Listed in Appendix B (Table S1).

Fryeella callophyllidoides (Hollenberg & I.A.Abbott) Filloramo & G.W.Saunders comb. nov. Fig. 3.5a-g

95

BASIONYM: Rhodymenia callophyllidoides Hollenberg & I.A.Abbott 1965 (New species and new combinations of marine algae from the region of Monterey, California.

Canadian Journal of Botany 43: 1184).

OBSERVATIONS OF EPITYPE SPECIMEN (UC1455019): Thalli 110-150 mm across; individual branch width 2-5 mm and typically consistent from base to apices, which are rounded (Fig. 3.5a). Blades 250-300 μm thick in cross section with an outer cortex of 1 (-

2) layers of small, round cells and 1 (-2) layers of ellipsoid inner cortical cells, which grade to 3-4 cell layers of large, colourless medullary cells (105-150 μm x 50-70 μm wide), blades of solid construction lacking hollow regions (Fig. 3.5b). Tetrasporangial sori appear as mottled nemathecia that develop submarginally and spread toward the blade center; cruciately divided tetrasporangia (30-45 μm long x 15-20 μm wide) or 2-3 celled paraphyseal filaments develop from adventitious growth of existing cortical cells

(Fig. 3.5c).

DIAGNOSTIC FEATURES OF GENETIC GROUP: Individual branch width consistent from base to the rounded apices (Fig. 3.5d). Blades 225-250 μm thick in cross section with an outer cortex of 1 (-2) layers of small, round cells and 1 (-2) layers of ellipsoid inner cortical cells and 3-4 cell layers of large, colourless medullary cells (55-75μm x 110-170

μm), solid construction (Fig. 3.5e). Tetrasporangial development (Fig. 3.5f,g) as observed for the epitype.

TYPE LOCALITY: Monterey Bay, California, United States.

DISTRIBUTION: Genetically verified collections from Monterey Bay, California, United

States and Bamfield, British Columbia, Canada.

REPRESENTATIVE COLLECTIONS: Listed in Appendix B (Table S1). 96

Gloiocladia fryeana (Setchell) Sánchez & Rodríguez-Prieto Fig. 3.6a-g

DIAGNOSTIC FEATURES OF GENETIC GROUP: Thalli flattened, broadly flabellate, 20-73 mm tall; irregularly dichotomously branched, branch widths (0.5-10 mm) variable within an individual, branches tapering slightly towards base; apices blunt to rounded (Fig. 3.6a, b). Blades 200-370 μm thick in cross section including a medulla composed of 1-3 layers of large, round or slightly ellipsoid colourless cells (largest cell typically central, 80-150

μm long x 70-110 μm wide and flanked by smaller cells) and a cortex of 3-4 celled filaments laxly arranged and arching toward the thallus surface (Fig. 3.6c,d).

Tetrasporangia in prominent nemathecia appearing as dark patches on a single surface of the thallus and spreading from base to just below the ultimate dichotomy (Fig. 3.6b), cruciately divided (37.5-50 μm long x 20-35 μm wide), borne on cortical cells, terminally on single-celled filaments or laterally on 5-6 celled paraphyseal filaments, which surround the developing tetrasporangia (Fig. 3.6e). Cystocarps strictly marginal (Fig.

3.6a,f), slightly coronate, 800-830 μm in diameter, ostiolate, tela arachnoidea present

(Fig. 3.6g). Male gametophytes not observed.

REPRESENTATIVE COLLECTIONS: Listed in Appendix B (Table S1).

Gloiocladia laciniata (J.Agardh) N.Sánchez & Rodríguez-Prieto Fig. 3.7a-k

OBSERVATIONS OF HOLOTYPE SPECIMEN (LD 25708): Thallus ~120 mm tall, irregularly dichotomous, individual branch width variable from base towards apices (2-10 mm wide)

(Fig. 3.7a). In cross section, blade ~300 μm thick with 2-3 layers of large medullary cells 97

(110-210 μm long x 70-120 μm wide), 1 inner cortical layer and a cortex of compact slightly arched filaments 3-4 cells long (Fig. 3.7b,c). Cystocarps marginal and scattered

(Fig. 3.7a) on both blade surfaces, distinctly coronate (Fig. 3.7d), 950-1000 μm in diameter, ostiolate, tela arachnoidea present (Fig. 3.7e).

DIAGNOSTIC FEATURES OF GENETIC GROUP: Thalli 20-180 mm tall, spreading, flabellate, irregularly dichotomously branched, individual branch width variable from base to apices

(1.3-11 mm wide) (Fig. 3.7f,g). In transverse section, plants were 250-440 μm thick with

2-3 layers of medullary cells (80-200 μm long x 90-124 μm wide), 1(-2) inner cortical layers and a cortex of compact slightly arched filaments 3-4 cells long (Fig. 3.7h,i).

Cystocarps scattered either on both blade surfaces (typically one side is more abundant) or marginal (Fig. 3.7f) or a combination of surface and marginal (Fig. 3.7g), distinctly coronate (Fig. 3.7j), 800-930 μm in diameter, ostiolate, tela arachnoidea present (Fig.

3.7k).

REPRESENTATIVE COLLECTIONS: Listed in Appendix B (Table S1).

Gloiocladia media (Kylin) Filloramo & G.W.Saunders comb. nov. Fig. 3.8a-j

BASIONYM: Fauchea media Kylin 1941 (Californische Rhodophyceen. Acta Universitatis

Lundensis 37: 27).

OBSERVATIONS OF HOLOTYPE SPECIMEN (LD 1613738): Cystocarpic plant ~ 29 mm tall, irregularly dichotomous (Fig. 3.8a). Blade ~360 μm thick in transverse section (Fig. 3.8b) with a dorsiventral cortex (3-4 celled compact filaments towards one surface and ~4-5 celled filaments on the opposite surface) (Fig. 3.8c,d); single inner cortical layer; and medulla of 2-3 layers of large colourless cells (100-200 μm long x 75-110 μm wide) (Fig. 98

3.8b). Cystocarps produced near thallus apices scattered on both blade surfaces, typically rounded (Fig. 3.8e), ~900 μm in diameter, ostiolate, sessile, tela arachnoidea present

(Fig. 3.8f).

DIAGNOSTIC FEATURES OF GENETIC GROUP: Thalli ~28 mm tall, irregularly dichotomously branched (Fig. 3.8g). In transverse section, 280-340 μm thick with a cortex 3-4 celled compact filaments towards one surface and ~4-5 celled filaments on the opposite surface; single inner cortical layer; and medulla of 2-3 layers of large colourless cells (130-270 μm long x 80-130 μm wide) (Fig. 3.8h). Cystocarps rounded, located towards ultimate portion of blade on both blade surfaces (Fig. 3.8i), 780-840 μm in diameter, ostiolate, sessile, tela arachnoidea present (Fig. 3.8j). Male gametophytes and tetrasporophytes not observed.

TYPE LOCALITY: Pacific Grove, California, USA.

DISTRIBUTION: Genetically confirmed only from Carmel and Monterey, California, USA.

REPRESENTATIVE COLLECTIONS: Listed in Appendix B (Table S1).

Gloiocladia vigneaultii Filloramo & G.W.Saunders sp. nov. Fig. 3.9a-h

DIAGNOSIS: Plants 37-75 mm tall, flabellate, irregularly dichotomously branched, individual branches 1.5-18 mm wide, tapering in width from base towards the apices

(Fig. 3.9a). Blades 370-450 μm thick in cross section with 2(-3) layers of medullary cells

(130-300 long μm x 70-200 μm wide) (Fig. 3.9b), a single inner cortical layer and a cortex of anticlinal, compact filaments of 6-8 cells (Fig. 3.9c); obvious cuticle ~15 μm thick (Fig. 3.9b,c). Cystocarps typically scattered over both blade surfaces (Fig. 3.9d), in some individuals predominantly marginal with few cystocarps on the blade surfaces (Fig. 99

3.9e), rounded to coronate (Fig. 3.9f), 900-1120 μm in diameter, ostiolate, tela arachnoidea present (Fig. 3.9f,g). Tetrasporangia in prominent nemathecia appearing as dark patches extending from the blade base to just below the ultimate dichotomy, on a single surface, mature tetrasporangia (35-45 μm long x 20-25 μm wide) cruciately divided and developing from cortical cells or laterally on paraphyses (5-6 cells long) (Fig.

3.9h). Male gametophytes not observed.

HOLOTYPE: G.W. Saunders & K. Dixon, 9 June 2012 (UNB, GWS030615), female (Fig.

3.9a-c,f,g); isotypes GWS030614, tetrasporophyte and GWS030616, tetrasporophyte

(Appendix B, Table S1).

TYPE LOCALITY: Islets west of Adam Rocks (52.11603, -131.23820), Gwaii Haanas,

British Columbia, Canada; subtidal (8 m) on invert.

HOLOTYPE COI-5P BARCODE: KU687640.

ETYMOLOGY: Named for Leandre Vigneault in recognition of his enthusiasm, encouragement and support of our investigation of the algal flora of Haida Gwaii.

Through his assistance we are developing a comprehensive understanding of the algal flora from that region.

DISTRIBUTION: Only known from British Columbia, Canada.

REPRESENTATIVE COLLECTIONS: Listed in Appendix B (Table S1).

Minium parvum R.L.Moe, Fig. 3.10a-d

OBSERVATIONS OF TETRASPOROPHYTES: Thalli appeared as dark patches on the surface of articulated coralline algae (Fig. 3.10a). In cross section, extensive basal holdfasts were

25-40 μm thick composed of a single layer of axially elongated cells that produced short, 100

erect filaments of 1-4 spherical cells that frequently became connected via secondary pit connections (Fig. 3.10b). The extensive holdfasts produce multiple reduced upright thalli, which typically spread laterally to overgrow the holdfast (Fig. 3.10c). The cells of the upright thalli were also joined by numerous secondary pit connections (Fig. 3.10b).

Extensive tetrasporangial nemathecia developed across the dorsal surface of the upright thalli via the adventitious growth of the outermost cortical cells (Fig. 3.10c).

Tetrasporangial initials originated from the outer cortical cells of the reduced upright thallus and when mature were cruciately divided (35-65 μm long x 17.5-28 μm wide)

(Fig. 3.10d). Cortical cells not bearing tetrasporangia divided to produce paraphyses (3-5 cells long), the cells of which became elongated and compressed between the developing sporangia (Fig. 3.10d).

REPRESENTATIVE COLLECTIONS: Listed in the Appendix B (Table S1).

Rhodymenia bamfieldensis Filloramo & G.W.Saunders sp. nov. Fig. 3.11a,b

DIAGNOSIS: Plants flabellate, irregularly to regularly dichotomously branched, erect, 53-

63 mm tall with a compressed stipe (up to 20 mm long) arising from a basal discoid holdfast bearing stolons typically giving rise to new thalli; individual blades typically

3.6-6 (-8) mm wide with broadly rounded apices (Fig. 3.11a). Blade thickness 116-250

μm at midthallus including 3-4 (-5) layers of large, round, colorless cells (47.5-80 μm in diameter) grading to 1-2 inner cortical layers of typically more elongated cells subtending

2 (-3) layers of darkly staining, rounded cortical cells (~5 μm in diameter) (Fig. 3.11b).

Reproductive material not observed.

101

HOLOTYPE: G.W. Saunders & K. Dixon, 26 June 2011 (UNB, GWS014391) (Fig.

3.11a,b); isotypes GWS014378, GWS014383 and GWS014388 (Appendix B, Table S1).

TYPE LOCALITY: Bordelais Island (48.81895, -125.23127), Bamfield, British Columbia,

Canada; subtidal (6 m) on invertebrate.

HOLOTYPE COI-5P BARCODE: KU687664.

ETYMOLOGY: Named for Bamfield, British Columbia, where this species was first collected — a centere for marine biological research in Canada.

DISTRIBUTION: Whidbey Island, Washington, USA to Bamfield and Sidney, British

Columbia, Canada.

REPRESENTATIVE COLLECTIONS: Listed in Appendix B (Table S1).

Rhodymenia californica Kylin Fig. 3.11c-g

DIAGNOSTIC FEATURES OF GENETIC GROUP: Plants flabellate, irregularly to regularly dichotomously branched, erect, up to 60 mm tall with a short stipe arising from a basal discoid holdfast that bears stolons; individual blades typically 0.5-4 mm wide with rounded apices (Fig. 3.11c). Blade thickness 112-283 μm at midthallus including 4-5 layers of large, round, colorless cells (41-50 μm in diameter) grading to 1-2 inner cortical layers of typically more elongated cells subtending 2 (-3) layers of darkly staining, rounded cortical cells (~5 μm in diameter) (Fig. 3.11d). Carpogonial branches and auxiliary cell branches not found. Cystocarps primarily located at branch apices, 500-520

μm in diameter, rounded, submarginal and protruding outwards, ostiolate (Fig. 3.11e).

Spermatangia located in sori appearing as light patches on blade surfaces at blade apices, spermatangia (~2.5 μm wide) developing from cells of the outer cortex (Fig. 3.11f). 102

Tetrasporangia located in superficial apical sori on one or both blade surfaces towards thallus apices, cruciately divided, 23-34 μm long x 9-15 μm wide with cortex surrounding tetrasporangia unmodified (Fig. 3.11g).

REPRESENTATIVE COLLECTIONS Listed in Appendix B (Table S1).

Rhodymenia pacifica Kylin Fig. 3.12a,b

DIAGNOSTIC FEATURES OF GENETIC GROUP: Plants flabellate, dichotomously branched, erect, 24-90 mm tall with terete stipes (2-30 mm long); discoid holdfast with short basal stolons; individual blades 4.1-12.6 mm wide with broadly rounded apices (Fig. 3.12a). In cross section, blades 162-250 μm thick with 3 (-4) rows of large, round, colourless medullary cells (67-76 μm in diameter) grading over 1-2 cell layers to a single outer cortical layer of small, round or oblong, darkly staining cells (~5-6 μm wide) (at times, an incomplete inner cortical layer present) (Fig. 3.12b). Male and female gametophytes and tetrasporophytes not observed in our collections.

REPRESENTATIVE COLLECTIONS: Listed in Appendix B (Table S1).

Rhodymenia rhizoides E.Y.Dawson Fig. 3.12c,d

DIAGNOSTIC FEATURES OF GENETIC GROUP: Plants broadly flabellate, dichotomously branched, erect, 19-68 mm tall with terete stipes (10-80 mm long) and small discoid holdfasts bearing abundant basal stolons that may give rise to new blades; individual blades 6-9 mm wide with rounded apices (Fig. 3.12c). In cross section, blades 144-170

μm thick featuring 2-3 layers of large, round, colourless medullary cells (35-56 μm in diameter) grading over 1-2 cell layers to a single outer cortical layer of small, darkly 103

staining cells (~5 μm in diameter) (at times, a second incomplete layer present) (Fig.

3.12d).

REPRESENTATIVE COLLECTIONS: Listed in Appendix B (Table S1).

Discussion

The only extensive floristic survey of the Rhodymeniales in British Columbia resulted in the recognition of 12 species in 9 genera (Hawkes & Scagel 1986). That study was completed at a time when molecular techniques were not applied for species diversity assessments. Over the past 20 years, advances in DNA-based identification have exposed the complications that phenotypic plasticity and convergent evolution bring to the task of distinguishing seaweed species based on morphology alone (Saunders 2005, 2008). The present study sought to reassess the number of rhodymenialean species present in British

Columbia using a contemporary molecular-assisted alpha taxonomic approach. In doing so, 16 species in 10 genera were resolved, confirming Fryeella garderni, Gloiocladia fryeana, Gloiocladia laciniata, Leptofauchea pacifica [first reported from British

Columbia by Dalen & Saunders (2007)], Lomentaria hakodatensis, Minium parvum,

Neogastroclonium subarticulatum, Rhodymenia californica, Rhodymenia pacifica and

Sparlingia pertusa and uncovering cryptic and overlooked taxa for Botryocladia,

Faucheocolax, Fryeella, Gloiocladia and Rhodymenia.

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Botryocladia

The species B. pseudodichotoma was described from Carmel Bay, California by

Farlow (1899) as Chrysymenia pseudodichotoma Farlow and later transferred to

Botryocladia by Kylin (1931). Although, B. pseudodichotoma has been widely reported from British Columbia (Collins 1913; Scagel 1967; Widdowson & Coon 1975; Hawkes et al. 1979; Lindstrom & Foreman 1978), Hawkes & Scagel (1986) postulated that specimens from British Columbia may not be conspecific with this species from

California owing to morphological differences between plants from each location. Our data validated their hypothesis as collections from British Columbia and northern

Washington were resolved as genetically distinct from Californian specimens (including collections from Monterey and Pacific Grove, which are near the type locality of Carmel

Bay). Owing to the previous, the northern species was described as the novel taxon, B. hawkesii.

While extensive collecting in British Columbia has resulted in frequent encounters with B. hawkesii, B. pseudodichotoma has not been collected suggesting that this species does not extend north to British Columbia. Extensive collecting in California is needed to determine if B. hawkesii extends to that flora (searching both COI-5P and rbcL data for B. hawkesii in GenBank did not yield matches to previously published sequences from California). Additional collection efforts in Washington and Oregon will contribute to a better understanding of the respective distributions of B. hawkesii and B. pseudodichotoma in the Pacific Northeast.

At present, biogeography is the most valuable character for distinction as morphological assessment failed to identify useful distinguishing features for B. hawkesii 105

and B. pseudodichotoma. Although Hawkes & Scagel (1986) indicated that their specimens from British Columbia were smaller than those from California and that the

British Columbian specimens typically consisted of a short stipe bearing a single vesicle or at most six vesicles vs. up to 100 vesicles in those from California, we found that overall specimen size and numbers of vesicles overlapped between the two species groups recognized herein. Unfortunately, we lacked tetrasporophytes and gametophytes of B. pseudodichotoma for comparison to reproductive material of B. hawkesii. Although details of reproductive features for B. pseudodichotoma exist in the literature (e.g.,

Bliding 1928; Abbott & Hollenberg 1976), the cryptic nature of these species and an incomplete knowledge of their respective distributions render those observations unreliable, and reproductive, genetically confirmed B. pseudodichotoma is needed to assess if there are indeed any useful reproductive features for delimiting B. hawkesii and

B. pseudodichotoma. Until that time, a combination of genetics and biogeography are the most reliable tools for identifying these taxa.

Fryeella

The monotypic genus Fryeella Kylin (1931) was based on Fauchea gardneri

Setchell (1901), which was described from Washington. This genus was defined in part by thalli with mucilage filled internal cavities separated by septa, and adventitious growth of the cortex in the formation of paraphyses and tetrasporangia (Kylin 1931; Le Gall et al. 2008). Our molecular analyses resolved some specimens (British Columbia n= 1;

California n= 2) as strongly allied to Fryella gardneri (Table 3.1). Subsequent morphological observations indicated adventitious growth of the cortex during 106

paraphyseal/tetrasporangial development (Fig. 3.5g), which is typical of the genus; however, the solid pseudoparenchymatous construction of the thalli (Fig. 3.5e) was more consistent with placement in Rhodymenia. Investigation of the literature uncovered,

Rhodymenia callophyllidoides Hollenberg & I.A.Abbott, which was described from

Monterey, California (Hollenberg & Abbott 1965). Interestingly, when R. callophyllidoides was described, it was noted that it lacked a stipe, which is atypical of

Rhodymenia, but consistent with Fryeella. Details of the tetrasporangia were lacking in the type description of R. callophyllidoides, described only as located in mottled, submarginal nemathecia that spread to the blade center (Hollenberg & Abbott 1965), which was consistent with development in the genetic group resolved in this study. This warranted a closer examination of the type. Although type material of R. callophyllidoides was previously mislocated (K.A. Miller pers. comm.), tetrasporangial epitype material (UC1455019) collected from Monterey and identified by I.A. Abbott as

R. callophyllidoides was provided on loan by Jepson Herbarium of the University of

California. Anatomical examination revealed a similar vegetative anatomy (Fig. 3.5b,e) and adventitious cortical growth for the formation of paraphyseas and tetrasporangia (Fig.

3.5c,g) as we observed in specimens of our genetic group. Owing to the previous, we transfered R. callophyllidoides to Fryeella as F. callophyllidoides (Hollenberg &

I.A.Abbott) Filloramo & G.W.Saunders comb. nov. Inclusion of F. callophyllidoides in

Fryeella challenges the feature of a hollow, mucilage-filled thalli as diagnostic of the genus; however, the utility of this character has been questioned for genus-level distinction in other rhodymenialean genera (e.g., Halichrysis includes taxa with both solid construction and hollow “regions”; Ballantine et al. 2007). One of our Californian 107

collections of F. callophyllidoides was divergent (COI-5P; 10 bp differences across 664 bp) from the British Columbian and the other Californian collection. Although some rhodymenialean taxa exhibit higher than usual intraspecific divergence (e.g., Saunders &

McDonald 2010; Filloramo & Saunders 2016b) future collection efforts in British

Columbia and California are necessary and further study is warranted to ascertain if two closely related species should be recognized for this genetic group.

Gloiocladia/Faucheocolax

Traditionally, G. fryeana and G. laciniata were the only species of Gloiocladia recognized in British Columbia. These taxa were distinguished according to cystocarp position, which was reportedly strictly marginal for G. fryeana and distributed over thallus surface for G. laciniata, and cystocarp shape (Setchell 1912; Sparling 1957;

Hawkes & Scagel 1986). When possible, field identifications were made for this study according to the previous criteria; however, the resolution of three genetic species groups for Gloiocladia in British Columbia called into question the utility of these features.

One of the genetic species groups featured female gametophytes with strictly marginal, slightly coronate cystocarps (Fig. 3.6a) and was assigned to G. fryeana as that criterion and the overall gross anatomy and vegetative construction were consistent with

Setchell’s (1912) original description, as well as Hawkes & Scagel’s (1986) concept of that species in British Columbia. Additionally, an rbcL sequence in GenBank reported as

G. fryeana from its type locality in San Juan Islands, Washington grouped with our

British Columbia collections. In the presence of female reproduction, G. fryeana may be distinguished from other Gloiocladia species in British Columbia according to its strictly 108

marginal cystocarps (Fig. 3.6a), thinner thalli in cross section (200-300 um) and short (2-

3 celled), arched, laxly arranged cortical filaments (Fig. 3.6c,d).

The two remaining Gloiocladia species in British Columbia included female gametophytes with cystocarps scattered on both blade surfaces, which was traditionally associated with G. laciniata. Owing to an inadequate protolog description, the cystocarpic holotype of G. laciniata was acquired on loan from the Lund University

Biological Museum to determine if it was a good morphological match to one of our two remaining British Columbia genetic species groups. Holotype material featured distinctly coronate cystocarps (Fig. 3.7d) scattered densely on the thallus margin, as well as scattered (sparsely) on both blade surfaces (Fig. 3.7a). Female gametophytes for one of our genetic groups were consistent with the previous observation and were assigned to G. laciniata. The other genetic group featured both rounded and coronate cystocarps located predominantly on the thallus surface and sparsely (if at all) on the thallus margin (Fig.

3.9a,d) and was described as the novel species G. vigneaultii. Interestingly, a single female gametophyte genetically assigned to G. vigneaultii featured predominantly marginal cystocarps (Fig. 3.9e) indicating greater variability for features of the cystocarp than in other Gloiocladia spp. in British Columbia. Vegetatively, G. laciniata and G. vigneaultii are similar in that the cortical filaments for each group are more densely arranged than those observed for G. fryeana; however, the former two species may be distinguished based on the number of cells in the cortical filaments as G. vigneaultii has more cells in the cortical filaments compared to G. laciniata (Fig. 3.9c vs. Fig. 3.7c,i).

Additionally, G. vigneaultii features an obvious, thick cuticle covering both blade surfaces (Fig. 3.9c). 109

Overall, we have identified some features, which when combined, are useful for distinguishing Gloiocladia species in British Columbia, primarily, the number of cells in the outer cortical filaments, arrangement of cortical filaments, blade thickness in cross section and secondarily, the location and shape of the cystocarps (see species key below).

Also included in our study was a single collection (GWS021443) of Gloiocladia from

California that was genetically distinct from the three Gloiocladia species recognized from British Columbia. Investigation of the literature revealed the species Fauchea media, which was described from Pacific Grove, California by Kylin (1941) to accommodate specimens that were intermediate in size between F. pygmaea (Setchell &

Gardner) Kylin (1-3 cm tall) and F. laciniata (8-15 cm tall). Soon after, Doty (1947) and then Abbott & Hollenberg (1976) reduced F. media and F. pygmaea, respectively, to synonymy with F. laciniata. Cystocarpic holotype material of F. media was provided on loan from Lund University Herbarium and compared to cystocarpic holotype material of

F. laciniata (= G. laciniata) and our single sample from California. Although F. media was considered qualitatively consistent with G. laciniata in all ways except thallus size; our observations of holotype material indicated inconsistencies in cystocarp shape and location between these species. While holotype material for G. laciniata featured distinctly coronate cystocarps distributed densely along the margin and sparingly on the thallus surface (Fig. 3.7a,d), the holotype specimen for F. media had smooth, round cystocarps that were located rarely on the margins and predominantly towards the upper half of the blade (Fig. 3.8a,e). The latter observations were consistent with those for specimen GWS021443 from California and the epithet, F. media was resurrected as

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distinct from G. laciniata and transferred to Gloiocladia (as G. media). Currently the distribution of this species is limited to California.

Key to species of Gloiocladia

1a. Cortical filaments laxly arranged and arching towards the thallus surface, blades 200-

300 μm in cross section (Fig. 3.6d); cystocarps strictly on blade margin (Fig.

3.6a)…..…………………………………………………………………………G. fryeana

1b. Cortical filaments compact and arising perpendicular from the thallus surface, blades

> 300 μm in cross section; cystocarps on both blade surfaces or both surfaces and blade margin…..………………………………………………………………………………... 2

2a. Blades 300-350 μm in cross section (Fig. 3.8b,h); cystocarps strictly rounded (Fig.

3.8e,i), produced on both blade surfaces toward apices…………...... G. media

2b. Blades > 350 μm in cross section; cystocarps rounded to distinctly coronate, covering both blade surfaces or both surfaces as well as blade margin...... …...... …. 3

3a. Cortex composed of 3-4 celled filaments, cuticle ~5 μm thick (Fig.

3.7c,i)………..………………………………………………………………... G. laciniata

3b. Cortex composed of 6-8 celled filaments subtending an obvious cuticle (~15 μm thick) (Fig. 3.8c)…...... …...... …...... G. vigneaultii

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Faucheocolax attenuata was described from Carmel Bay, California by Setchell

(1923) as parasitic on Fauchea laciniata (= Gloiocladia laciniata). At this time, we cannot accurately determine the number of parasitic species for Faucheocolax. The current data suggest three with two of those being specific to G. laciniata. One parasitic species group was resolved (based on COI-5P, rbcL and ITS) as genetically identical to its host species, which was G. laciniata (Table 3.1; Figure 3.3). A second parasitic species included collections that were genetically distinct (based on COI-5P, rbcL and

ITS) from their host (genetically assigned to G. laciniata) and were related to the

Californian species G. media (Table 3.1; Figure 3.3). The last parasitic species group included two genetically identical specimens from Washington that were resolved (based on ITS) by Goff et al. (1996) as genetically distinct from their two host species.

Comparison of our data for F. attenuata and Gloiocladia spp. to the previous indicated that the parasite species in Goff et al. (1996) was not a molecular match to either of our parasite genetic groups. Further, the host specimen Goff et al. (1996) identified as G. fryeana is G. laciniata, while their host specimen identified as G. fryeana is G. vigneaultii extending the host range of their parasite to our new species. A source of uncertainty is the possibility that our genetic group that resolved as identical to its host is actually not representative of a parasitic genetic group, but rather is sequence derived from host DNA. Owing to the previous, future studies aimed at eliminating host DNA contamination are needed to accurately clarify the number of species parasitic on their respective Gloiocladia hosts. Our collections that resolved distantly from their host (G. laciniata) and allied to G. media likely represent an alloparasite, while our other parasite collections that resolved as genetically identical to their host G. laciniata represent a 112

recently diverged adelphoparasite. It is challenging to establish which parasite lineage is likely representative of true F. attenuata. Indeed, before any formal taxonomic changes are made, more work is needed to better understand host-parasite diversity in the

Northeast Pacific. Regardless to the number of parasitic species and their relative host specificity, these species should be assigned to the same genus (Fig. 3.3).

Minium

Current family-level distinction in the Rhodymeniales is based, in part, on the ontogeny of the tetrasporangia (Dalen & Sunders 2007; Le Gall et al. 2008; Filloramo &

Saunders 2016a). The most recent phylogenetic assessment of the Rhodymeniales supported inclusion of the monospecific genus Minium within the Fryeellaceae

(Filloramo & Saunders 2016a); however, that study lacked anatomical observations to determine if the vegetative and reproductive features of Minium were consistent with those considered diagnostic of the Fryeellaceae. Most members of the Fryeellaceae

(Fryeella, Hymenocladiopsis and Pseudohalopeltis) feature adventitious growth in tetrasporangial development whereby the outer cortical cells swell and either convert to tetrasporangia or divide to produce 3-4 celled paraphyses with a basal cell that becomes elongated between the enlarging tetrasporangia (Le Gall et al. 2008; Saunders &

McDonald 2010). In his original description of Minium, Moe (1979) did not report adventitious growth of the cortex during tetrasporangial differentiation; however, this fine distinction in tetrasporangial development is relatively new (Le Gall et al. 2008) and would not have been considered at that time. In addition, Moe (1979) not surprisingly 113

viewed Minium as a crust with reproductive features formed in an extensive nemathecia in analogous comparison to the crustose Peyssonneliaceae. Moe (1979) reported a “basal crustose layer” composed of branched filaments that were generated from a monostromatic layer of cells. In Moe’s interpretation, this basal layer thickened to produce nemathecia (the cells of which were joined by secondary pit connections) for both the gametophyte and tetrasporophyte generations. Owing to our molecular results, we view Moe’s “basal crustose layer” as equivalent to an extensive holdfast and his nemathecia as reduced upright thalli. The development of the upright thallus and onset of reproduction appear to occur in quick succession as the diminutive upright thalli were always female gametophytes with developed carposporophytes or the tetrasporophyte generation with well-developed tetrasporangial nemathecia (male gametophytes not observed). In our interpretation, postfertilization events and development of the carposporophyte do not occur within a “nemathecium”, but within the upright (although diminutive) axes, which is typical of Rhodymeniales. Vegetative development of the tetrasporophyte was fundamentally similar to the carposporophyte, but included the production of a true fryeellacean nemathecium (including tetrasporangia and associated paraphyses) on the dorsal surface of the diminutive upright axes. Although details were challenging to decipher, the absence of secondary pit connections among cells of the nemathecium compared to the abundance of secondary pit connections among cells of the reduced upright thallus suggests that tetrasporgangia and paraphyses develop as a result of adventitious growth of the outermost cells of the reduced upright rather than from a direct transition of those outer cells. Owing to reproductive similarities between Minium and Fryeella, as well as their close affiliation in phylogenetic analyses (Fig. 3.3), it would 114

not be inappropriate to subsume the former into the latter; however, due to the unique gross morphology of Minium, we continue to recognize it as a distinct genus in the

Fryeellaceae.

Rhodymenia

Rhodymenia californica and R. pacifica were both described from California by

Kylin (1931) who distinguished them according to thallus size and individual branch width (R. pacifica being taller with broader individual blades compared to R. californica).

For the present study, those criteria were used to field identify specimens as either R. californica or R. pacifica. The resolution of cryptic complexes for each of those species indicated that Kylin’s diagnostic features were taxonomically unreliable. Interestingly, genetic data resolved R. californica and R. pacifica as more closely related than either is to their overlooked counterpart (Fig. 3.2), which indicates that similarities among the complexes were derived at least in part by convergence.

Both genetic groups assigned to R. californica were consistent with the prototype description of R. californica (Kylin 1931). One of the genetic species groups included collections from the type locality of that species, Pacific Grove, CA and was consequently deemed representative of R. californica, while the second group was rare and limited to southern British Columbia and northern Washington and was described as

R. bamfieldensis sp. nov. A comprehensive molecular-assisted floristic survey of

California and Oregon is required to determine the southern distribution of R. bamfieldensis relative to R. californica (genetically confirmed from British Columbia,

Oregon and California). Although our collections of R. californica significantly 115

outnumber those of R. bamfieldensis, it is possible that some of the southern British

Columbia and northern Washington specimens that were attributed to R. californica by

Hawkes & Scagel (1986) were in fact R. bamfieldensis.

The only observed difference between R. bamfieldensis and R. californica was individual branch width, which was typically less for the latter species. Although this feature may be helpful in the field, species identifications should be confirmed by genetic data as thinner collections of R. bamfieldensis and thicker collections of R. californica overlap in size. Included in our collections of R. californica were the first records from

British Columbia of female and male gametophytes (Fig. 3.11e,f). Interestingly, all gametophytic collections of this species were collected from Haida Gwaii in northern

British Columbia. Unfortunately, we lacked reproductive material of R. bamfieldensis for comparison. Future collections of reproductive R. bamfieldensis, if they exist, may provide useful characters for distinguishing it from R. californica.

One of our genetic species groups field identified as R. pacifica was anatomically consistent with Kylin’s (1931) protolog description and also included topotype material.

As a result, the aforementioned group was attributed to R. pacifica. The second genetic group was morphologically assigned to R. rhizoides, which was described from San

Diego, California by Dawson (1941) who considered the prominent stipes and abundant stolons of R. rhizoides as justification for distinguishing that species from R. pacifica.

Dawson (1963) later rejected the significance of those differences and synonymized R. rhizoides with R. pacifica suggesting that this species was highly variable in morphology.

Our anatomical observations herein indicate that Dawson’s initial segregation of these species was warranted as collections attributed to Rhodymenia rhizoides were 116

distinguished from those assigned to R. pacifica according to the following criteria: prominent stipes, abundance of stolons, fewer medullary cell layers, smaller medullary cells and thinner blades in cross section. Recognition of R. rhizoides in British Columbia represents a northern range extension for this species. Traditionally, R. pacifica was reported from northern British Columbia to northern Baja Mexico (Guiry & Guiry 2016); however, some of those reports may be based on R. rhizoides. Future molecular studies are needed to clarify the distribution of R. pacifica relative to R. rhizoides in the northeast

Pacific. At present, R. rhizoides has a disjunct distribution between central California and northern British Columbia (only two collections were from southern British Columbia;

Appendix B, Table S1). If that distribution pattern holds, R. rhizoides adds to a growing number of species that may have migrated to northern British Columbia from California on the “kelp conveyor” (i.e., the northern migration of Californian species growing on substrata that is carried along with kelp rafts to northern British Columbia on the winter

Davidson Current; Saunders 2014).

Key to species of Rhodymenia

1a. Mature plants usually under 60 mm in height; individual branches mostly less than 6 mm wide..……..…………..……………………..……………………..….……………... 2

1b. Mature plants typically 20-90 mm in height; individual branches 4-13 mm wide...... 3

2a. Individual branches 3.6-6 mm wide (Fig. 3.11a,b).…………..…..… R. bamfieldensis

2b. Individual branches 0.5-4 mm wide (Fig. 3.11c-g).……………...…….. R. californica 117

3a. Blades 162-250 μm in cross section with 3-4 layers of large round medullary cells 67-

76 μm in diameter; stolons not prominent (Fig. 3.12a,b).……...... ….………… R. pacifica

3b. Blades 144-170 μm in cross section with 2-3 layers of large round medullary cells 35-

56 μm in diameter; stolons prominent and abundant (Fig. 3.12c,d)...... ….. R. rhizoides

Acknowledgements

We thank all those listed in Appendix B, Table S1 for their collection efforts in British

Columbia, California and Washington. Additional thanks to T. Moore and former

Saunders’ lab members for generating some of the molecular data used in our analyses.

Thank you to Leandre Vigneault and Lynn Lee of Marine Toad Enterprises for dive support in Haida Gwaii. We also thank Dr. Norm Sloan, Parks Canada and the Haida

Nation, especially the staff of the Gwaii Haanas National Park Reserve and Haida

Heritage Site, for their encouragement and support of our efforts to understand the algal flora from this locale. Thank you to the Bamfield Marine Sciences Centre for hosting some of the fieldwork for this study. Sincerest gratitude to C.W. Schneider for his assistance with Latin nomenclature, M. Hawkes for his thoughtful correspondence regarding type collections of Gloiocladia, K.A. Miller for organizing a generous loan of

California Gloiocladia samples as well as type material from the University and Jepson

Herbaria of the University of California, Berkeley and U. Arup for providing type material from the Botanical Museum at Lund University Biological Museum. This

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research was supported through funding to the Canadian Barcode of Life Network from

Genome Canada through the Ontario Genomics Institute, Natural Sciences and

Engineering Research Council of Canada (NSERC) and other sponsors listed at www.BOLNET.ca. Support was also provided by the Canada Research Chair Program, the Canada Foundation for Innovation and the New Brunswick Innovation Foundation.

References

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Table 3.1 Intraspecific COI-5P divergence and distance to nearest neighbor for rhodymenialean species included in this study.

Max Distance intraspecific to nearest divergence neighbor Species (%) Nearest species (%) Botryocladia hawkesii Botryocladia (n= 27) 0.31 pseudodichotoma 5.13 Botryocladia pseudodichotoma (n= 7)* 0.15 Botryocladia hawkesii 5.13 Faucheocolax attenuata (n= 2) 0.30 Gloiocladia media 2.67 Faucheocolax attenuata (n= 3) † 0 Gloiocladia laciniata 0 Fryeella callophyllidoides (n= 3) 1.53 Fryeella gardneri 6.50 Fryeella gardneri Fryeella (n= 63) 0.61 callophyllidoides 6.50 Gloiocladia fryeana (n= 21) 0.46 Gloiocladia media 13.14 Gloiocladia media (n= 1)* N/A Faucheocolax attenuata 2.67 Gloiocladia laciniata Faucheocolax attenuata 0 (n= 74) † 0.76 Gloiocladia vigneaultii 3.73 Gloiocladia vigneaultii (n= 14) 0.76 Gloiocladia laciniata 3.73 Leptofauchea pacifica Neogastroclonium (n= 36) 0.30 subarticulatum 17.76 Lomentaria hakodatensis (n= 20) 0 Fryeella gardneri 20.46 Minium parvum (n= 6) 0 Fryeella gardneri 9.24 Neogastroclonium subarticulatum Fryeella (n= 31) 0.93 callophyllidoides 16.87 Rhodymenia bamfieldensis (n= 7) 0 Rhodymenia rhizoides 3.26 Rhodymenia californica (n= 138) 0.30 Rhodymenia pacifica 7.18 Rhodymenia pacifica (n= 16) 0.30 Rhodymenia californica 7.18

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Rhodymenia rhizoides Rhodymenia (n= 27) 0.63 bamfieldensis 3.26 Sparlingia pertusa (n= 81) 0.77 Rhodymenia californica 17.34

* Species collected exclusively from California.

† Some specimens of Faucheocolax attenuata were resolved as genetically identical to their host species Gloiocladia laciniata.

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Figure 3.1 Simplified schematic of ITS1 for representatives of Faucheocolax attenuata and Gloiocladia species. The asterisk (*) indicates the F. attenuata genetic group that was genetically distinct from its host species, while the single cross () refers to GenBank data (U30366 & U30365) assigned to F. attenuata and is included here for comparison. The double cross (‡) refers to the F. attenuata genetic group that was genetically identical to its host, G. laciniata.

For all species, ITS1 included a 5’ variable region (gray) followed by a series of repetitive elements (~160 bp), which occurred as a complete unit (gray and white horizontal stripes), as a complete unit interrupted by an indel (white with dash indicating indel) or as only a portion of the 5’ (black) or 3’ (black and white diagonal stripes) end. Additional indels indicated by black line.

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Figure 3.2 Phylogenetic tree resulting from RAxML analysis of the concatenated

COI-5P and rbcL alignment for representatives of Botryocladia and Rhodymenia

(general biogeography included in parentheses) and select rhodymeniaceaen taxa as the outgroup. Bootstrap support values appended with asterisks indicating full branch support and no value (-) indicating branch support below 50%. Taxa in bold font represent species emphasized in the current study. Country/Province/State abbreviations are as follows: “BC”= British Columbia, Canada; “CA”= California,

United States; “OR”= Oregon, United States; and, “WA”= Washington, United

States.

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128

Figure 3.3 Phylogenetic tree resulting from RAxML analysis of the concatenated

COI-5P and rbcL alignment for representatives of the Faucheaceae and

Fryeellaceae (general biogeography included in parentheses). Bootstrap support values appended with asterisks indicating full branch support and no value (-) indicating branch support below 50%. GenBank accession numbers are cited for data obtained from GenBank. Taxa in bold font represent species emphasized in the current study. Country/Province/State abbreviations are as follows: “BC”= British

Columbia, Canada; “CA”= California, United States; and, “WA”= Washington,

United States.

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Figure 3.4 Botryocladia hawkesii Filloramo & G.W.Saunders sp. nov. a) Habit of vegetative holotype (GWS009487). Scale Bar = 16 mm. b) Vegetative anatomy, cross section at mid-vesicle (GWS003120). Scale Bar = 40 m. c) Surface view of gland cells borne on medullary cell (GWS004852). Scale Bar = 50 m. d) Cross section of mature male gametophyte showing spermatangial mother cells borne on cortical cells. Gland cells (gc) also indicated on a medullary cell (GWS009461). Scale Bar =

47 m. e) Habit of female gametophyte. (GWS009082). Scale rules are in millimeters. f) Transverse section showing an ostiolate cystocarp that is protruding inwards. Arrow indicates developing ostiole (GWS009082). Scale Bar = 255 m. g)

Transverse section of tetrasporangial sorus showing cruciately divided tetrasporangia in an unmodified cortex (GWS009081). Scale Bar = 72 m.

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Figure 3.5 Fryeella callophyllidoides (Hollenberg & I.A.Abbott) Filloramo &

G.W.Saunders comb. nov. a) Habit of tetrasporangial epitype (UC1455019). Scale

Bar = 7 cm. b) Cross section of vegetative portion of tetrasporangial epitype

(UC1455019). Scale Bar = 110 μm. c) Cross section of tetrasporangial sorus showing developing tetrasporangia embedded among paraphyseal filaments (UC1455019).

Scale Bar = 75 μm. d) Habit of vegetative specimen from Monterey Bay, California

(GWS014349). Scale Bar = 18 mm. e) Cross section of vegetative specimen

(GWS014349). Scale Bar = 87 μm. f) Habit of tetrasporophyte showing tetrasporangial sori (arrows) (GWS022358). Scale Bar = 13 mm. g) Cross section of tetrasporangial sorus (GWS022358). Scale Bar = 80 μm.

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Figure 3.6 Gloiocladia fryeana. a) Habit of female gametophyte showing marginal cysocarps (GWS008730). Scale bar = 20 mm. b) Habit of tetrasporophyte

(GWS019620). Scale bar = 20 mm. c) Enlarged image of the cortical cell filaments

(GWS10426). Scale bar = 23 μm. d) Cross section of vegetative plant (GWS010426).

Scale bar = 128 μm. e) Cross section of tetrasporophyte showing developing tetrasporangia on cortical cell (arrow), terminally on single-celled filament

(arrowhead) or laterally on paraphyseal filaments (double arrowhead)

(GWS028558). Scale bar = 65 μm. f) Slightly coronate, ostiolate cystocarps exclusively on thallus margin. (GWS008730). Scale bar = 1.6 mm. g) Cross section of cystocarp with visible tela arachnoidea surrounding the developing carposporangia.

(GWS008730). Scale bar = 283 μm.

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Figure 3.7 Gloiocladia laciniata. a) Habit of cystocarpic holotype specimen

(LD 25708). Scale bar = 37 mm. b) Cross section of vegetative portion of the cystocarpic holotype specimen (LD 25708). Scale bar = 102 μm. c)

Enlarged image showing the cortical cell filaments. Scale bar = 12 μm. d)

Distinctly coronate cystocarp located marginally on holotype specimen

(LD 25708). Scale bar = 558 μm. e) Cross section of a cystocarp showing tela arachnoidea as well as ostiole (LD 25708). Scale bar = 231 μm. f) Habit of female gametophyte from British Columbia showing predominantly marginally located cystocarps with a few scattered on the blade surface

(GWS004324). Scale bar = 43 mm. g) Habit of female gametophyte from

British Columbia showing cystocarps scattered on both the blade surface and margins (GWS027923). Scale bar = 22 mm. h) Cross section of vegetative specimen (GWS021106). Scale bar = 95 μm. i) Enlarged image showing the cortical cell filaments. Scale bar = 19 μm. j) Distinctly coronate cystocarps located marginally and on thallus surface.

(GWS027923). Scale bar = 900 μm. k) Cross section of cystocarp showing ostiole and tela arachnoidea surrounding the developing carposporangia

(GWS027923). Scale bar = 268 μm.

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Figure 3.8 Gloiocladia media (Kylin) Filloramo & G.W.Saunders comb. nov. a)

Habit of cystocarpic holotype specimen (arrow) from Pacific Grove, California

(LD1613738). Scale bar = 28 mm. b) Cross section of vegetative portion of cystocarpic holotype (LD1613738). Scale bar = 99 μm. c) Enlarged image showing the cortical cell filaments of the dorsal cortex. Scale bar = 9 μm. d) Enlarged image showing the cortical cell filaments of the ventral cortex. Scale bar = 11 μm. e)

Round, ostiolate cystocarps scattered over blade surface of holotype specimen

(LD1613738). Scale bar = 241 μm. f) Cross section of a cystocarp of the holotype specimen showing ostiole and tela arachnoidea surrounding the developing carposporangia (LD1613738). Scale bar = 196 μm. g) Habit of cystocarpic specimen from Monterey, California (GWS021443). Scale rules are in millimeters. h) Cross section of vegetative portion of cystocarpic specimen (GWS021443). Scale bar = 74

μm. i) Round, ostiolate cystocarps scattered over blade surface. (GWS021443). Scale bar = 1000 μm. j) Cross section of cystocarp showing ostiole and tela arachnoidea surrounding the developing carposporangia (GWS021443). Scale bar = 209 μm.

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Figure 3.9 Gloiocladia vigneaultii Filloramo & G.W.Saunders sp. nov. a) Habit of the holotype specimen with cystocarps scattered on blade surface and margins

(GWS030615). Scale bar = 17 mm. b) Cross section of vegetative portion of cystocarpic holotype showing obvious cuticle on both blade surfaces (GWS030615).

Scale bar = 88 μm. c) Enlarged image showing cortical cell filaments (GWS030615).

Scale bar = 20 μm. d) Cystocarpic specimen with coronate cystocarps scattered on blade surface. (GWS036877). Scale bar = 20 mm. e) Cystocarpic specimen with exclusively marginal cystocarps (GWS008496). Scale bar = 16 mm. f) Developing cystocarps on both blade surfaces of the holotype specimen, tela arachnoidea visible

(GWS030615). Scale bar = 400 μm. g) Cross section of cystocarp showing ostiole and tela arachnoidea surrounding the developing carposporangia (GWS030615). Scale bar = 187 μm. h) Cross section of tetrasporophyte showing developing and mature cruciately divided tetrasporangia (GWS027428). Scale bar = 91 μm.

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Figure 3.10 Minium parvum. a) Tetrasporangial specimen (arrows) (GWS019580) on

Calliarthron tuberculosum. Scale bar = 12 mm. b) Transverse section of extensive basal holdfast, which is growing on C. tuberculosum. Secondary pit connections

(arrowheads) visible between cells of neighboring upright filaments (GWS020962).

Scale bar = 27 μm. c) Transverse section of reduced erect thallus extending from the holdfast with an extensive tetrasporangial sorus on the upper surface. Secondary pit connections (arrowhead) among cells of the upright thallus as well as apically borne tetrasporagnia. Arrows indicate the portion of the reduced thallus growing over the basal holdfast. Scale bar = 142 μm. d) Mature cruciately divided tetrasporangia (arrow) born apically on filaments. Paraphyses composed of small spherical cells (arrowheads) borne on cells that become elongated and compressed between the developing tetrasporangia (GWS020962). Scale bar = 60 μm.

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Figure 3.11 Rhodymenia spp. a) R. bamfieldensis Filloramo & G.W.Saunders sp. nov. vegetative habit of the holotype (GWS014391). Scale bar = 46 mm. b)

Transverse section of R. bamfieldensis sp. nov. at thallus midpoint of vegetative holotype (GWS014391). Scale bar = 86 μm. c) Vegetative habit of R. californica

(GWS022228). Scale Bar = 27 mm. d) Transverse section of vegetative specimen of

R. californica (GWS022284). Scale bar = 92 μm. e) Cross section of cystocarp of R. californica showing developing carposporophyte (GWS038588). Scale bar = 231 μm. f) Cross section of spermatangial sorus of R. californica showing sporangia developing from outer cortical cells (GWS038591). Scale bar = 66 μm. g) Cross section of tetrasporophyte of R. californica showing developing and mature cruciately divided tetrasporangia in an unmodified cortex (GWS003239). Scale bar

= 47 μm.

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Figure 3.12 Rhodymenia spp. a) Vegetative habit of R. pacifica (GWS022080). Scale bar = 46 mm. b) Transverse section of vegetative specimen of R. pacifica

(GWS021330). Scale bar = 125 μm. c) Vegetative habit of R. rhizoides (GWS022378).

Scale bar = 46 mm. d) Transverse section of vegetative specimen of R. rhizoides

(GWS022378). Scale bar = 80 μm.

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Chapter 4 A re-examination of the genus Leptofauchea (Faucheaceae,

Rhodymeniales) with clarification of species in Australia and the

northwest Pacific

Citation for published manuscript: Filloramo, G.V. & Saunders, G.W. (2015). A re- examination of the genus Leptofauchea (Faucheaceae, Rhodymeniales) with clarification of species in Australia and the northwest Pacific. Phycologia, 54: 375-384.

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Abstract

Recently collected material from Coffs Harbour, New South Wales, Australia was identified as the generitype of Leptofauchea, L nitophylloides. This facilitated a re- examination of Leptofauchea and its constituent species to assess monophyly. Multigene phylogenetic analysis of COI-5P, LSU and rbcL solidly allied species assigned to

Leptofauchea in previous studies with the generitype, establishing for the first time monophyly for this genus. In addition to L. nitophylloides, COI-5P barcode analyses revealed a second Australian species of Leptofauchea from the Cocos (Keeling) Islands,

Indian Ocean, described as L. cocosana sp. nov. Further, COI-5P barcode analyses resolved collections assignable to Leptofauchea from South Korea as two genetic species groups. One of these groups was L. leptophylla while the second genetic species group warranted description of L. munseomica sp. nov. GenBank entries for the two species of

Leptofauchea previously reported in the northwest Pacific (L. leptophylla described from

Japan and L. rhodymenioides described from the Caribbean) were resolved as conspecific

(a cryptic complex at best) and morphologically assigned to L. leptophylla.

Introduction

The red algal order Rhodymeniales is composed of six families differentiated by the number of cells in the carpogonial branch, tetrasporangial cleavage patterns and the ontogeny of the tetrasporangial nemathecia (Le Gall et al. 2008). The Faucheaceae was established by Saunders et al. (1999) and accommodates genera with three-celled carpogonial branches, typically well-developed tela arachnoidea in the cystocarp,

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cruciate tetrasporangia and adventitious cortical growth during nemathecial development

(Dalen & Saunders 2007; Le Gall et al. 2008). The genus Leptofauchea Kylin (1931) was included in the Faucheaceae based on the Australian species L. nitophylloides (J. Agardh)

Kylin (Dalen & Saunders 2007).

Dalen & Saunders' (2007) recent monograph of Leptofauchea added considerably to Kylin’s (1931) original description of the genus by distinguishing the following diagnostic criteria: a medulla of 2-3 cell layers grading abruptly in size to an incomplete layer of inner cortical cells subtending (1-) 2-3 (-4) irregular layers of small pigmented cells, a well-developed tela arachnoidea in the cystocarp, and adventitious cortical growth in the formation of cruciately divided tetrasporangia and paraphyses. In addition to the generitype, Dalen & Saunders (2007) included L. chiloensis Dalen et G.W.

Saunders from Chile, L. pacifica E.Y. Dawson from Mexico and L. rhodymenioides W.R.

Taylor from the Netherlands Antilles in this genus. Although Dawson’s (1963) description of Mexican material as L. auricularis E.Y. Dawson was consistent with the vegetative construction typical of Leptofauchea, archival material was not available for comparison; therefore the taxonomic assignment could not be confirmed (Dalen &

Saunders 2007). Dalen & Saunders (2007) excluded L. anastomosans (Weber-van Bosse)

R.E. Norris et Aken from Indonesia, which had been transferred to the genus

Asteromenia (Saunders et al. 2006). In the same study, Dalen & Saunders (2007) further questioned the generic- and species-level assignment of plants from South Africa attributed to L. anastomosans by Norris & Aken (1985), which featured vegetative construction consistent with Leptofauchea, but lacked tela arachnoidea in the cystocarp.

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Other species excluded by Dalen & Saunders (2007) included L. brasiliensis A.B. Joly from Brazil characterized by a single layered medulla and tetrasporangia produced in chains similar to members of the Phyllophoraceae (Schneider & Searles 1976; Dalen &

Saunders 2007), to which it has recently been assigned (Schneider et al. 2011), and L. earleae Gavio et Fredericq from the Gulf of Mexico, which featured a monostromatic medulla and lacked tela arachnoidea in the cystocarp (Gavio & Fredericq 2005).

At the time of the Dalen & Saunders (2007) monograph, the only Leptofauchea species included in molecular analyses were L. chiloensis, L. pacifica and an unidentified species from Western Australia represented by a single specimen. These species resolved as a distinct lineage in the Faucheaceae; however, without molecular representation of the generitype, monophyly of the genus was uncertain (Dalen & Saunders 2007). In the interim, subsequent molecular and morphological studies increased the number of species assigned to Leptofauchea. Leptofauchea coralligena Rodríguez-Prieto et De Clerck was described from the Mediterranean Sea (Rodríguez-Prieto & De Clerck 2009). In addition,

L. leptophylla (Segawa) Suzuki, Nozaki, Terada, Kitayama, Hashimoto & Yoshizaki was recognized based on a poorly known Japanese faucheacean species previously assigned to Gloiocladia J. Agardh (Suzuki et al. 2012).

Prior to the transfer of L. leptophylla to Leptofauchea, some specimens from northeastern Japan were attributed to the Caribbean species L. rhodymenioides (Suzuki et al. 2010) greatly extending its reported range (Taylor 1942; Guiry & Guiry 2014). Suzuki et al. (2010) did not clarify if the recent collections were compared to type material, nor was material from near the type locality included in their molecular analyses. The

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disjunct biogeographical pattern between the Caribbean type and the cold-water Japanese collections warranted a reassessment of this determination.

For the present study we used COI-5P to assign recent Leptofauchea collections from Australia and South Korea to genetic species groups. Subsequent morphological assessment identified one of these genetic groups as the generitype, L. nitophylloides.

When included in multigene analyses with novel and previously described Leptofauchea species this material provided the necessary genetic data to assess monophyly of this genus for the first time.

Materials and Methods

All specimens used in this study were collected in the subtidal by SCUBA (collection information included in Appendix C, Table S1). Plants were pressed or dried in silica to serve as vouchers with subsamples preserved in silica gel for molecular analyses. Sample processing and DNA extraction followed Saunders & McDevit (2012) with amplification of the 5’ end of the cytochrome c oxidase subunit 1 gene (COI-5P), the ribulose-1, 5- biphosphate carboxylase large subunit (rbcL) gene and the nuclear large-subunit ribosomal RNA (LSU) gene as outlined in Saunders & Moore (2013). Primer pair combinations for COI-5P are recorded with accessions in the Barcode of Life Database

(BOLD) (http://www.boldsystems.org/) and GenBank. Amplified PCR products were sequenced by the Génome Québec Innovation Centre. Raw data were edited using

Sequencher™ 5.0 (Gene Codes Corporation, Ann Arbor, MI, USA) and aligned in

Geneious R7 version 7.1.7 (Biomatters, http://www.geneious.com/).

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Using BOLD, COI-5P data were searched and subsequently subjected to a barcode gap analysis to determine intraspecific variation and distance to nearest neighbors. LSU and rbcL data were generated for one representative from each genetic species group resolved during the COI-5P analyses with faucheacean species from the genera

Gloiocladia, Gloioderma and Webervanbossea included as outgroup taxa (Appendix C,

Table S1; Table 4.1). Additional sequence data were acquired from GenBank for all available Leptofauchea species (Table 4.1). Single gene alignments were analyzed using

RAxML 7.2.8 (Stamatakis et al. 2008) in Geneious R7 version 7.1.7 under a GTR+I+G model with partitioning by codon (COI-5P, rbcL) and nodal support determined by 1000 rounds of bootstrap resampling. All genes resolved congruent phylogenetic relationships and were combined to generate a multigene alignment (18 taxa, 4779 bp). Taxa listed in

Table 4.1 were represented by COI-5P, LSU and rbcL data with the exception of L. chiloensis (lacked COI-5P), L. coralligena (lacked COI-5P and rbcL) and L. earleae

(lacked COI-5P and LSU). Maximum likelihood (ML) analysis was performed using

RAxML 7.2.8 (Stamatakis et al. 2008) in Geneious R7 version 7.1.7 under a GTR+I+G model with partitioning by gene and then by codon for COI-5P and rbcL. Robustness was assessed using 1000 bootstrap replicates.

Morphological analyses were completed by rehydrating algal tissue in 5% formaldehyde in seawater, and preparing sections using a freezing microtome (CM18250,

Leica, Heidelberg, Germany). Sections were stained with 1% aniline blue solution in 6%

5N HCl and mounted in 50% corn syrup with 4% formaldehyde. Features were observed

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using a Leica DM5000B light microscope and photographed with a Leica DFC480 digital camera.

Results

COI-5P Barcode results

Barcode gap analyses resolved the single Australian collections from the Cocos (Keeling)

Islands and Coffs Harbour, New South Wales and seven South Korean collections from

Jeju as four distinct genetic species groups (Table 4.2). The specimen from Coffs

Harbour was morphologically assigned to the generitype L. nitophylloides (below) and was distantly related to its nearest neighbor L. pacifica (10.82%) (Table 4.2). The collection from the Cocos was described as the novel species L. cocosana (below) and was most closely related (11.02%) to L. munseomica, a new species described herein from South Korea (Table 4.2). The second South Korean genetic species group was representative of the Asian species L. leptophylla and its nearest neighbor was

Leptofauchea sp._1WA (9.63%) (Table 4.2).

Phylogenetic results

Single and multigene analyses were used to explore relationships within Leptofauchea.

Initial rbcL analysis resolved our South Korean isolate of L. leptophylla (GWS018540) with the GenBank entries for L. leptophylla (AB693120) and L. rhodymenioides

(AB383124); however, AB693120 resolved on an anomalously long branch. Observation of this latter rbcL alignment revealed that the sequence AB693120 was chimeric with the

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first ~640 bp matching Champia sp. while the final ~730 bp were almost identical to our sequence for South Korean L. leptophylla (a single substitution at a third codon position).

Comparison of our sequence for South Korean L. leptophylla to the GenBank entry for L. rhodymenioides (AB383124) revealed two base pair differences (one at a second codon position and the other at a third codon position) over 1362 bp of the rbcL. As second codon positions are conservative, this difference may represent an error, indicating that only a single or, at most, two differences were present for the rbcL of these specimens.

The low divergence among the three rbcL sequences suggested conspecificity. The LSU data resolved similar results. Over a total alignment of 2753 bp, the LSU sequence for our South Korean isolate of L. leptophylla (GWS018362) had 6 bp differences when compared to the GenBank entry for L. leptophylla (AB677823) with two of these uncertain (at highly conserved sites). Comparison of the LSU sequence for our L. leptophylla specimen from South Korean to the GenBank entry for L. rhodymenioides

(AB677824) revealed only 2 bp differences, both within variable regions. Overall, the observed differences in rbcL and LSU data likely reflect population level differences or at best two closely related species groups for L. leptophylla in the northwest Pacific accounting for previous reports of L. leptophylla and L. rhodymenioides (discussed below). Owing to the previous, we included LSU and rbcL data only from our South

Korean representative of L. leptophylla in the multigene phylogeny.

Maximum likelihood analysis of the multigene alignment established monophyly for the genus Leptofauchea in placing species currently assigned to this genus in a fully supported group with the generitype L. nitophylloides (Fig. 4.1). Relationships among

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species of Leptofauchea were resolved with variable support. Leptofauchea chiloensis, L. coralligena, L. nitophylloides and L. pacifica formed distinct lineages while L. cocosana and L. munseomica were fully resolved as sister species and allied to the fully supported group comprised of L. earleae, L. leptophylla and Leptofauchea sp._1WA (Fig. 4.1).

Morphological results

Leptofauchea nitophylloides (J. Agardh) Kylin

Our specimen of L. nitophylloides was a diminutive plant (15-20 mm tall) with rounded to attenuate apices (Fig. 4.2a). Thallus thickness in cross-section was 70-80 µm. It had a medulla of two layers of large, elongated cells (35-60 µm long x 13-25 µm wide), an incomplete inner cortical layer, and an outer cortex comprised of a single layer of small cells (to 5 µm long x 2.5 µm wide) (Fig. 4.2b). In surface view, the cortical layer was complete, although underlying medullary cells were visible (Fig. 4.2c). Reproductive structures were not observed.

Leptofauchea cocosana Filloramo & G.W. Saunders sp. nov. Fig. 4.3

DESCRIPTION: Thallus of flat, prostrate blades, ~28 mm in dimension; individual blades ~8 mm in length and 4 mm broad at base, typically narrowing at the apex (~1.5-

2.0 mm wide) and forming secondary attachments to each other as well as the substratum

(Fig. 4.3a). Cross-sections at mid-thallus were 65-95 µm wide with 2 (-3) layers of large

(30-50 µm long x 17.5-30.0 µm wide), thin-walled, axially elongated medullary cells

(Fig. 4.3b) These supported a cortex of smaller, round cells (7-16 µm in diam.), typically

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with an incomplete second layer of small, scattered, darkly pigmented cells as seen in surface view (Fig. 4.3c). Female reproductive anatomy and early post-fertilization events were not observed. Cystocarps (570 um in diameter) distributed at blade margins (Fig.

4.3a), smooth, ampulliform, ostiolate, with well-defined tela arachnoidea (Fig. 4.3d).

Spermatangia and tetrasporangia unknown.

HOLOTYPE: GWS037755 (cystocarpic) deposited in Connell Memorial Herbarium

(UNB). Images of the holotype shown in Fig. 4.3. Collected from Winter Wall, south of

Horsburgh Island, Cocos (Keeling) Islands, Australia, Indian Ocean (-12.08788ºS,

96.83701ºE), 12 May 2013; from 15 m on reef flat; leg. K. Dixon and G.W. Saunders.

Holotype DNA barcode: GenBank KR140330, COI-5P.

ETYMOLOGY: Named for the type locality, Cocos (Keeling) Islands, Australia.

DISTRIBUTION: Thus far only known from the Cocos (Keeling) Islands, Australia.

Leptofauchea leptophylla (Segawa) Suzuki, Nozaki, Terada, Kitayama, Hashimoto &

Yoshizaki

South Korean collections of L. leptophylla were 25-40 mm tall and forked 3-4 times with mostly rounded/obtuse, at times attenuate apices (Fig. 4.4a,b). Some blades were constricted below the apices (Fig. 4.4b). At mid-thallus, blades were 130-150 µm thick featuring 2 (-3) layers of large, polygonal to elliptical medullary cells (55-90 µm long x

40-80 µm wide) with abundant secondary pit connections (Fig. 4.4c). Small, axially elongated cells formed a single layer between the medullary cells and cortex (Fig. 4.4c).

The cortex is comprised of 2 (-3) cell layers; a continuous innermost layer of cells 7.5 µm

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in diam. and 1 (-2) outer layers of smaller irregularly arranged cells (2-4 µm in diam.)

(Fig. 4.4c). Among the South Korean collections was a single tetrasporophyte with tetrasporangia formed in adventitious nemathecial sori. These appeared as bands or patches that extended to the thallus margin, and present from mid-thallus to branch apices confined to a single blade surface (Fig. 4.4d). Mature tetrasporangia (50-55 µm long x

25-30 µm wide) were cruciately divided (Fig. 4.4e). The initials arose directly on modified cortical cells (Fig. 4.4f), either terminal on single celled filaments or lateral on

5-8 celled paraphyses (to 100 µm long x 5 µm wide) (Fig. 4.4g). Male and female plants were not represented in our South Korean collections.

Leptofauchea munseomica Filloramo & G.W. Saunders sp. nov. Fig. 4.5

DESCRIPTION: Thallus flat, prostrate, 30-42 mm in dimension. Individual blades flabellate, regularly dichotomously branched, 2-3 mm broad throughout. Branch apices typically rounded, at apices narrowing and becoming attenuate (0.5-1.5 mm) (Fig. 4.5a).

Cross-sections at mid-thallus were 130-180 µm thick with 3 (-4) layers of large, round, thick-walled (~2.5 µm) medullary cells (30-65 µm in diam.) commonly separated by intercellular spaces (Fig. 4.5b). Medulla graded into a single inner cortical layer of smaller cells (15-22 µm long x 7.5-12 µm wide) subtending an outer cortex of 1-2 layers, with the outermost commonly incomplete (Fig. 4.5b). Reproductive structures not observed.

HOLOTYPE: GWS018532 deposited in Connell Memorial Herbarium (UNB). Images of holotype shown in Fig. 4.5. Collected from the channel between Little and Big Munseom

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Islands, Jeju, South Korea, northwestern Pacific Ocean (33.22751ºN, 126.56802ºE), 19

May 2010; from 16 m on rock; leg. G.W. Saunders and H-G. Choi. Holotype DNA barcode: GenBank HQ544094, COI-5P.

ISOTYPES: GWS018533, GWS018537 and GWS018674 deposited in Connell

Memorial Herbarium (UNB).

ETYMOLOGY: Named for the type locality, Munseom Islands, Jeju, South Korea.

DISTRIBUTION: Thus far only known from Jeju, South Korea.

Discussion

Dalen & Saunders (2007) clarified the distinguishing features of the genus Leptofauchea, which were revisited and modified slightly based on observations herein. Useful characters for genus-level diagnosis include a medulla comprised of 2-4 cell layers grading abruptly in size to a thin cortex typically of 2-3 cell layers, well-developed tela arachnoidea in smooth, ostiolate cystocarps and well-defined tetrasporangial nemathecia with both cruciately divided tetrasporangia and paraphyses formed by adventitious growth of the cortical cells. When comparing individual Leptofauchea species to the generitype, aspects of vegetative and female reproductive anatomy were all consistent; however, Kylin (1931) lacked tetrasporophytes when he described L. nitophylloides.

Millar (1999) collected plants from Norfolk Island that he considered the first encounter with L. nitophylloides since the type collection. He noted terminal, cruciately divided tetrasporangia located in oval sori. Millar’s (1999) report lacked details of tetrasporangial

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ontogeny as the taxonomic utility of this feature was not acknowledged at that point (Le

Gall et al. 2008); however, his description (Millar 1999, p. 510, Fig. 31) was inconsistent relative to other species attributed to Leptofauchea (Dalen & Saunders 2007). This called into question the monophyly of Leptofauchea. Regardless, Dalen & Saunders (2007) retained the genus pending the inclusion of L. nitophylloides in molecular analyses. In retrospect, the tetrasporangia described for Millar's (1999) Norfolk Island collections were more consistent with the genus Halopeltis, which is species rich in Australia

(Saunders & McDonald 2010; Schneider et al. 2012) but not recognized at the time of

Millar’s 1999 publication. Species of Halopeltis feature oval to oblong tetrasporangial sori formed by the swelling of intercalary and terminal outer cortical cells without adventitious growth (Saunders & McDonald 2010). Despite numerous collecting trips to

Australia by the second author, including Norfolk Island, only three collections, one each from the Cocos (Keeling) Islands, Coffs Harbour, New South Wales and Abrolhos Island,

Western Australia, have been assignable to Leptofauchea. By comparison, over 250 specimens of Halopeltis representing 15 genetic species groups distributed widely throughout Australia were collected (GWS, unpublished data). Indeed, a collecting trip by the second author has uncovered four Halopeltis spp. (total of 13 specimens) from

Norfolk Island and one or more of these likely accounts for Millar’s (1999) tetrasporophytic observations of L. nitophylloides.

Collected about 500 km north of the type locality, our plant of Leptofauchea nitophylloides had more attenuate apices and was not as robust in height or thallus thickness compared to the type collection; however, the general appearance and number

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of cortical and medullary cell layers were consistent with observations in Kylin (1931) and Dalen & Saunders (2007). Similar phenotypic variation was noted among individuals for L. pacifica (Dawson & Neushul 1966; Abbott & Hollenberg 1976; Dalen & Saunders

2007) and L. coralligena (Rodríguez-Prieto & De Clerck 2009). Thus, variation in robustness and consequently blade thickness and medullary cell size is not unexpected within species of Leptofauchea (Dalen & Saunders 2007) and we consider our collection assignable to L. nitophylloides. Accordingly, it is the first record for this species since the type collection. This collection provided the necessary genetic data to confirm

Leptofauchea as a monophyletic lineage in which we resolved the following species as members of the genus: L. chiloensis, L. cocosana sp. nov., L. coralligena, L. leptophylla,

L. munseomica sp. nov., L. nitophylloides and L. pacifica. Leptofauchea earleae is discussed below as a special case while bona fide L. auricularis and L. rhodymenioides have yet to be included in molecular studies (below).

Previously, only Leptofauchea nitophylloides and Leptofauchea sp._1WA were recognized in Australia; however, we added L. cocosana from the Cocos (Keeling)

Islands. Two of the Australian species are differentiated by their patterns of vegetative construction (Figs 4.2b, 4.3b). Compared to L. nitophylloides (Fig. 4.2b), L. cocosana lacks an inner cortical layer. It also features a single layered cortex of larger, round cells and an incomplete secondary layer of smaller, scattered cells (Fig. 4.3b) that creates a distinct appearance in surface view (Fig. 4.3c).

In acquiring data from GenBank, we discovered that the rbcL entry for

Leptofauchea leptophylla (AB693120) from Japan was a chimeric sequence of a

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Champia sp. and L. leptophylla. Previous comparison of this chimeric sequence to the rbcL GenBank entry for putative L. rhodymenioides (AB383124) from Japan falsely suggested two genetically distinct entities in that flora. Our reassessment of those data

(using only the correct portion of the chimeric sequence) revealed their conspecificity to each other, as well as to our sequence for L. leptophylla from South Korea. Variation among the rbcL GenBank entries for L. leptophylla, the incorrectly attributed L. rhodymenioides and our South Korean representative of L. leptophylla was typical of intraspecific divergence reported for red algae (Freshwater et al. 2010). Accordingly, we conclude that these entities are conspecific, and morphologically assignable to L. leptophylla. Evaluation of LSU data was largely consistent with rbcL; however, the

GenBank entry of L. leptophylla (AB677823) from southern Japan was slightly more divergent relative to our South Korean representative of L. leptophylla and the GenBank entry incorrectly attributed to L. rhodymenioides (AB677824) from northeastern Japan.

The slight variation in rbcL and LSU data likely reflects population level differences, at most two very closely related species groups, and further research is warranted.

Consistent with our molecular findings, the anatomical features reported for Japanese collections attributed to L. rhodymenioides (Suzuki et al. 2010) reflected the authors’ later observations for L. leptophylla (Suzuki et al. 2012) and those presented here for collections from South Korea (Appendix C, Table S1; Fig. 4.4). When compared to the protologue of L. rhodymenioides, the Japanese collections attributed to this species

(Suzuki et al. 2010) differed in terms of overall thallus construction and corresponding medullary cell sizes, thallus thickness and size of tetrasporangia (Taylor 1942; Dalen &

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Saunders 2007; Suzuki et al. 2010). The disjunct distribution of L. rhodymenioides in the cold-temperate waters of northeastern Japan so far from its type locality in the Caribbean, further challenges this species determination.

In addition to Leptofauchea leptophylla, we also recognized L. munseomica in the northwest Pacific, thus far known only from South Korea. This new species differs from

L. leptophylla in overall habit and vegetative construction. While blades of L. munseomica may be as thick as L. leptophylla in cross section, it has more medullary layers of smaller cells (Figs 4.4c, 4.5b). Additionally, blades in L. munseomica are typically uniform in width throughout their length (Fig. 4.5a), whereas mature thalli of L. leptophylla typically narrow toward the apices or are constricted below the apices (Fig.

4.4b). However, as habit varies for many Leptofauchea species, anatomical differences are more reliable diagnostic features.

Dalen & Saunders (2007) questioned placement of Leptofauchea earleae in

Leptofauchea based on its uncharacteristic monostromatic medulla, lack of tela arachnoidea, and weak association with the Lomentariaceae rather than the Faucheaceae in the phylogenetic analyses of Gavio & Fredericq (2005). However, our more robust single and multigene analyses resolved L. earleae deep within Leptofauchea. The only other monostromatic species of Leptofauchea ever reported, L. brasiliensis, was recently transferred to the genus Archestenogramma in the Phyllophoraceae (Schneider et al.

2011); this would leave L. earleae as the only remaining monostromatic member of

Leptofauchea. Additional analyses are needed to confirm that the GenBank sequence

HQ400570 corresponds to the voucher used for morphological observations and on

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which this species is based. If the same collection was in fact used for molecular and morphological analyses, L. earleae would represent a relatively recent speciation (Fig.

4.1) with remarkably atypical vegetative and reproductive generic features. Alternatively,

HQ400570 may represent rbcL for the collection (LAF-27-5-00-9-3) assigned to L. rhodymenioides in Gavio & Fredericq (2005). Although the vegetative anatomy depicted for LAF-27-5-00-9-3 is typical of Leptofauchea, it is inconsistent with the protologue of

L. rhodymenioides as described by Taylor (1942). As a consequence, LAF-27-5-00-9-3 may represent a novel species of Leptofauchea in the Gulf of Mexico. Both of these anomalies require clarification. Additional molecular investigation is also needed to clarify the taxonomic position of South African plants attributed to L. anastomosans by

Norris & Aken (1985). These were characterized by a Leptofauchea-like vegetative anatomy but, similar to L. earleae, they lacked tela arachnoidea in the cystocarp (Dalen

& Saunders 2007). Resolution of these South African plants as a novel Leptofauchea species, and clarification of the anomalies previously described for L. earleae could challenge the reliability of tela arachnoidea as a useful diagnostic feature of the genus.

Further study of both of these entities is necessary.

Leptofauchea auricularis and L. rhodymenioides were the only species traditionally assigned to Leptofauchea that lacked molecular representation in our study.

Dalen & Saunders (2007) accepted L. rhodymenioides in Leptofauchea based on vegetative and reproductive observations of type material. Despite the congruence of

Dawson’s (1963) type description of L. auricularis with Leptofauchea, the status of this species could not be confirmed without material available for study (Dalen & Saunders

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2007). The taxonomic status of L. auricularis and L. rhodymenioides should be considered tentative until both are included in molecular analyses.

Acknowledgements

Without the collection assistance of H-G. Choi in South Korea and K. Dixon in Australia, this project would not have been possible. We also thank the following collectors for their contributions to this project: A. Celona, B. Clarkston, M. Cowan, R. Cowan, J.P.

Danko, G.T. Kraft and M. Vicinanza. Additional thanks to M. Brooks, J. Dalen, C. Lane,

L. Le Gall, D. McDevit, T. Moore and M. Surette for generating some of the sequence data used in our analyses. Thank you to C.W. Schneider for his assistance with Latin nomenclature. We thank S. Fredericq at the University of Louisiana at Lafayette for providing silica-dried material of L. earleae for analysis. This research was supported through funding to the Canadian Barcode of Life Network from Genome Canada through the Ontario Genomics Institute, Natural Sciences and Engineering Research Council of

Canada (NSERC) and other sponsors listed at www.BOLNET.ca. Support was also provided by the Canada Research Chair Program, the Canada Foundation for Innovation and the New Brunswick Innovation Foundation.

References

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DALEN J.L. & SAUNDERS G.W. 2007. A review of the red algal genus Leptofauchea (Faucheaceae, Rhodymeniales) including a description of L. chiloensis sp. nov. Phycologia 46: 198-213. DAWSON E.Y. 1963. Marine red algae of Pacific Mexico. Part 6. Rhodymeniales. Nova Hedwigia 5: 437-478, 19 pls. DAWSON E.Y. & NEUSHUL M. 1966. New records of marine algae from Anacapa Island, California. Nova Hedwigia 12: 173-187. GAVIO B. & FREDERICQ S. 2005. New species and new records of offshore members of the Rhodymeniales (Rhodophyta) in the northern Gulf of Mexico. Gulf of Mexico Science 1: 58-83. FRESHWATER D.W., TUDOR K., O’SHAUGHNESSY K. & WYSOR B. 2010. DNA barcoding in the red algal order Gelidiales: comparison of COI with rbcL and verification of the “barcoding gap”. Cryptogamie, Algologie 31: 435-449. GUIRY M.D. & GUIRY G.M. 2014. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org. KYLIN H. 1931. Die Florideenordnung Rhodymeniales. Lunds Universitets Årsskrift. Ny Följd, Andra Afdelningen 2. 27: 1-48. LE GALL L., DALEN J.L. & SAUNDERS G.W. 2008. Phylogenetic analyses of the red algal order Rhodymeniales supports recognition of the Hymenocladiaceae fam. nov., Fryeellaceae fam. nov., and Neogastroclonium gen. nov. Journal of Phycology 44: 1556-1571. MILLAR A.J.K. 1999. Marine benthic algae of Norfolk Island, South Pacific. Australian Systematic Botany 12: 479-547. NORRIS R.E. & AKEN M.E. 1985. Marine benthic algae new to South Africa. South African Journal of Botany 51: 55-65. RODRÍGUEZ-PRIETO C. & DE CLERCK O. 2009. Leptofauchea coralligena (Faucheaceae, Rhodophyta), a new species from the Mediterranean Sea. European Journal of Phycology 44: 107-121. SAUNDERS G.W., STRACHAN I.M. & KRAFT G.T. 1999. The families of the order Rhodymeniales (Rhodophyta): a molecular-systematic investigation with a description of Faucheaceae fam. nov. Phycologia 38: 23-40. SAUNDERS G.W., LANE C.E., SCHNEIDER C.W. & KRAFT G.T. 2006. Unraveling the Asteromenia peltata species complex with clarification of the genera Halichrysis and Drouetia (Rhodymeniaceae, Rhodophyta). Canadian Journal of Botany 84: 1581-1607. SAUNDERS G.W. & MCDONALD B. 2010. DNA barcoding reveals multiple overlooked Australian species of the red algal order Rhodymeniales (Florideophyceae), with resurrection of Halopeltis J. Agardh and description of Pseudohalopeltis gen. nov. Botany 88: 639-667. SAUNDERS G.W. & MCDEVIT D.C. 2012. Methods for DNA barcoding photosynthetic protists emphasizing the macroalgae and diatoms. Methods in Molecular Biology 858: 207-222.

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SAUNDERS G.W. & MOORE T.E. 2013. Refinements for the amplification and sequencing of red algal DNA barcode and RedToL phylogenetic markers: a summary of current primers, profiles and strategies. Algae 28: 31-43. SCHNEIDER C.W. & SEARLES R.B. 1976. North Carolina marine algae. VII. New species of Hypnea and Petroglossum (Rhodophyta, Gigartinales) and additional records of other Rhodophyta. Phycologia 15: 51-60. SCHNEIDER C.W., CHENGSUPANIMIT T. & SAUNDERS G.W. 2011. A new genus and species from the North Atlantic Archestenogramma profundum (Phyllophoraceae, Rhodophyta), with taxonomic resolution of the orphaned Leptofauchea brasiliensis. European Journal of Phycology 46 416-441. SCHNEIDER C.W., FRESHWATER, D.W. & SAUNDERS G.W. 2012. First report of Halopeltis (Rhodophyta, Rhodymeniaceae) from the non-tropical Northern Hemisphere: H. adnata (Okamura) comb. nov. from Korea, and H. pellucida sp. nov. and H. willisii sp. nov. from the North Atlantic. Algae 27: 95-108. SEGAWA S. 1941. New or noteworthy algae from Izu. Scientific Papers of the Institute of Algological Research, Faculty of Science, Hokkaido Imperial University 2: 251- 271. STAMATAKIS A., HOOVER P. & ROUGEMONT J. 2008. A rapid bootstrapping algorithm for the RAxML web-servers. Systematic Biology 57: 758-771. SUZUKI M., HASHIMOTO T., NAKAYAMA T. & YOSHIZAKI M. 2010. Morphology and molecular relationships of Leptofauchea rhodymenioides (Rhodymeniales, Rhodophyta), a new record for Japan. Phycological Research 58: 116-131. SUZUKI M., NOZAKI H., TERADA R., KITAYAMA T., HASHIMOTO T. & YOSHIZAKI M. 2012. Morphology and molecular relationships of Leptofauchea leptophylla comb. nov. (Rhodymeniales, Rhodophyta) from Japan. Phycologia 51: 479-488. TAYLOR W.R. 1942. Caribbean marine algae of the Allan Hancock Expedition, 1939. Allan Hancock Atlantic Expedition, 2. 1-193.

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Table 4.1 Collection details for samples included in molecular analyses. GenBank accessions in bold were determined for this study.

Species VOUCHER COI-5P LSU rbcL Gloiocladia fryeana (Setchell) Sánchez & Rodríguez-Prieto GWS001381 KR085174 EU624157 ND GWS001421 ND ND KR085187 Gloiocladia halymenioides (Harvey) R.E. Norris GWS000469 HQ919528 DQ873283 KR085192 Gloiocladia laciniata (J. Agardh) Sánchez & Rodríguez-Prieto B048GWS KR0851701 EU624158 ND GWS020495 KR085172 ND KR085189 Gloiocladia repens (C. Agardh) Sánchez & Rodríguez-Prieto G0332 ND AF419143 ND GWS001639 KR140326 ND KR140335 Gloioderma australe J. Agardh G0306 JX969722 DQ873282 JX969786 Leptofauchea chiloensis Dalen & G.W. Saunders GWS000503 ND DQ873285 KR140338 Leptofauchea cocosana Filloramo & G.W. Saunders GWS037755 KR140330 KR140334 KR140340 Leptofauchea coralligena Rodríguez-Prieto & De Clerck OCD1501 ND EU418774 ND Leptofauchea earleae Gavio & Fredericq LAF-26-5-00-1-1 ND ND HQ400570 Leptofauchea leptophylla (Segawa) Mas.Suzuki, Nozaki, R. GWS018362 KR1403291 KR140333 ND Terada, Kitayama, Tetsu.Hashimoto & Yoshizaki

GWS018363 KR140328 ND ND GWS018540 KR140327 ND KR140336 TNS-AL-174121 ND AB677823 AB6931202 Leptofauchea munseomica Filloramo & G.W. Saunders GWS018532 HQ5440941 KR140331 KR140337 GWS018533 HQ544095 ND ND GWS018537 HQ544099 ND ND GWS018674 HQ544137 ND ND Leptofauchea nitophylloides (J.Agardh) Kylin GWS032631 KR085173 KR085179 KR085190

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Species VOUCHER COI-5P LSU rbcL Leptofauchea pacifica E.Y. Dawson JD032 AY970566 DQ873286 ND GWS010140 HM9161761 ND KR085195 Leptofauchea rhodymenioides W.R. Taylor3 TNS-AL-161637 ND AB677824 AB383124 Leptofauchea sp._1WA G0400 HM915831 DQ873287 KR085194 Webervanbossea sp._1splachnoides GWS016349 HM918169 KR085175 KR085185 Webervanbossea sp._1TAS GWS015310 HM917690 KR085182 KR085193 Webervanbossea splachnoides (Harvey) De Toni GWS002435 HM916011 DQ873288 KR085186 Webervanbossea tasmanensis Womersley GWS000922 HM9158871 ND KR085196 GWS029664 KR085171 KR085178 ND 1 Where more than one voucher was used for molecular data, the COI-5P sequence used in multigene is noted.

2 Chimeric sequence deposited in GenBank for L. leptophylla. Only the last ~730 bp were representative of L. leptophylla and used in our analyses.

3 This collection is molecularly similar to and morphologically representative of L. leptophylla as described herein.

ND= Data not determined for this study.

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Table 4.2 Intraspecific COI-5P divergence and distance to nearest neighbor for species of Leptofauchea included in this study.

Max Distance to intraspecific nearest neighbor Species divergence (%) Nearest species (%) L. cocosana (n=1) N/A L. munseomica 11.02

L. leptophylla (n= 3) 0.15 L. sp._1WA 9.63

L. munseomica (n=4) 0.15 L. cocosana 11.02

L. nitophylloides N/A L. pacifica 10.82

(n=1)

L. pacifica (n=30) 0.31 L. nitophylloides 10.82

L. sp. _1WA (n=1) N/A L. leptophylla 9.63

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Figure 4.1 Phylogenetic tree resulting from RAxML analysis of the combined alignment with bootstrap support values

appended. Asterisks (*) indicate full nodal support while dashes (-) indicate nodal support below 70%.

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Figure 4.2 Leptofauchea nitophylloides collected from Coffs

Harbour, New South Wales, Australia (GWS032631). a) Habit of vegetative specimen. Scale bar = 1 cm. b) Vegetative anatomy, cross section at mid-thallus. Scale bar = 40 µm. c) Surface view at mid-thallus, underlying medullary cells visible (arrow). Scale bar = 50 µm.

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Figure 4.3 Images of holotype for Leptofauchea cocosana sp. nov. (GWS037755). a)

Habit of cystocarpic holotype with cytocarps (arrows) on thallus margins. Scale bar

= 5.7 mm. b) Vegetative anatomy, cross section at mid-thallus. Scale bar = 43 µm. c)

Surface view at mid-thallus; underlying medullary cells visible (arrow) with outer cortical cell indicated (arrow head). Scale bar = 30 µm. d) Protuberant cystocarp with well-defined tela arachnoidea (arrow). Scale bar = 190 µm.

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Figure 4.4 Leptofauchea leptophylla from Jeju, South Korea. a) Habit of vegetative specimen with rounded apices (GWS018362). Scale bar = 1.7 cm. b) Habit of tetrasporangial plant with attenuate apices and constrictions below the apices

(GWS018540). Scale bar = 2.5 cm. c) Vegetative anatomy, cross section at mid- thallus (GWS018362). Scale bar = 37 µm. d) Tetrasporangial thallus showing nemathecia at mid-thallus (left) and branch apex (right) (GWS018540). Scale bar =

1 mm. e) Cruciately divided tetrasporangia (GWS018540). Scale bar = 48 µm. f)

Transverse section of tetrasporangial nemathecium showing tetrasporangial initial

(arrow) borne on modified cortical cell (GWS018540). Scale bar = 15 µm. g)

Transverse section of tetrasporangial nemathecium showing tetrasporangial initials positioned on single-celled filaments (arrow heads) or laterally on paraphyseal cells

(arrow) (GWS018540). Scale bar = 19 µm.

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Figure 4.5 Images of holotype for Leptofauchea munseomica sp. nov. (GWS018532). a) Habit of vegetative holotype. Scale bar = 1 cm. b) Cross section at mid-thallus.

Scale bar = 34 µm.

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Chapter 5 Molecular-assisted alpha taxonomy of the genus Rhodymenia

(Rhodymeniaceae, Rhodymeniales) from Australia reveals overlooked

species diversity

Citation for published manuscript: Filloramo, G.V. & Saunders, G.W. (2016).

Molecular-assisted alpha taxonomy of the genus Rhodymenia (Rhodymeniaceae,

Rhodymeniales) from Australia reveals overlooked species diversity. European Journal of Phycology, 51: 354-368.

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Abstract

A previously published DNA barcode survey of red macroalgae in Australia revealed significant cryptic and overlooked diversity for the genus Rhodymenia with recognition of R. novahollandica, R. prolificans, R. stenoglossa, R. wilsonis and an additional four uncharacterized genetic species groups. Since that study, increased sampling effort in

Australia has warranted reassessment and reinvestigation of the number of genetic species groups attributed to Rhodymenia and their respective taxonomic affiliations.

Using molecular-assisted alpha taxonomy employing the DNA barcode (COI-5P), the present study resolved 188 Australian specimens in 12 genetic species groups assignable to the genus Rhodymenia. Four of these groups were attributed to the previously recognized species (above), whereas some collections from Lord Howe Island were attributed to the New Zealand species R. novazelandica expanding its biogeographical range. The following seven genetic groups were inconsistent with existing species of

Rhodymenia and established as novel taxa: R. compressa sp. nov., R. contortuplicata sp. nov., R. gladiata sp. nov., R. insularis sp. nov., R. lociperonica sp. nov., R. norfolkensis sp. nov. and R. womersleyi sp. nov. Although morphological and biogeographical features were adequate for distinguishing some species of Rhodymenia from Australia,

DNA sequencing in combination with morphology and biogeography provided the most reliable means of identification.

Introduction

Prior to the advent of molecular techniques, red algal taxonomists relied on vegetative and reproductive criteria for identifying organisms. This approach presented challenges

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in the morphologically simple red algae for which accurate identification was further complicated by evolutionary convergence and phenotypic plasticity (Saunders 2005).

Current red algal taxonomy has overcome such complications by employing the more objective technique of molecular-assisted alpha taxonomy (MAAT), which uses differences in gene sequence data to delineate genetic species groups that are subsequently analyzed morphologically and assigned to existing species or established as novel taxa (Saunders 2005; Cianciola et al. 2010). The 5’ region of the mitochondrial cytochrome c oxidase I gene (COI-5P; DNA barcode) has been championed as a “DNA barcode” (i.e., a short, standardized region of DNA employed for diagnosing species based on genetic differences) among the red algae owing to its effectiveness in discriminating between closely related species (Saunders 2005; Saunders 2008; Le Gall

& Saunders 2010). Since its inception, MAAT employing the COI5P region has proven to be an effective and reliable method for assessing red algal species boundaries that has radically altered perspectives on diversity (e.g., Clarkston & Saunders

2010; Saunders & McDonald 2010; Clarkston & Saunders 2013; Hind et al. 2014;

Saunders & Millar 2014; Schneider et al. 2015).

The red algal genus Rhodymenia is the type genus of the family Rhodymeniaceae in the order Rhodymeniales. Greville (1830) described Rhodymenia based on ten species including the generitype, R. palmetta (Stackhouse) Greville, which is presently regarded as a synonym of R. pseudopalmata (J.V. Lamouroux) P.C. Silva (Silva 1952). Currently, there are an estimated 62 species of Rhodymenia worldwide (Guiry & Guiry 2015).

Members of this genus are characterized by erect or prostrate fronds that are commonly stipitate and develop from a discoid or stoloniferous base (Dawson 1941; Sparling 1957;

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Hawkes & Scagel 1986). Blades of Rhodymenia are typically flat, slightly cartilaginous, simply or dichotomously divided, and, at times, bear marginal or apical proliferations

(Dawson 1941; Hawkes & Scagel 1986). Vegetative construction is multiaxial featuring a cortex of small, pigmented cells and a compact medulla of large, colourless, thin- walled cells (Dawson 1941; Sparling 1957). Cystocarps located apically or scattered on the blade and are commonly hemispherical with protruding ostioles (Hawkes & Scagel

1986). Carpogonial branches may be either three- or four-celled (Sparling 1957).

Spermatangia are located in small subapical sori or large irregular patches scattered over the blade surface and are produced from the cells of the outer cortex (Sparling 1957;

Hawkes & Scagel 1986). Cruciately divided tetrasporangia develop directly from preexisting cortical cells and are located in subapical sori, scattered on the blade or formed in special proliferations (Sparling 1957; Guiry 1977; Hawkes & Scagel 1986; Le

Gall et al. 2008).

Historically, classification of Rhodymenia species has been based largely on reproductive criteria, particularly features of the tetrasporangia (Agardh 1876). In the most comprehensive taxonomic survey of Rhodymenia, Dawson (1941) divided the genus into sections based primarily on the location and distribution of the tetrasporangia

(at or below the apices; scattered or in sori) and the degree to which the tetrasporangia modify the cortex, and secondarily, based on the branching habit of the stipe, presence or absence of stolons, nature of the margins, apex shape, and overall height and thickness of the thallus in transverse section. According to those criteria, 54 species of Rhodymenia were recognized including four species from Australia: R. foliifera Harvey described from King George Sound, Western Australia, R. linearis J. Agardh described from “in

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mari Australi”, R. prolificans Zanardini described from Georgetown, Tasmania and R. stenoglossa J. Agardh described from “Nova Holl. aust” (Dawson 1941). In addition to the latter two taxa, Womersley (1996) subsequently reported the following Rhodymenia species from Australia: R. australis Sonder (including R. foliifera as a synonym) described from Western Australia, R. cuneata Harvey described from the east coast of

Tasmania, R. halymenioides (J. Agardh) Womersley described from Orford, Tasmania,

R. leptophylla J. Agardh described from Bay of Islands, New Zealand, R. obtusa

(Greville) Womersley described from Cape of Good Hope, South Africa and R. verrucosa Womersley described from Gabo Island, Victoria. Although Womersley

(1996) did not record R. linearis, it was later reported from Queensland, Australia increasing the total number of Rhodymenia species reported from Australia to ten

(Phillips 1997; Phillips 2002; Bostock & Holland 2010).

A recent DNA barcode (COI-5P) survey of southern Australia resolved collections field identified to the genus Rhodymenia as 20 genetic species groups within four genera and two families in the Rhodymeniales (Saunders & McDonald 2010). As a result of that study, the genus Halopeltis J. Agardh was resurrected to accommodate four species traditionally assigned to Rhodymenia [R. australis, R. cuneata (including R. halymenioides) and R. verrucosa]. The novel genus Pseudohalopeltis G.W. Saunders and its only species, P. tasmanensis G.W. Saunders, were established for some collections from Tasmania that resolved as a novel lineage in the Fryeellaceae while the novel species Microphyllum robustum G.W. Saunders was described to accommodate some rhodymenialean collections from New South Wales (Saunders & McDonald 2010).

Within Rhodymenia sensu stricto the novel species R. novahollandica G.W. Saunders

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(described from Port Philip Heads, Victoria) was reported widely from Victoria with some collections from Western Australia and Tasmania indicating a wider distribution while R. prolificans and R. stenoglossa were confirmed from Tasmania and Victoria, respectively (Saunders & McDonald 2010). The species R. wilsonis, which had been synonymized previously with R. obtusa (Womersley 1996), was resurrected for specimens from Tasmania and Victoria, which resolved separately from specimens of R. obtusa from its type locality in South Africa (Saunders & McDonald 2010). The same study also resolved two divergent genetic species groups that were loosely assigned to R. leptophylla, as well as two other groups (from South Australia and Victoria, respectively) that were uncharacterized owing to low sample sizes (Saunders &

McDonald 2010). Although the study by Saunders & McDonald (2010) was not specifically aimed at assessing Rhodymenia biodiversity in Australia, it indirectly highlighted the extensive overlooked and misunderstood species diversity for this genus from that locale.

In the current study we further explored and assessed the findings of Saunders &

McDonald (2010) by completing a comprehensive MAAT survey restricted to the genus

Rhodymenia from Australia. Our analyses provide new insight into the species diversity and biogeographical distributions of Rhodymenia species from this locale with the recognition of twelve species where five were previously reported.

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Materials and Methods

A total of 189 samples field identified to the genus Rhodymenia predominantly from

Victoria (n= 73) and Tasmania (n= 51) with some collections from Western Australia

(n= 14), South Australia (n= 12), mainland New South Wales (n= 17), Lord Howe Island

(n= 11) and Norfolk Island (n= 10), in addition to a single specimen from New Zealand, were included in this study (Appendix D, Table S1). All collections were deposited in the Connell Memorial Herbarium (UNB; Thiers 2015). Samples were collected in the subtidal by SCUBA or in the intertidal as drift or attached specimens. The collections were preserved as pressed material or dried in silica to serve as vouchers with subsamples dried in silica for subsequent molecular analysis. Genomic DNA was extracted following Saunders & McDevit (2012) with amplification of both the 5’ end of the mitochondrial cytochrome c oxidase subunit 1 gene (COI-5P; DNA barcode) and the nuclear internal transcribed spacer region of the ribosomal cistron (ITS) according to

Saunders & Moore (2013). Primer pair combinations for COI-5P are recorded on

GenBank and the Barcode of Life Database (BOLD) website

(http://www.boldsystems.org/). Amplicons were sequenced by the Génome Québec

Innovation Centre and raw data were edited and aligned using Geneious R7 version 7.1.7

(http://www.geneious.com; Kearse et al. 2012).

The DNA barcode alignment (664bp) was analyzed in BOLD using a barcode gap analysis to determine intraspecific variation and nearest neighbor distances. Genetic species groups exhibiting high intraspecific and/or low interspecific variation based on

COI-5P analysis were further analyzed with amplification of either the full ITS region

(~700 bp) or only the 3’ end of the ITS2 (~222 bp). Generated ITS sequences were 181

searched in BOLD and GenBank and examined using a neighbor-joining analysis in

Geneious R7.

For phylogenetic assessment, a single COI-5P sequence for each genetic species

group uncovered here was combined in an alignment with additional Rhodymenia spp.

COI-5P data (Appendix D, Table S1; GenBank numbers included with accessions in

figure). Data from Botryocladia spp. were included as the outgroup (Filloramo &

Saunders in press). The COI-5P alignment was analyzed using RAxML under a

GTR+I+G model with partitioning by codon and support determined by 1000 rounds of

bootstrap resampling.

Anatomical observations were completed by rehydrating algal tissue in 5% formalin and seawater and then using a freezing microtome (CM18250, Leica,

Heidelberg, Germany) to prepare sections that were stained with 1% aniline blue solution in 6% 5N hydrochloric acid and mounted in 50% corn syrup with 4% formalin.

Vegetative and reproductive anatomy were observed using a Leica DM5000B light microscope and photographed with a Leica DFC480 digital camera. Plates were assembled using Adobe Photoshop Elements 11 (2012).

Results

Molecular Results

The COI-5P region was successfully amplified for 189 collections. The 188 specimens from Australia resolved as 12 genetic species groups assigned to the genus Rhodymenia while a single specimen from New Zealand resolved as distinct from the Australian 182

species groups and was morphologically assigned to R. leptophylla (Table 5.1).

Maximum intraspecific divergence was 0.61% excepting R. novahollandica (1.65%,

Table 5.1; represented by collections from Western Australia, South Australia, Victoria and Tasmania, Fig. 5.1), R. wilsonis (2.40%, Table 5.1; represented by specimens from

South Australia, Victoria and Tasmania; Fig. 5.1) and the novel species R. womersleyi described herein (1.06%, Table 5.1; predominantly from New South Wales with a single representative from South Australia, Fig. 5.1). Interspecific variation ranged from 3.09% to 8.66% except between R. prolificans and R. stenoglossa (1.09%; Table 5.1). A cluster of specimens exclusively from Norfolk Island (Fig. 5.1) was described as the novel species R. norfolkensis (below) and resolved closest (4.90%, Table 5.1) to the novel species R. insularis (below) known only from Lord Howe Island (Fig. 5.1). Two collections from New South Wales (Fig. 5.1) recognized herein as the novel species R. compressa were resolved as a distantly related lineage (8.66%, Table 5.1) to a group of specimens exclusively from Lord Howe Island (Fig. 5.1) that were morphologically assigned to the New Zealand species R. novazelandica. A group of collections from

Western Australia (Fig. 5.1) were described as the novel species R. lociperonica (below) and were most closely related (6.20%, Table 5.1) to R. norfolkensis. Specimens assigned to R. novazelandica were most closely resolved (6.56%, Table 5.1) to R. lociperonica.

The novel species R. womersleyi (below) was distantly related (7.55%, Table 5.1) to another newly recognized species exclusively from Tasmania (Fig. 5.1) described herein as R. contortuplicata. The latter species was distinct (3.09%, Table 5.1) from its nearest neighbor R. prolificans. A unique lineage comprised of collections from New South

Wales, Victoria and Tasmania (Fig. 5.1) was described as the novel species R. gladiata

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(below) and was most closely related (6.58%, Table 5.1) to the single collection from

New Zealand identified as R. leptophylla.

Although COI-5P intraspecific variation for R. gladiata was typical of within species variation rates, analysis of the COI-5P alignment indicated that specimens from

Tasmania and Victoria were genetically more similar when compared to collections from

New South Wales. Uni-directional reads of ITS2 data for samples of R. gladiata from each of the previous three locales were essentially identical (0-1 bp differences out of

222 bp) and representative of a single species group (Table 5.1).

The ITS was also amplified to further assess the high COI-5P intraspecific variation for R. womersleyi (Table 5.1). The ITS data were challenging to interpret owing to significant base pair heterogeneity within individuals (data not shown); however, where the data were interpretable the number of base pair substitutions was low and only one ITS type was evident. Owing to the previous, collections of R. womersleyi were recognized as a single species while acknowledging high intraspecific

COI-5P variation at the population level.

The ITS was amplified for exploring the low interspecific variation between R. prolificans and R stenoglossa (Table 5.1); however, ITS amplification of the single representative of R. stenoglossa (GWS001555) was not successful and the status of R. prolificans and R. stenoglossa as distinct species was not resolved.

In RAxML analysis the Australian Rhodymenia genetic species were resolved in two clades (Fig. 5.2). The first clade included R. wilsonis, which was allied to its namesake R. obtusa from South Africa in addition to R. contortuplicata, R. novahollandica, R. prolificans, R. stenoglossa and R. womersleyi, which were associated 184

with Rhodymenia species from South Korea, the Northeast Pacific, Chile and France

(Fig. 5.2). The second clade included R. compressa, R. gladiata, R. insularis, which was strongly allied to an unknown Rhodymenia species from Hawaii, R. lociperonica, R. norfolkensis and R. novazelandica, as well as Rhodymenia entities from New Zealand, the Northwest Atlantic and France (Fig. 5.2).

Taxonomic Results

We continue to recognize the four species R. novahollandica, R. prolificans, R. stenoglossa and R. wilsonis attributed to Rhodymenia by Saunders & McDonald (2010).

A single vegetative sample collected from a shallow tidal pool in Pahi, New Zealand was morphologically (Fig. 5.3a) and anatomically (Fig. 5.3b) assignable to R. leptophylla J.

Agardh, and we consider this species absent from Australia. Four collections from Lord

Howe Island were, however, morphologically (Fig. 5.3c) and anatomically (Fig. 5.3d) consistent with R. novazelandica E.Y. Dawson extending the range of this New Zealand species to Australia. The remaining seven genetic species groups were described as novel species.

Rhodymenia compressa Filloramo & G.W. Saunders, sp. nov. (Figs 5.4a,b)

DESCRIPTION: Thalli 2.5-5 cm high with numerous linear, cylindrically compressed blades that are sparsely and irregularly dichotomously branched, individual branches

0.3-0.5 mm wide with attenuate apices (Fig. 5.4a); discoid, crustose holdfast typically fusing with neighboring holdfasts to form a coalesced base, stolons not observed. Axes

280-320 µm in diameter in transverse section with 5 (-7) medullary layers of colourless,

185

axially elongated or rounded cells (70-100 µm long x 40-80 µm wide), 1-2 inner cortical layer of smaller oblong cells subtending 2 (-3) outer cortical layers of smaller cells, the outermost cells columnar in shape (Fig. 5.4b). Reproductive structures not observed.

HOLOTYPE: UNB GWS032782, on a rock at 4 m depth, G.W. Saunders and K. Dixon,

12 December 2012.

COI-5P BARCODE: GenBank accession KT781957.

ISOTYPE: Specimen UNB GWS032780 (Appendix D, Table S1).

TYPE LOCALITY: Mutton Bird Island (South), Coffs Harbour, New South Wales,

Australia.

ETYMOLOGY: Latin for “compressed” in recognition of the compressed blades found in this species.

DISTRIBUTION: Known only from the type locality (Fig. 5.1).

COMMENTS: This novel Rhodymenia species is distinguished from other Australian species of Rhodymenia owing to its unique linear, whip-like, compressed axes.

Rhodymenia compressa may be confused with species of Cephalocystis that are also characterized by sparsely branched and terete thalli (Millar et al. 1996). Described from

New South Wales (Millar et al. 1996) and subsequently reported from Norfolk Island

(Millar 1999), the species C. leucobotrys A.J.K. Millar, G.W. Saunders, I.M. Strachan &

Kraft is thicker in cross section and has a greater number of medullary cell layers.

Although, R. compressa is known only from the type locality in New South Wales (Fig.

5.1), it is possible that this species has a wider biogeographical distribution making it difficult to distinguish from another species of Cephalocystis, C. furcellata (J. Agardh)

A.J.K. Millar, G.W. Saunders, I.M. Strachan & Kraft, described from Victoria and 186

reported in South Australia and Tasmania (Womersley 1996). Although superficially similar, the axes of R. compressa are thinner and less cartilaginous than those of C. furcellata.

Rhodymenia contortuplicata Filloramo & G.W. Saunders, sp. nov. (Figs 5.4c,d)

DESCRIPTION: Thalli 2.8-4.6 cm in height with flat, unbranched or regularly dichotomously branched (1-3 times) blades, 2.6-7.5 mm wide typically narrowing at the apices (Fig. 5.4c); stipes ca. 11-30 mm long with stolons extending from discoid holdfast or above from the stipe. Blades 130-175 µm thick in transverse section with 3-4 medullary layers of large, colorless, axially elongated cells (50-70 µm long x 32.5-47.5

µm wide) flanked by a single layer of smaller colorless medullary cells (25-28 µm long x

20-22.5 µm wide), an incomplete single inner cortical layer of smaller darkly staining cells subtending a single layer of small, round, darkly staining outer cortical cells (Fig.

5.4d). Reproductive structures not observed.

HOLOTYPE: UNB GWS015201, on rock wall at 6-10 m depth, G.W. Saunders & K.

Dixon, 21 January 2010.

COI-5P BARCODE: GenBank accession HM917629.

TYPE LOCALITY: Burying Ground Point, Tasmania, Australia.

ETYMOLOGY: Latin meaning “tangled” in recognition for the often-tangled habit of the blades and stolons.

DISTRIBUTION: Known only from Tasmania (Fig. 5.1).

MATERIAL EXAMINED: Listed in Appendix D, Table S1.

187

COMMENTS: Rhodymenia contortuplicata is distinguished from other species of

Rhodymenia in Australia by its commonly entangled stipes and stolons. Currently

Rhodymenia contortuplicata is known only from Tasmania (Fig. 5.1) where it may be confused with the species R. novahollandica; however, the latter species is taller and thicker in cross section with more medullary and cortical cell layers.

Rhodymenia gladiata Filloramo & G.W. Saunders, sp. nov. (Figs 5.4e,f)

DESCRIPTION: Thalli 0.5-3 cm high with numerous flat, undivided to sparsely and irregularly branched (typically no more than 2 dichotomies) blades arising from a clumped base of fused holdfasts, prominent stolons commonly giving rise to new blades

(Fig. 5.4e); individual blades 0.5-2 mm wide with attenuated or rounded apices (Fig.

5.4e). Blades 80175 µm thick in transverse section with 2-4 (-5) tightly packed layers of round medullary cells (17.5-38 µm in diameter) and a single inner cortical layer subtending 1-2 outer cortical layers of smaller darkly staining cells (Fig. 5.4f).

Reproductive structures not observed.

HOLOTYPE: UNB GWS032615, growing on invert at 8 m depth, G.W. Saunders & K.

Dixon, 9 December 2012.

COI-5P BARCODE: GenBank accession KT781956.

TYPE LOCALITY: Split Solitary Island (Northwest), Coffs Harbour, New South Wales,

Australia.

ETYMOLOGY: Derived from the Latin gladius (sword), due to the typically undivided, narrow habit of the individual blades that are reminiscent of swords.

DISTRIBUTION: New South Wales, Victoria and Tasmania (Fig. 5.1).

188

MATERIAL EXAMINED: Listed in Appendix D, Table S1.

COMMENTS: This taxon is distinguished from other Rhodymenia species by its narrow, lanceolate blades. Rhodymenia gladiata inhabits the subtidal from New South Wales,

Victoria and Tasmania making it one of the more widely distributed Rhodymenia species currently known in Australia (Fig. 5.1). Across that range, specimens from New South

Wales were thinner in cross section (80-90 µm) than those from Tasmania and Victoria

(125-175 µm) with 2-3 medullary cell layers and a single outer cortical layer in the former specimens and 4-5 medullary cell layers and 1-2 outer cortical layers in the latter.

Rhodymenia insularis Filloramo & G.W. Saunders, sp. nov. (Figs 5.4g,h)

DESCRIPTION: Thalli 1-6 cm in height with flat dichotomously branched blades, individual blades 0.5-3 mm wide with typically rounded apices, at times attenuate (Fig.

5.4g); stipes 10-15 mm long with stolons extending from above the discoid holdfast (Fig.

5.4g). Blades 170-190 µm thick in transverse section with 4-5 medullary layers of round to axially elongated cells (35-52.5 µm in diameter) grading in size to 1-2 inner cortical layers of smaller oblong cells (6.5-9 µm) subtending a single layered outer cortex of small, round darkly staining cells (Fig. 5.4h). Reproductive structures not observed.

HOLOTYPE: UNB GWS002043, growing on rock wall at 24 m depth, G.W. Saunders,

1 February 2004.

COI-5P BARCODE: GenBank accession HM033147.

TYPE LOCALITY: Islands off Balls Pyramid, Lord Howe Island, New South Wales,

Australia.

189

ETYMOLOGY: Latin for “island” in recognition of the type locality of the remote Balls

Pyramid, ~ 20 km southeast of Lord Howe Island.

DISTRIBUTION: Known only from Lord Howe Island and nearby Balls Pyramid (Fig.

5.1).

MATERIAL EXAMINED: Listed in Appendix D, Table S1.

COMMENTS: Rhodymenia insularis is currently known only from Balls Pyramid and nearby Lord Howe Island and is superficially similar to R. novazelandica, the only other species of Rhodymenia we collected from Lord Howe Island (Fig. 5.1). Although both species feature dichotomously divided blades extending from elongated stipes and long stolons from which new blades typically arise, R. insularis has more medullary cell layers and is thicker in cross section than R. novazelandica.

Rhodymenia lociperonica Filloramo & G.W. Saunders, sp. nov. (Figs 5.5a,b)

DESCRIPTION: Thalli 1-4.5 cm in height with flat, dichotomously branched blades, individual blades 1-2 mm wide with rounded at times attenuate apices (Fig. 5.5a); stipes

3-4 mm long with stolons extending from above the holdfast (Fig. 5.5a). Blades 240-250

µm thick in transverse section with (-3) 4 medullary layers of round cells (51-65 µm in diameter) and a single at times two inner cortical layers of elongated cells subtending a single layered outer cortex of small, round darkly staining cells (Fig. 5.5b). Reproductive structures not observed.

HOLOTYPE: UNB GWS025412, growing on a rock at 2.5 m depth, G.W. Saunders &

K. Dixon, 13 November 2010.

COI-5P BARCODE: GenBank accession KT781938. 190

ISOTYPES: Specimens UNB GWS025411 and UNB GWS025430 (Appendix D, Table

S1).

TYPE LOCALITY: Pt. Peron, Western Australia, Australia.

ETYMOLOGY: Named after the type locality Pt. Peron.

DISTRIBUTION: Known only from Western Australia (Fig. 5.1).

MATERIAL EXAMINED: Listed in Appendix D, Table S1.

COMMENTS: Rhodymenia lociperonica is known from Pt. Peron and Canal Rocks in

Western Australia where the only other species of Rhodymenia uncovered from Western

Australia, R. novahollandica, has also been collected (Fig. 5.1). Both species inhabit the subtidal and grow on invertebrates or rocks. Although R. lociperonica and R. novahollandica may be confused in the field, R. lociperonica has a thicker medulla and thinner cortex than R. novahollandica.

Rhodymenia norfolkensis Filloramo & G.W. Saunders, sp. nov. (Figs 5.5c,d)

DESCRIPTION: Thalli clumped, sprawling and decumbent, 2-8 cm in dimension with flat regularly dichotomously branched blades arising from stipes (12.5-18 mm long) attached to substrate by a small discoid holdfast, stolons typically giving rise to new stipitate blades (Fig. 5.5c); individual blades 1-4 mm wide with typically broadly rounded apices, at times attenuate (Fig. 5.5c). Blades 105-160 µm thick in transverse section with typically 3-4 layers of medullary cells (40-55 µm long x 22.5-42.5 µm wide) grading to a single inner cortical layer of smaller, elongate darkly staining cells

(10 µm long x 6-7.5 µm wide) subtending a single outer cortical layer of round darkly staining cells (Fig. 5.5d). Reproductive structures not observed.

191

HOLOTYPE: UNB GWS034326, growing on rock at 17 m depth, G.W. Saunders & K.

Dixon, 27 November 2012.

COI-5P BARCODE: GenBank accession KT781979.

ISOTYPES: Specimens UNB GWS032489, UNB GWS034321, UNB GWS034329,

UNB GWS034333, UNB GWS034352 deposited in UNB (Appendix D, Table S1).

TYPE LOCALITY: Little Organ, Norfolk Island, Australia.

ETYMOLOGY: Named for its status as the only species of Rhodymenia currently known from Norfolk Island.

DISTRIBUTION: Known only from Norfolk Island (Fig. 5.1).

MATERIAL EXAMINED: Listed in Appendix D, Table S1.

COMMENTS: Rhodymenia norfolkensis is distinguished primarily by biogeography as the only confirmed species of Rhodymenia from Norfolk Island (Fig. 5.1). Rhodymenia norfolkensis is superficially similar to its closest relative R. insularis (Table 5.1) from

Lord Howe Island and nearby Balls Pyramid. However, R. norfolkensis is thinner in cross section with fewer medullary cell layers.

Rhodymenia womersleyi Filloramo & G.W. Saunders, sp. nov. (Figs 5.5e-h)

DESCRIPTION: Thalli 0.8-4.3 cm in height with flat, flabellate, regularly dichotomously branched blades, individual blades 0.8-3.5 mm wide with rounded apices, stipes 0.5-1 mm long, attached to substrate by discoid holdfast, stolons typically extending from above the holdfast (Fig. 5.5e). Blades 130-150 µm thick in transverse section with 2-3 layers of large round medullary cells (35-50 µm in diameter) grading to a single inner cortical layer of smaller axially elongated cells subtending a single outer

192

cortical layer of small, round darkly staining cells (at times a second incomplete layer present) (Fig. 5.5f). Spermatangial sori formed at blade apices on both surfaces, spermatangia 3 µm long x 2.5 µm wide (Fig. 5.5g). Tetrasporangial sori formed at blade apices on a single blade surface, cruciately divided tetrasporangia (35-40 µm long x 18-

20 µm wide) borne on cells of the inner cortex and embedded in the cortex (Fig. 5.5h).

Cystocarps unknown.

HOLOTYPE: UNB GWS032769, growing on invert at 5 m depth, by G.W. Saunders &

K. Dixon, 11 December 2012. Images of the holotype shown in Figs 5.5e,h.

COI-5P BARCODE: GenBank accession KT781966.

ISOTYPES: Specimens UNB GWS032741 (male gametophyte) and UNB GWS032742

(tetrasporophyte) (Appendix D, Table S1).

TYPE LOCALITY: Mutton Bird Island (North), Coffs Harbour, New South Wales,

Australia ETYMOLOGY: Named after H.B.S. Womersley in recognition of his significant contributions to understanding the red algal flora of southern Australia including the genus Rhodymenia.

DISTRIBUTION: South Australia and New South Wales (Fig. 5.1).

MATERIAL EXAMINED: Listed in Appendix D, Table S1.

COMMENTS: Rhodymenia womersleyi was collected from the subtidal (0.5-5 m) and low intertidal pools and is distinguished from other Australian species of Rhodymenia by its extremely short stipe and bushy, turf-like appearance. Although this species is predominantly encountered in New South Wales, a single collection from South

Australia suggests a wider distribution (Fig. 5.1).

193

Discussion

Although a variety of vegetative criteria have been used to differentiate species of

Rhodymenia, members of this genus can exhibit extensive variability in habit and thallus thickness in cross section, which complicates accurate species-level identification

(Sparling 1957; Womersley 1996). Owing to the previous, greater emphasis has been placed on features of the tetrasporangia for delineating Rhodymenia species (Agardh

1876; De Toni 1900; Dawson 1941; Sparling 1957). Although effective when reproductive material is acquired, reliance on tetrasporangial features (or any reproductive character) becomes problematic when fertile material is lacking. Of the twelve Australian genetic species groups resolved in the current study (a total of 188 individuals), tetrasporophytes were represented in only three species: R. novahollandica

(n= 11), R. prolificans (n= 2) and R. womersleyi (n= 3). While R. novahollandica featured tetrasporangial sori on both blade surfaces, R. prolificans and R. womersleyi had tetrasporangial sori on a single blade surface. The latter two species were differentiated based on tetrasporangia size with smaller tetrasporangia for R. prolificans when compared to R. womersleyi. Indeed, when reproductive material was collected, features of the tetrasporangia were a reliable means of species identification; however, the scarcity of fertile material for most of our genetic species groups limited our ability to identify collections using these features.

By using MAAT, we objectively assigned all of our Australian collections to one of twelve genetic species groups in the genus Rhodymenia. While five of these groups were assigned to previously established Rhodymenia species, the remaining seven were distinct from existing species and described as novel taxa. 194

Most of the genetic species groups that we resolved were characterized by typical red algal COI-5P intraspecific variation (0-0.6%; Saunders 2005); however, COI-5P divergence was uncharacteristically high for R. novahollandica (1.65% maximum divergence, Table 5.1), R. wilsonis (2.4% maximum divergence, Table 5.1) and the novel species R. womersleyi (1.06% maximum divergence, Table 5.1). Our findings for R. novahollandica and R. wilsonis were consistent with Saunders & McDonald (2010) who reported that within each respective species COI-5P intraspecific variation was high and characterized by a biogeographical split between mainland Australian and Tasmanian collections. Subsequent assessment of the internal transcribed spacer (ITS) indicated that the mainland and Tasmanian populations for both R. novahollandica and R. wilsonis represent single species groups (Saunders & McDevit 2012). For each species group, high COI-5P intraspecific variation likely resulted from isolation between the mainland and Tasmanian populations followed by a collapse in those boundaries and the reoccurrence of successful interbreeding or past incomplete lineage sorting (Saunders &

McDevit 2012).

Rhodymenia womersleyi was represented by a single collection from South

Australia as well as collections from Sydney, New South Wales (n= 2) and Coffs

Harbour, New South Wales (n= 6) (Fig. 5.1). Collections from Sydney and Coffs

Harbour were resolved as two slightly divergent clusters (6-7 bp differences). The single

South Australian representative of R. womersleyi was more similar to the Sydney

collections (3 bp differences) than to those from Coffs Harbour (5-6 bp differences).

Despite the variation among representatives of this species, analysis of ITS data indicated

a single ITS type (Appendix D, Table S1). Owing to the previous, the observed COI-5P

195

variation reflects population level differences possibly retained from a previous period of isolation.

Although collections assigned to R. gladiata were resolved with low intraspecific variation (0.46%, Table 5.1), analysis of the COI-5P alignment (664 bp) indicated a biogeographical split between collections from New South Wales (n= 6; 0-1 bp differences) and those from Victoria (n= 7) and Tasmania (n= 4) (0 bp differences; Fig.

5.1). Between these two clusters there were 2-3 bp differences, which is a typical amount of variation within a single species; however, we also observed morphological differences between the New South Wales specimens and those in the Victoria/Tasmania cluster, which were thicker in cross section and had a greater number of medullary cells layers. Further assessment of this complex using ITS was warranted to determine if a single species or two closely related species should be recognized. The ITS data for representatives of each cluster were essentially identical (0-1 bp differences across ~700 bp) and indicated a single species with anatomical differences possibly resulting from abiotic factors (e.g., salinity, temperature, water motion).

Saunders & McDonald (2010) originally questioned the distinction of R. prolificans and R. stenoglossa after resolving low interspecific divergence based on COI-5P. The current study attempted to assess further these species using the ITS; however, amplification of this marker failed for our single collection of R. stenoglossa. Despite the previous, we propose that these entities should be maintained as distinct species based on biogeography (Fig. 5.1) as well as morphological differences in overall habit and vegetative construction. Whereas R. stenoglossa had narrow, linear blades and a cortex comprised of 2-3 outer layers and a single inner layer, collections of R. prolificans had 196

numerous marginal proliferations, a cortex of two layers and lacked an inner cortical layer.

The species R. leptophylla was originally described from New Zealand by J.

Agardh (1878) and has been widely reported from Australia including from Lord Howe

Island (Millar & Kraft 1993), mainland New South Wales (Millar 1990; Millar & Kraft

1993; Womersley 1996), Norfolk Island (Millar 1999), Queensland (Lewis 1984; Cribb

1996; Womersley 1996; Phillips 2002; Bostock & Holland 2010), South Australia

(Womersley 1996) and Tasmania (Womersley 1996). Womersley (1996) noted that while some of the Australian specimens assigned to this species agreed well with those from

New Zealand, the plants from southern Australia were variable in size and branching.

Saunders & McDonald (2010) temporarily assigned two divergent genetic groups (one from Lord Howe Island and the other from Tasmania) to R. leptophylla according to their overall gross morphology (notably the occurrence of slender stipes and basal stolons). In an effort to assess if these morphologies represented a single species group and to clarify the geographical boundaries of R. leptophylla, we included in our DNA barcode analysis a New Zealand specimen consistent in habitat and morphology (Appendix D, Table S1;

Figs 5.3a,b) with the protologue of R. leptophylla. This collection failed to resolve with any of the Australian Rhodymenia genetic species groups (Table 5.1; Fig. 5.2). Owing to the previous, R. leptophylla could not be confirmed in Australia despite its reportedly wide distribution (Fig. 5.1). While it is possible that R. leptophylla exists in Australia but was not represented in our collections, this seems unlikely owing to the extensive sampling efforts in Australia by the second author over the past 25 years. If R. leptophylla does indeed extend into Australia, it is likely not as widespread as previously 197

reported. Previous reports of this species from Australia were more likely representatives

of some of the novel species described herein, (e.g., R. contortuplicata and R. insularis)

many of which feature the slender stipes and basal stolons historically recognized as

typical of R. leptophylla.

Although R. leptophylla was not confirmed in Australia, another New Zealand

species, R. novazelandica, was assigned to some specimens from Lord Howe Island (Fig.

5.1). Recognition of this species in Australia has increased its known biogeographical

range. Ultimately, molecular analysis of topotype material for this species will confirm if

our collections indeed represent R. novazelandica; however, without any inconsistencies

in morphology between the Lord Howe Island specimens and the type description of R.

novazelandica, we consider our collections as representative of this species.

Observation of collection information indicated that closely related species of

Rhodymenia inhabited similar ecological niches. However, those species were typically biogeographically distinct. For example, although R. prolificans and R. stenoglossa inhabit exposed areas in the shallow intertidal, in our experience, the former is limited to

Tasmania while the latter is restricted to Victoria (Fig. 5.1). Similarly, R. insularis and R. norfolkensis were collected from reef flats in the subtidal; however, R. insularis has only been encountered from Lord Howe Island while R. norfolkensis has only been collected from Norfolk Island (Fig. 5.1). The previous indicates that allopatry, rather than ecological separation, has likely played a significant role in speciation of Rhodymenia in

Australia.

Overall, MAAT using COI-5P provided clarification of species boundaries for the genus Rhodymenia in Australia. Moving forward, we advocate for a combination of 198

molecular, morphological and biogeographical data to identify Australian Rhodymenia species. MAAT assessments from less sampled locations in Australia (i.e., South

Australia, Western Australia & Queensland) are needed as is a comprehensive molecular-assisted floristic survey of New Zealand species of Rhodymenia to determine the extent to which that country shares Rhodymenia spp. with Australia and possibly facilitate interesting studies regarding red algal speciation and distribution.

Key to species of Rhodymenia in Australia

1a. Blades compressed oval in cross section; New South Wales ..……..….. R. compressa

1b. Blades flat in cross section ………………………………...... 2

2a. Blades predominantly linear or with few (1-2 times) dichotomies …………...... 3

2b. Blades branched with more than 1-2 dichotomies ...………………………………… 4

3a. Stolons present, blades typically with predominantly lanceolate apices; plants 5-30

mm tall; New South Wales, Victoria or Tasmania ……………………...... R. gladiata

3b. Stolons absent; apices rounded; plants 100-250 mm tall; Victoria….… R. stenoglossa

4a. Blades with marginal or surface proliferations ...………………………………….… 5

4b. Blades lacking marginal or surface proliferations ...……………………………….... 6

199

5a. Blades with numerous marginal proliferations (sterile or fertile); numerous blades from clumped crustose holdfast; lacking mid-rib at lower blade; typically growing at low tide level or in tide pools; Tasmania .……………………………….…....… R. prolificans

5b. Blades with surface proliferations; blades developing from single discoid non- crustose holdfast; slight mid-rib at lower blade; inhabiting tide pools, low intertidal or subtidal (1-15 m); South Australia, Tasmania or Victoria ..………………… R. wilsonis

6a. Collected from mainland Australia or Tasmania ………………………………..…. 7

6b. Collected from Lord Howe Island or Norfolk Island ……..…………….…………. 10

7a. Blades arising from elongated stipes (11-30 mm long) that form an entangled network with stolons; Tasmania ….…...... ……………………...... R. contortuplicata

7b. Blades developing from less prominent stipes that do not become entangled with stolons …………………………………………………………………………………… 8

8a. Blades 240-250 um thick in cross section at midthallus; ~4 layers of round medullary cells (51-65 um in diameter) ……………………..…...………………….. R. lociperonica

8b. Blades < ~240 um in cross section at midthallus………………………………….… 9

9a. Blades 180-220 um in cross section; 4-6 medullary layers of compact cells, 1-2 inner cortical layers and 2-3 outer cortical layers; tetrasporangial sori band-shaped; widely distributed from South Australia, Tasmania, Victoria and Western

Australia……………………………………………………………….. R. novahollandica 200

9b. Blades 130-150 um in cross section with 2-3 layers of medullary cells, a single inner cortical layer and single outer cortical layer; tetrasporangial sori oval-shaped; collected from New South Wales or South Australia………………………………... R. womersleyi

10a. Collected from Lord Howe Island ……………………………………………...… 11

10b. Collected from Norfolk Island; plants clumped, sprawling and decumbent; blades

105-160 um in cross section with 3-4 layers of medullary cells ………. R. norfolkensis

11a. Blades 170-190 um thick in cross section at midthallus; 4-5 layers of round medullary cells (35-52.5 um in diameter)…...……………………………… R. insularis

11b. Blades 100-160 um thick in cross section at midthallus; 2-3 layers of round medullary cells (30-40 um in diameter) ...…………………………….... R. novazelandica

Acknowledgements

The following collectors are thanked for their contributions to this project: F. Gurgel, J.

Huisman, G.T. Kraft, L. Le Gall, C. Sanderson and R. Withall. Additional thanks to T.

Birch, B. Herman, A. Johnson, L.G.K. Kraft, C. Lane, L. Le Gall, B. McDonald, D.

McDevit, T. Moore, A. Savoie and M. Wolf for generating some of the DNA barcode data used in analyses. Special thanks to C.W. Schneider for his assistance with Latin nomenclature. This research was supported through funding to the Canadian Barcode of

Life Network from Genome Canada through the Ontario Genomics Institute, Natural

Sciences and Engineering Research Council of Canada (NSERC) and other sponsors

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listed at www.BOLNET.ca. Support was also provided by the Canada Research Chair

Program, the Canada Foundation for Innovation and the New Brunswick Innovation

Foundation.

References

Agardh, J.G. (1876). Species genera et ordines algarum, seu descriptions succinctae specierum, generum et ordinum, quibus algarum regnum constituitur. Volumen tertium: de Florideis curae posteriores. Part 1. pp. [ii*-iii*], [i]-[vii], [1]-724. Lipsiae [Leipzig]: C.W.K. Gleerup. Bostock, P.D. & Holland, A.E. (2010). Census of the Queensland Flora. Queensland Herbarium Biodiversity and Ecosystem Sciences, Department of Environment and Resource Management, Brisbane. Cianciola, E.N., Popolizio, T.R., Schneider, C.W. & Lane, C.E. (2010). Using molecular-assisted alpha taxonomy to better understand red algal biodiversity in Bermuda. Diversity, 2: 946-958. Clarkston, B.E. & Saunders, G.W. (2010). A comparison of two DNA barcode markers for species discrimination in the red algal family Kallymeniaceae (Gigartinales, Florideophyceae), with a description of Euthora timburtonii sp. nov. Botany, 88: 119-131. Clarkston, B.E. & Saunders, G.W. (2013). Resolving species diversity in the red algal genus Callophyllis (Kallymeniaceae, Gigartinales) in Canada using molecular assisted alpha taxonomy. European Journal of Phycology, 48: 27-46. Cribb, A.B. (1996). Seaweeds of Queensland. A Naturalist's Guide. The Queensland Naturalists' Club Inc, Brisbane. Dawson, E.Y. (1941). A review of the genus Rhodymenia with descriptions of new species. Allan Hancock Pacific Expeditions, 3: 123-181. De Toni, G.B. (1900). Sylloge algarum omnium hucusque cognitarum. Vol. IV. Florideae. Sectio II. pp. [i-iv], 387-774 + 775-776 [Index]. Patavii [Padova]: Sumptibus auctoris. Greville, R.K. (1830). Algae britannicae, or Descriptions of the Marine and Other Inarticulated Plants of the British islands, Belonging to the order Algae; with Plates Illustrative of the Genera. McLachlan & Stewart; Baldwin & Cradock, Edinburgh & London. Guiry, M.D. (1977). Studies on marine algae of the British Isles. 10. The genus Rhodyemania. British Phycological Journal, 12: 385-425. Guiry, M.D. & Guiry, G.M. (2015). AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org; searched on 05 January 2015. Hawkes, M.W. & Scagel, R.F. (1986). The marine algae of British Columbia and northern Washington: division Rhodophyta (red lagae), class Rhodophyceae, order Rhodymeniales. Canadian Journal of Botany, 64: 1549-1580. 202

Hind, K.R., Gabrielson, P.W. & Saunders, G.W. (2014). Molecular-assisted alpha taxonomy reveals pseudocryptic diversity among species of Bossiella (Corallinales, Rhodophyta) in the eastern Pacific Ocean. Phycologia, 53: 443-456. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Mentjies, P. & Drummond, A. (2012). Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28: 1647-1649. Le Gall, L. & Saunders, G.W. (2010). DNA barcoding is a powerful tool to uncover algal diversity: a case study of the Phyllophoraceae (Gigartinales, Rhodophyta) in the Canadian flora. Journal of Phycology, 46: 374-389. Le Gall, L.L., Dalen, J.L. & Saunders, G.W. (2008). Phylogenetic analyses of the red algal order Rhodymeniales supports recognition of the Hymenocladiaceae fam. nov., Fryeellaceae fam. nov., and Neogastroclonium gen. nov. Journal of Phycology, 44: 1556-1571. Lewis, J.A. (1984). Checklist and bibliography of benthic marine macroalgae recorded from northern Australia I. Rhodophyta. Department of Defense. Defense Science and Technology Organisation. Materials Research Laboratories, Melbourne, Victoria, Report MRL-R-912. Millar, A.J.K. (1990). Marine red algae of the Coffs Harbour region, northern New South Wales. Australian Systematic Botany, 3: 293-593. Millar, A.J.K. (1999). Marine benthic algae of Norfolk Island, South Pacific. Australian Systematic Botany, 12: 479-547. Millar, A.J.K. & Kraft, G.T. (1993). Catalogue of marine and freshwater red algae (Rhodophyta) of New South Wales, including Lord Howe Island, South-Western Pacific. Australian Systematic Botany, 6: 1-90. Millar, A.J.K., Saunders, G.W., Strachan, I.M. & Kraft, G.T. (1996). The morphology, reproduction and small-subunit rRNA gene sequence of Cephalocystis (Rhodymeniaceae, Rhodophyta), a new genus based on Cordylecladia furcellata J. Agardh from Australia. Phycologia, 35: 48-60. Phillips, J.A. (1997). Algae. In: Queensland Plants: Names and Distribution (Henderson, R.J.F., editor), 223-240. Queensland Herbarium, Department of Environment, Queensland. Phillips, J.A. (2002). Algae. In: Names and distribution of Queensland plants, algae and lichens (Henderson, R.J.F., editor), 228-244. Queensland Government Environmental Protection Agency, Brisbane. Saunders, G.W. (2005). Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 360: 1870-1888. Saunders, G.W. (2008). A DNA barcode examination of the red algal family Dumontiaceae in Canadian waters reveals substantial cryptic species diversity. 1. The foliose Dilsea-Neodilsea complex and Weeksia. Botany, 86: 773-789. Saunders, G.W. & McDevit, D.C. (2012). Acquiring DNA sequence data from dried archival red algae (Florideophyceae) for the purpose of applying available names to contemporary genetic species: a critical assessment. Botany, 90: 191-203. 203

Saunders, G.W. & McDonald, B. (2010). DNA barcoding reveals multiple overlooked Australian species of the red algal order Rhodymeniales (Florideophyceae), with resurrection of Halopeltis J. Agardh and description of Pseudohalopeltis gen. nov. Botany, 88: 639-667. Saunders, G.W. & Millar, K.R. (2014). A DNA barcoding survey of the red algal genus Mazzaella in British Columbia reveals overlooked diversity and new distributional records: descriptions of M. dewreedei sp. nov. and M. macrocarpa sp. nov. Botany, 92: 223-231. Saunders, G.W. & Moore, T.E. (2013). Refinements for the amplification and sequencing of red algal DNA barcode and RedToL phylogenetic markers: a summary of current primers, profiles and strategies. Algae, 28: 31-43. Schneider, C.W., Cianciola, E.N., Popolizio, T.R., Spagnulo, D.S. & Lane, C.E. (2015). A molecular-assisted alpha taxonomic study of the genus Centroceras (Ceramiaceae, Rhodophyta) in Bermuda reveals two novel species. Algae, 30: 5-33. Silva, P.C. (1952). A review of nomenclatural conservation in the algae from the point of view of the type method. University of California Publications in Botany, 25: 241- 323. Sparling, S.R. (1957). The structure and the reproduction of some members of the Rhodymeniaceae. University of California Publications in Botany, 29: 319-96. Thiers, B. (2015). Index Herbariorum: A global directory of public herbaria and associated staff. New York Botanical Garden's Virtual Herbarium. http://sweetgum.nybg.org/science/ih/; searched on 04 January 2015. Womersley, H.B.S. (1996). The Marine Benthic Flora of Southern Australia – Part IIIB- Gracilariales, Rhodymeniales, Corallinales and Bonnemaisoniales. Vol. 5. Australian Biological Resources & the State Herbarium of South Australia, Canberra & Adelaide.

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Table 5.1 Intraspecific COI-5P divergence and distance to nearest neighbor for

Australian Rhodymenia species included in this study.

Species Max Nearest neighbor Distance to intraspecific species nearest divergence (%) neighbor (%) R. compressa (n= 2) 0.15 R. novazelandica 8.66

R. contortuplicata (n= 7) 0 R. prolificans 3.09

R. gladiata (n= 17) 0.46 R. leptophylla 6.58

R. insularis (n= 9) 0.30 R. norfolkensis 4.90

R. leptophylla (n= 1)a N/A R. gladiata 6.58

R. lociperonica (n= 4) 0.15 R. norfolkensis 6.20

R. norfolkensis (n= 10) 0.15 R. insularis 4.90

R. novahollandica (n= 92) 1.65 R. prolificans 5.86

R. novazelandica (n= 4) 0 R. lociperonica 6.56

R. prolificans (n= 11) 0.61 R. stenoglossa 1.09

R. stenoglossa (n= 1) N/A R. prolificans 1.09

R. wilsonis (n= 22) 2.40 R. prolificans 5.86

R. womersleyi (n= 9) 1.06 R. contortuplicata 7.55

a single collection from New Zealand.

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Figure 5.1 Map (not drawn to scale) of Australia and New Zealand showing Rhodymenia species distributions. Symbols refer to the general collection location of a species and do not correspond to sample size. Background map from www.conceptdraw.com.

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Figure 5.2 Phylogenetic tree resulting from RAxML analysis of COI-5P data with bootstrap values appended. Australian taxa are in bold font. Asterisks denote full branch support and dashes (-) indicate support below 50%. Abbreviations indicate the Australian state(s) where each Rhodymenia species was collected (NSW= New

South Wales, SA= South Australia, TAS= Tasmania, VIC= Victoria, WA= Western

Australia).

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Figure 5.3 Australian and New Zealand Rhodymenia spp. a) Habit of vegetative R. leptophylla (GWS002130). b) Cross section at mid-thallus of vegetative R. leptophylla

(GWS002130). c) Habit of vegetative R. novazelandica (GWS022832). d) Cross section at mid-thallus of vegetative R. novazelandica (GWS022832). Scale bars: a) 11 mm; b) 53 µm; c) 25 mm; d) 85 µm.

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Figure 5.4 Australian Rhodymenia spp. a) Habit of vegetative holotype of R. compressa (GWS032782). b) Cross section at mid-thallus of vegetative holotype of R. compressa (GWS032782). c) 9. Habit of vegetative holotype of

R. contortuplicata (GWS015201). d) Cross section at mid-thallus of vegetative holotype of R. contortuplicata (GWS015201). e) Habit of vegetative holotype of R. gladiata (GWS032615). f) Cross section at mid- thallus of vegetative holotype of R. gladiata (GWS032615). g) Habit of vegetative holotype of R. insularis (GWS002043). h) Cross section at mid- thallus of vegetative holotype of R. insularis (GWS002043). Scale bars: a) 11 mm; b) 72 µm; c) 24 mm; d) 44 µm; e) 27 mm; f) 29 µm; g) 25 mm; h) 46

µm.

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Figure 5.5 Australian Rhodymenia spp. a) Habit of vegetative holotype of R. lociperonica (GWS025412). b) Cross section at mid-thallus of vegetative holotype of

R. lociperonica (GWS025412). c) Habit of vegetative holotype of R. norfolkensis

(GWS034326). d) Cross section at mid-thallus of vegetative holotype of R. norfolkensis (GWS034326). e) Habit of vegetative holotype of R. womersleyi

(GWS032769). f) Cross section at mid-thallus of the vegetative holotype of R. womersleyi (GWS032769). g) Cross section of male sorus of R. womersleyi with elongate spermatangial mother cells bearing oval spermatangia (GWS032741). h)

Cross section of tetrasporangial sorus of R. womersleyi with modified outer cortical cells and a cruciately divided tetrasporangium borne on cell of the inner cortex.

(GWS032474). Scale bars: a) 13 mm; b) 60 µm; c) 26 mm; d) 56 µm; e) 8 mm; f) 42

µm; g) 18 µm; h) 43 µm.

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Chapter 6 General Conclusion

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Species provide the foundation of biology and yet, many of the Earth’s species are poorly understood. The overwhelming magnitude of Earth’s biodiversity can make the discovery and characterization of life on earth seem like a daunting or even impossible task (Hebert et al. 2003); however, the biological effects of global climate change have emphasized the need for accurate assessment and documentation of biodiversity so that it may be properly managed and conserved (Costello et al. 2013). Unfortunately, previous studies of biological diversity have been hindered by traditional comparative morphological approaches, which have failed to account for phenotypic plasticity or evolutionary convergence, and thus, rendered those assessments unreliable (Hebert et al.

2003).

The advent of molecular tools, specifically DNA barcoding, has offered a more objective and accessible solution for assessing species diversity and establishing a sustainable means of discovering and delimiting species (Padial et al. 2010; Pečnikar &

Buzan 2014). Similarly, molecular tools have transformed the ability to understand species in a larger evolutionary context and provided a revolutionary approach for resolving the tree of life (Hajibabaei et al. 2007). Over the past two decades the use of molecular data for establishing evolutionary hypotheses and assessing species diversity has become the rule rather than the exception (De Clerck et al. 2013). Indeed, it is rare to encounter phylogenetic or large-scale biodiversity assessments that are not based on genetic data and recently published species descriptions are almost always accompanied by representative DNA sequences (Hajibabaei et al. 2007). To accommodate ever- increasing amounts of sequence data, internet-based public global databases (e.g., the

Barcode of Life Data systems and GenBank) were established. Those repositories 215

facilitate comparison of molecular data at a global scale for consistency in species identifications across studies and act as a central “hub” for housing additional information about an organism (e.g., morphological, biogeographical and environmental data) so that species can be assessed beyond a molecular context.

Employment of integrative approaches (i.e., combining molecular data with the morphology, biogeography or ecology of a species) coupled with publically available genetic databases and increased access to valuable taxonomic information (e.g., species names databases, online species descriptions, digitized herbaria, online pictures of holotype specimens and digitized historical literature) has motivated reassessment of classically defined taxonomy and systematics to obtain a more objective and accurate perspective of organismal evolution and diversity (Padial et al. 2010). The overarching theme of this PhD thesis was reassessing the current understanding of evolutionary relationships and species diversity for the red algal order Rhodymeniales using a contemporary integrative approach combining molecular tools and morphological observations to overcome the deficits of traditional approaches based solely on morphological assessment. The integrative approach implemented for this study has provided an improved understanding of rhodymenialean systematics and given insight into overlooked and cryptic rhodymenialean diversity for a more comprehensive understanding of species limits, distributions and diagnostic features of taxa from two routinely surveyed locales, Australia and western Canada.

In Chapter 2, I aimed to expand upon previously published phylogenetic studies

(Saunders et al. 1999; Le Gall et al. 2008) of the Rhodymeniales to clarify subordinal relationships within this order and create a more robust framework for lower-level 216

rhodymenialean taxonomy. I assessed phylogenetic relationships using the most comprehensive rhodymenialean dataset to date in terms of the number of markers used and number of taxa represented. By implementing a site-stripping technique that systematically removed more quickly evolving sites, I was able to resolve sufficient phylogenetic signal at interfamilial nodes to provide reasonable support for subordinal relationships for the first time. As part of this study, I addressed outstanding issues of polyphyly for some rhodymenialean genera (Erythrymenia and Lomentaria) and established the phylogenetic affinities of taxa (Binghamiopsis, Chamaebotrys and

Minium) missing from previous phylogenetic studies. Further, inclusion of the historically misunderstood genus Drouetia in molecular analyses enabled a reinvestigation of its phylogenetic position. Subsequent morphological observations of holotype material for the generitype species, Drouetia coalescens, clarified tetrasporangial development for taxa assigned to Drouetia and three novel Australian

Drouetia species (D. aggregata sp. nov., D. scutellata sp. nov., and D. viridescens sp. nov.) were described.

In Chapter 3 I assessed species diversity for the Rhodymeniales in British

Columbia using MAAT employing COI-5P and rbcL-3P DNA barcoding, which identified species previously reported from this locale and uncovered overlooked diversity for the genera Botryocladia, Fryeella, Gloiocladia and Rhodymenia. To accommodate those overlooked taxa, I described three novel species (B. hawkesii sp. nov., G. vigneaultii sp. nov. and R. bamfieldensis sp. nov), resurrected R. rhizoides, and recognized F. callophyllidoides comb. nov. for the misclassified Rhodymenia callophyllidoides. I also provided preliminary molecular data suggesting that there may 217

be more species of the parasitic genus Faucheocolax than currently recognized. Lastly, I reassessed anatomical development of the monospecific genus Minium, suggesting that

M. parvum represents a diminutive blade rather than a crust.

In Chapter 4 I used MAAT to identify recently collected material from Australia as the generitype species of Leptofauchea, L. nitophylloides. The previous enabled a reinvestigation of the genus Leptofauchea and its constituent species to assess monophyly. To do so, I used multigene phylogenetic analysis of COI-5P, LSU and rbcL, which demonstrated that species assigned to Leptofauchea were solidly allied with the generitype, establishing monophyly for this genus for the first time. Genetic analyses also indicated that GenBank entries for two previously reported species of Leptofauchea in the northwest Pacific (L. leptophylla described from Japan and L. rhodymenioides described from the Caribbean) are conspecific and best assigned to L. leptophylla. The COI-5P data also warranted description of the novel taxa L. cocosana sp. nov. from Australia and L. munseomica sp. nov. from South Korea.

In Chapter 5 I surveyed Australian species of Rhodymenia using MAAT. The

COI-5P data resolved 12 genetic species groups where five were previously reported. I confirmed the presence of four previously recognized Australian Rhodymenia species, extended the biogeographical range of a New Zealand species into Australia and, after determining that the remaining genetic groups were not consistent with existing concepts of Rhodymenia, described seven novel Australian Rhodymenia species. I emphasized that morphological features and biogeography were adequate for distinguishing only some species of Rhodymenia and recommended DNA sequencing in combination with morphology and biogeography as the most reliable means of identification. 218

Where previous systematic and taxonomic assessments of the Rhodymeniales have been complicated by the limitations of traditional morphology-based approaches, the integrative taxonomic approach employed here (Chapters 2-5) has provided a more objective perspective of rhodymenialean evolutionary relationships and species diversity.

The tools employed for this thesis are widely applicable and have the potential to provide a more reliable context for assessing evolutionary relatedness and biodiversity for a wide variety of eukaryotic organisms.

Future Research

Scientific research is inspired by a question and more often than not investigation of that inquiry motivates additional avenues for research. While this thesis has clarified evolutionary relationships and species diversity for the Rhodymeniales, it has also raised other research questions that warrant future consideration.

This thesis employed small-scale genetic datasets to address evolutionary relationships among taxonomic groups (Chapters 2-5). However, over the past decade, next-generation sequencing methods have provided efficient and relatively cost-effective access to genome-scale datasets (Lemmon & Lemmon 2014). Recent next-generation sequencing studies have demonstrated that transcriptome sequence data provide a rich set of characters for resolving evolutionary relationships among eukaryotic taxa (Hittinger et al. 2010; Grant & Katz 2014; Lemmon & Lemmon 2014). Further, transcriptome data have been successful for resolving highly supported deep phylogenetic relationships previously challenging to resolve with certainty using traditional PCR-based methods

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(Kocot et al. 2011; Wen et al. 2013). Owing to the previous, it would be of interest to reinvestigate subordinal relationships for the Rhodymeniales using transcriptome data to assess if the relationships resolved herein (Chapter 2; Figure 2.1) based on a six-gene concatenated dataset will hold. I expect that transcriptome data will reinforce the relationship of the Fryeellaceae (branch D; Figure 2.1) relative to the Faucheaceae and

Lomentariaceae (branch F; Figure 2.1) as those relationships have been resolved consistently with full statistical support using small-scale datasets. While I suspect that the relationships among the Champiaceae, Hymenocladiaceae and Rhodymeniaceae will also be consistent with my results (branches C & I; Figure 2.1), I suspect that the support for those relationships will be more robust when based on transcriptome data. Although there remain numerous challenges that must be addressed relative to large-scale data collection and analysis (Lemmon & Lemmon 2014), transcriptome data hold great potential to serve as a valuable source for improving accuracy and statistical support of red algal evolutionary relatedness and addressing fundamental questions of red algal biology (e.g., biogeographic origin of lineages, mechanisms of evolution and reproductive isolation, and ecological adaptations; Shaffer & Purugganan 2013).

Ideally, all currently known taxa assigned to a particular group would be included in a phylogenetic context to provide the most comprehensive understanding of phylogenetic affinities and relationships; however, that may be impossible for practical reasons (e.g., lack of material, inability to acquire adequate sequence data; Wiens 2006).

The phylogenetic assessment completed for Chapter 2 includes the greatest representation

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of rhodymenialean genera to date; however, some genera (i.e., Agardhinula, Binghamia,

Cenacrum, Grammephora and Sciadophycus)1 historically assigned to the

Rhodymeniales were lacking. Most of those genera are monospecific (excepting

Binghamia and Sciadophycus) with limited reports of species assigned to those genera since their description (Guiry & Guiry 2016). In the absence of recently collected material the phylogenetic affinities of those groups will likely remain incertae sedis; however, sequencing DNA from historical holotype material has been successful in recent studies (Hind et al. 2014; Hind et al. 2015; Hughey & Miller 2016) and may offer a reasonable alternative for resolving the phylogenetic position of rare taxa as long as appropriate experimental controls are included to reduce the potential of a positive result from contamination (Saunders & McDevit 2012). Once the phylogenetic affinities of the aforementioned genera are established, morphological assessment will be critical to determine if the currently recognized features for family-level distinction hold.

Phylogenetic analyses (Chapter 2) resolved the genus Chrysymenia as polyphyletic with full resolution of C. wrightii as sister to Botryocladia leptopoda (rendering this genus polyphyletic as well) (Figure 2.1). Restoring monophyly for Botryocladia and

Chrysymenia was not possible owing to a lack genetic data for the generitype species of

1 Since the publication of the phylogenetic assessment completed for this thesis, genetic data for the generitype of Sciadophycus, S. stellatus, has been made public and inclusion of that sequence (rbcL) in a larger phylogenetic context has confirmed its position within the Rhodymeniaceae where it was originally placed (Hughey & Miller 2016).

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those genera. As a result of the gross morphological differences between species assigned to Botryocladia (mucilage filled vesicles borne on solid stalks) and those attributed to

Chrysymenia (completely hollow vesicular thallus lacking solid stalks), I suspect that molecular data will support Botryocladia and Chrysymenia as separate genera. Molecular data are also needed for the generitype of the genus Cryptarachne, which was established as a subgenus of Chrysymenia Harvey (1853) to accommodate taxa with “Chrysymenia- like” thalli (hollow and gelatinous saccate thalli) and internal rhizoides in the medullary cavity. Based on those criteria, Kylin (1931) elevated Cryptarachne to the genus level.

The distinction of Chrysymenia and Cryptarachne has been debated in the literature

(accepted by Taylor 1960, Ben Maïz et al. 1987; rejected by Okamura 1930, 1936,

Yamada & Segawa 1953) and at present, Cryptarachne is considered a synonym of

Chrysymenia (Guiry & Guiry 2016). At present, I follow Abbott & Littler (1969) who, after observing collections examined by Taylor (1960) and Okamura (1936) and reporting internal rhizoides were present in some areas of the thallus but absent in other parts of the same thallus, suggested “that the presence or absence of internal rhizoids is a very poor characteristic to distinguish these otherwise very closely related morphological entities”. Thus, I suspect that the molecular data for generitype taxa for Chrysymenia and

Cryptarachne will resolve a monophyletic lineage, supporting Cryptarachne as a synonym of Chrysymenia. All speculation aside, a comprehensive taxonomic assessment of Botryocladia, Chrysymenia and Cryptarachne including their respective generitype taxa is warranted to clarify the species appropriately assigned to each genus and finally lay to rest the Chrysymenia-Cryptarachne debate.

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Based on our Australian collections, overlooked diversity and cryptic complexes were resolved for numerous genera (Ceratodictyon, Chamaebotrys, Chrysymenia,

Rhodymenia, Semnocarpa and Webervanbossea; Figure 2.1); however, a full taxonomic assessment of those groups was outside the scope of this thesis (excepting Rhodymenia,

Chapter 5). Some taxa assigned to the previously mentioned genera that remain unidentified at the species level were included in phylogenetic analyses (Chapter 2;

Figure 2.1) to help break-up long branches and add to the robustness of the dataset.

Future detailed morphological analyses may accurately assign those genetic groups to the appropriate existing species or, if needed, establish them as novel taxa. Uncovering extensive overlooked and cryptic diversity for the Rhodymeniales in Australia is attributed to increased sampling efforts and routine DNA barcoding assessments of that country’s red algal flora. The resolution of seven overlooked species for the genus

Rhodymenia (Chapter 5) puts into perspective the potential extent of underestimated diversity for other genera assigned to the Rhodymeniales as well as red algae in general, not only from Australia but from other areas across the globe.

At present, we have an incomplete understanding of the distributional boundaries along the west coast of North America for the British Columbian rhodymenialean species reported herein (Chapter 3) and comparable comprehensive molecular-assisted alpha taxonomic surveys are needed from Washington, Oregon and California. Of particular interest will be whether the disjunct distributions between central California and northern

British Columbia for Rhodymenia rhizoides hold as recent algal studies have hypothesized that species with that disjunct distribution pattern may have migrated to northern British Columbia from California by the “kelp conveyor” (Saunders 2014). 223

Owing to the previous, a better understanding of rhodymenialean biodiversity along the west coast of North America has the potential to provide greater insight into migration patterns of algal species in the northeast Pacific.

The historic isolation of Australia and New Zealand from other land masses has resulted in high levels of endemism in each respective locale, which are both regarded as biodiversity “hotspots” (Myers et al. 2000). At present, there is an absence in the literature of studies comparing the red algal floras of Australia and New Zealand from a molecular perspective. As a result, the extent to which those locales have a shared red algal flora is largely unknown. While comprehensive molecular-assisted floristic surveys of red algae are needed from New Zealand, our improved understanding of species diversity for the genus Rhodymenia in Australia (Chapter 5) will provide a convenient starting point for those comparative studies.

A common fear that persists among biologists is that species will become extinct before they can be discovered, properly described and conserved; however, there is some hope as recent studies have shown that the number of taxonomists actively describing species has reached an all-time high and current extinction rates may not be as high as previously predicted (Stork 2010; Costello et al. 2013). In regards to the status of red algal taxonomy, there has been an overall increase in description rates of red algal species since the end of the 20th century, which, not surprisingly, coincides with increased application of molecular tools to taxonomic studies (De Clerck et al. 2013). Similarly, the employment of genetic data for inferring red algal relationships at all taxonomic levels has increased substantially over the past 20 years (e.g., Le Gall & Saunders 2007;

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Verbruggen et al. 2010; Dixon & Saunders 2013; Hind & Saunders 2013; Saunders et al.

2016).

This thesis has demonstrated the utility, power and value of combining molecular data with morphological, biogeographical and ecological information for a reliable perspective of evolutionary relationships and more accurate understanding of species diversity. One of the overarching goals of assessing species diversity and reconstructing evolutionary relationships stems from a long-lasting desire to complete a thorough inventory of life on earth and then assemble a comprehensive evolutionary tree to determine how those organisms evolved over time (Burki 2014). The molecular techniques (DNA barcoding with COI-5P, small-scale multigene phylogenetics, site stripping analyses) employed for this thesis have broad applicability among eukaryotic taxa and moving forward continued application of the techniques employed herein will undoubtedly provide a more comprehensive understanding of the world in which we live so that it may be maintained and conserved appropriately.

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Dixon, K.R., & Saunders, G.W. (2013). DNA barcoding and phylogenetics of Ramicrusta and Incendia gen. nov., two early diverging lineages of the Peyssonneliaceae (Rhodophyta). Phycologia, 52: 82-108. Grant, J.R., & Katz, L.A. (2014). Building a phylogenomic pipeline for the eukaryotic tree of life-addressing deep phylogenies with genome-scale data. PLoS Currents, 6. Guiry, M.D. & Guiry, G.M. (2016). Algaebase. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebaseorg; searched on 1 May 2016. Hajibabaei, M., Singer, G.A., Hebert, P.D., & Hickey, D.A. (2007). DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. TRENDS in Genetics, 23: 167-172. Harvey, W.H. (1853). Nereis boreali-americana; or, contributions towards a history of the marine algae of the atlantic and pacific coasts of North America. Part II. Rhodospermeae. Smithsonian Contributions to Knowledge, 5: 1-258. Hebert, P.D., Cywinska, A. & Ball, S.L. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London B: Biological Sciences, 27: 313-321. Hind, K.R., & Saunders, G.W. (2013). A molecular phylogenetic study of the tribe Corallineae (Corallinales, Rhodophyta) with an assessment of genus‐level taxonomic features and descriptions of novel genera. Journal of Phycology, 49: 103-114. Hind, K.R., Gabrielson, P.W., Lindstrom, S.C. & Martone, P.T. (2014). Misleading morphologies and the importance of sequencing type specimens for resolving coralline taxonomy (Corallinales, Rhodophyta): Pachyarthron cretaceum is Corallina officinalis (Note). Journal of Phycology, 50: 760-764. Hind, K.R., Miller, K.A., Young, M., Jensen, C., Gabrielson, P.W., & Martone, P.T. (2015). Resolving cryptic species of Bossiella (Corallinales, Rhodophyta) using contemporary and historical DNA. American Journal of Botany, 102: 1912-1930. Hittinger, C.T., Johnston, M., Tossberg, J.T., & Rokas, A. (2010). Leveraging skewed transcript abundance by RNA-Seq to increase the genomic depth of the tree of life. Proceedings of the Natural Academy of Sciences USA, 107: 1476-1481. Hughey, J.R., & Miller, K. A. (2016). Molecular phylogenetic analysis of Sciadophycus stellatus (Rhodymeniales, Rhodophyta) supports its placement in the family Rhodymeniaceae. Phytotaxa, 245: 297-300. Kocot, K.M., Cannon, J.T., Todt, C., Citarella, M.R., Kohn, A.B., Meyer, A., ... & Halanych, K.M. (2011). Phylogenomics reveals deep molluscan relationships. Nature, 477: 452-456. Kylin, H. (1931). Die Florideenordung Rhodymeniales. Acta Universitatis Lundensis 27: 1-48. Le Gall, L., & Saunders, G.W. (2007). A nuclear phylogeny of the Florideophyceae (Rhodophyta) inferred from combined EF2, small subunit and large subunit ribosomal DNA: establishing the new red algal subclass Corallinophycidae. Molecular Phylogenetics and Evolution, 43: 1118-1130. Le Gall, L., Dalen, J.L. & Saunders, G.W. (2008). Phylogenetic analyses of the red algal order Rhodymeniales supports recognition of the Hymenocladiaceae fam. nov., 226

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227

Appendix A

Table S1. List of species and voucher numbers used in this study. GenBank sequences in bold were generated for this study.

Generitype species are noted with an asterisk. ND indicates data not generated for this study. Percent (%) complete is based

on the number of included sites out of the 8714 bp alignment. Collection details for all specimens are available on the BOLD

website in the public dataset DS-RHDYPHY.

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete

HALYMENIALES

Aeodes nitidissima J. GWS015461 KU722626 ND HM917788 ND ND KU722690 KU726700 74 Agardh*

GWS001525 ND ND ND ND KU722574 ND ND

Cryptonemia undulata G0081 KU722630 ND ND GQ471902 AF419133 ND ND 93 Sonder

GWS014849 ND ND HM917444 ND ND KU501338 KU382069

Glaphyrosiphon GWS015935 KU722631 ND HM917953 ND ND KU722691 KU726703 75 intestinalis (Harvey) Leister & W.A. GWS001524 ND ND ND ND KU722596 ND ND Nelson*

Gelinaria ulvoidea GWS000932 ND ND ND GQ471903 KU722566 ND ND 83 Sonder*

228

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete GWS011421 ND ND HM915271 ND ND KU722711 KU726724

Grateloupia GWS015115 KU722625 ND HM917590 ND ND KU722689 KU726699 75 subpectinata Holmes

G0126 ND ND ND ND KU722570 ND ND

Halymenia plana GWS001572 ND ND HM916222 GQ471906 GQ471914 ND ND 83 Zanardini

GWS015387 ND ND ND ND ND KU722698 KU726713

Isabbottia ovalifolia GWS001334 ND ND HM915678 EF033563 EF033616 ND ND 72 (Kylin) Balakrishnan*

GWS002736 ND ND ND ND ND ND KU726704

Prionitis sternbergii (C. GWS001168 ND ND HM915707 EF033565 EF033617 ND ND 72 Agardh) J. Agardh

GWS021741 ND ND ND ND ND ND JX969790

Tsengia sp._2TAS GWS015837 KU722643 ND HM917898 ND KU722584 KU722703 KU726717 74

Zymurgia GWS000966 KU722650 ND HM916232 EF033566 DQ343698 KU501336 KU382065 93 chondriopsidea (J. Agardh) Lewis & Kraft*

SEBDENIALES

229

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Crassitegula imitans GWS001076 KU722632 ND JN641180 ND DQ343700 ND ND 83 G.W. Saunders & Kraft

GWS002089 ND ND JN641182 EF033581 ND KM360020 KM360043

GWS022917 ND ND ND ND ND ND KU382068

Crassitegula sp._1WA GWS025666 ND ND JN641183 ND JN602192 KU722683 KU726695 64

Lesleigha hawaiiensis Kraft G0436 ND ND JN641187 ND DQ343699 ND KU726734 53 & G.W. Saunders* Sebdenia flabellata CL031201 ND ND JN641197 ND JN602196 ND ND 74 (J. Agardh) P.G. Parkinson BDA0051 KU722655 ND ND ND ND KU722717 KU726730

Sebdenia sp. GWS002074 ND ND ND EF033579 JN602197 ND ND 72

GWS023442 ND ND ND ND ND ND KU382048

RHODYMENIALES

CHAMPIACEAE

Champia gigantea M.J. 17092000-10- ND ND ND EU624164 EU624159 ND ND 48 Wynne 01MW Champia zostericola GWS016179 KU722652 KU934266 ND KU722677 KU722606 KU722714 KU726728 99 (Harvey) Reedman & Womersley

230

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Chylocladia verticilata G0210 KU722627 KU382030 KU382030 EF033573 DQ343712 KU501329 KU382046 100 (Lightfoot) Bliding* 1 Coelothrix irregularis CL020502 ND ND ND EU624165 EU624160 ND KU382050 100 (Harvey) Børgesen*

BDA0233 KU722624 ND ND ND ND KU501327 ND

BDA0328 ND KU934256 ND ND ND ND ND

Dictyothamnion G0231 ND ND JN641186 EU624166 EU624161 ND ND 93 saltatum A. Millar*

GWS032638 KU722633 ND ND ND ND KU722692 KU726705

Gastroclonium ovatum GWS001831 KU722640 KU707861 KU707861 EU624168 EU624162 KU722699 KU726714 100 (Hudson) Papenfuss* 1 Neogastroclonium GWS002940 KU722635 KU687571 KU687571 EU624169 EU624163 KU722693 KU687838 100 subarticulatum 1 (Turner) L. Le Gall, J. Dalen & G.W. Saunders*

FAUCHEACEAE

Faucheocolax attenuata B053a ND ND KU687720 ND EU624156 ND ND 53 Setchell

GWS001423 ND ND ND ND ND ND KU687886

Gloiocladia fryeana GWS001381 ND ND ND EU624170 EU624157 ND ND 93 (Setchell) Sánchez &

231

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Rodríguez-Prieto GWS001421 KU722636 ND ND ND ND KU722694 KR085187

Gloiocladia furcata G0153 ND ND ND EU624171 DQ873280 ND ND 49 J. Agardh* Gloiocladia GWS000469 ND ND HQ919528 EF033574 DQ873283 KU722721 KR085192 82 halymenioides (Harvey) Norris Gloiocladia laciniata B048GWS ND ND KR085170 ND EU624158 ND ND 53 (J. Agardh) Sánchez & Rodríguez-Prieto GWS020495 ND ND ND ND ND ND KR085189

Gloiocladia repens (C. GWS001639 KU722621 KU934253 ND KU382080 ND KU501324 KR140335 100 Agardh) Sánchez & Rodríguez-Prieto G0332 ND ND ND ND AF419143 ND ND

Gloioderma australis J. G0306 KU722664 KU934272 ND EU624172 DQ873282 KU722725 JX969786 100 Agardh* Leptofauchea chiloensis GWS000503 ND ND ND EU624173 DQ873285 ND KR140338 64 Dalen & G.W. Saunders Leptofauchea GWS018362 ND ND KR140329 KU722675 KR140333 ND ND 93 leptophylla (Segawa) Suzuki, Nozaki, Terada, GWS018540 KU722623 ND ND ND ND KU722687 KR140336 Kitayama, Testsu.Hashimoto &Yoshizaki

232

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Leptofauchea GWS018532 ND ND HQ544094 ND KR140331 ND KR140337 53 munseomica Filloramo & G.W. Saunders Leptofauchea GWS032631 ND ND KR085173 ND KR085179 KU722704 KR085190 63 nitophylloides (J. Agardh) Kylin* Leptofauchea pacifica GWS010140 KU722665 KU934273 ND KU382089 ND KU501342 KR085195 100 E.Y. Dawson

JD032 ND ND ND ND DQ873286 ND ND

Leptofauchea sp._1WA G0400 ND ND HM915831 ND DQ873287 ND KR085194 53

Webervanbossea GWS002435 ND ND HM916011 EU624175 DQ873288 ND KR085186 72 splachnoides (Harvey) De Toni fil.* Webervanbossea GWS016349 KU722620 KU934252 ND ND KR085175 KU722682 KR085185 81 sp._1splachnoides Webervanbossea GWS015310 KU722663 ND HM917690 ND KR085182 KU722724 KR085193 74 sp._1TAS Webervanbossea GWS029664 ND ND ND ND KR085178 ND ND 72 tasmanensis Womersley GWS000922 ND ND HM915887 EU624176 ND ND KR085196

FRYEELLACEAE

Hymenocladiopsis CCMP455 KU722660 KU934271 ND EU624203 EU624144 KU501339 KU382071 99 prolifera (Reinsch) M.J. Wynne*

233

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Fryeella gardneri G0335 ND ND ND ND EF033622 ND ND 100 (Setchell) Kylin*

GWS001131 KU722629 KU934257 ND EF033578 ND KU501330 JX969776

Minium parvum R.L. GWS019350 KU722622 KU934255 ND KU722667 KU722550 KU722685 KU687822 100 Moe* Pseudohalopeltis GWS001980 KU722638 KU934259 ND ND ND KU722696 KU726712 100 tasmanensis G.W. Saunders* GWS001481 ND ND ND EU624204 EU624145 ND ND

HYMENOCLADIACEAE

Asteromenia GWS001059 KU722647 ND ND ND DQ068299 KU501334 KU382059 100 anastomosans (Weber- van Bosse) G.W. GWS001079 ND KU934270 ND EU624182 ND ND ND Saunders, C.E. Lane, C.W. Schneider & Kraft

Asteromenia GWS001252 ND ND AY970560 EU624183 DQ068297 ND ND 56 bermudensis G.W. Saunders, C.E. Lane, C.W. Schneider & Kraft Erythrymenia obovata KZNb2348 ND KU707862 KU707862 ND DQ068296 KU722702 KU726716 70 F. Schmitz ex. Mazza* 1 Hymenocladia GWS000423 ND ND ND EU624202 DQ068294 ND ND 82 chondricola (Sonder) J.

234

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Lewis GWS015238 ND ND HM917652 ND ND KU501326 KU382045

Hymenocladia usnea GWS016695 ND ND HM918318 ND KU722617 ND ND 37 (R. Brown ex Turner) J. Agardh* Perbella minuta G0273 ND ND ND ND DQ068295 ND ND 93 (Kylin) Filloramo & G.W. Saunders* GWS002411 ND ND ND EU624201 ND ND ND

GWS015600 KU722659 ND HQ919321 ND ND KU722722 KU726733

LOMENTARIACEAE

Binghamiopsis GWS021625 ND ND KM254698 ND KU722597 KU722709 KU726721 64 caespitosa , J.A. West & Hommersand* Ceratodictyon GWS001072 ND ND ND EF033575 EF033620 ND ND 49 intricatum (C. Agardh) R.E. Norris Ceratodictyon sp._1LH GWS001071 KU722644 KU934263 ND KU722673 KU722586 KU722705 KU726718 100

Ceratodictyon sp._2LH GWS023565 ND ND KU707864 ND ND ND ND 64

GWS023732 ND ND ND ND KU722551 KU722686 KU726697

Ceratodictyon GWS000379 ND ND ND EU624177 DQ873290 ND ND 49 spongiosum Zanardini*

235

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Fushitsunagia catenata G0201 ND ND ND EU624178 EU624155 ND ND 93 (Harvey) Filloramo & G.W. Saunders* GWS008890 ND ND HM918837 ND ND ND KU726735

GWS025507 KU722661 ND ND ND ND KU722723 ND

Lomentaria articulata GWS001821 ND ND ND ND EU624154 ND KU726701 53 (Hudson) Lyngbye*

GWS014682 ND ND KU707860 ND ND ND ND

Lomentaria clavellosa GWS017928 KU722651 ND HM915127 ND KU722603 KU722713 KU726726 74 (Lightfoot ex Turner) Gaillon Lomentaria divaricata RI0265 KU941948 ND KU941949 ND KU941950 KU941951 KU941952 74 (Durant) M.J. Wynne Lomentaria GWS010698 ND ND HM917323 ND ND ND KU687860 83 hakodatensis Yendo

GWS013643 ND ND ND ND KU722564 ND ND

GWS020588 KU722634 ND ND KU722669 ND ND ND

Lomentaria orcadensis GWS001885 ND ND GQ497311 EU624179 DQ873292 KU501333 KU382058 93 (Harvey) Collins ex W.R. Taylor GWS018011 KU722648 ND ND ND ND ND ND

Semnocarpa GWS000325 ND ND ND EU624180 DQ873293 ND ND 72 corynephora (J.

236

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Agardh) Huisman, GWS024812 ND ND KU707879 ND ND ND KU726731 Foard & Kraft*

Semnocarpa minuta GWS015196 KU722641 KU934261 ND KU722672 KU722580 KU722700 KU726715 100 Huisman, Foard & Kraft Semnocarpa sp._2Aus GWS024777 KU722654 ND ND ND ND KU722716 ND 75

GWS024795 ND ND KU707856 ND KU722572 ND KU726709

Semnocarpa sp._3Aus GWS025110 ND ND KU707858 KU722670 KU722576 ND KU726711 72

Stirnia prolifera M.J. 14092000-07- ND KU382037 KU382037 EU624181 DQ873294 KU501340 KU382072 89 Wynne* 09MW 1

RHODYMENIACEAE

Botryocladia leptopoda GWS001073 ND ND ND EU624185 DQ345756 ND ND 71 (J. Agardh) Kylin

GWS008828 ND ND HM918826 ND ND ND KU726723

Botryocladia GWS008823 KU722639 KU934260 ND KU722671 KU722578 KU722697 KU687846 100 pseudodichotoma (Farlow) Kylin Cephalocystis G0133 ND ND ND ND EF033621 ND ND 100 furcellata (J. Agardh) A. Millar, G.W. GWS000958 KU722656 KU934268 ND EF033577 ND KU722718 JQ907550 Saunders, I.M. Strachan & Kraft*

237

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Chamaebotrys sp._1LH GWS023292 ND ND KU707875 ND KU722605 ND KU726727 53

Chamaebotrys GWS024505 ND ND KU707876 KU722678 KU722608 KU722715 KU726729 83 boergesenii (Weber-van Bosse) Huisman* Chrysymenia ornata (J. G0281 KU722662 ND KU382038 EU624186 DQ343670 KU501341 KU382073 93 Agardh) Kylin Chrysymenia wrightii GWS018576 ND KU934254 ND ND ND KU722684 ND 89 (Harvey) Yamada

MH004 ND ND ND ND ND ND KU726707

GWS000306 ND ND ND EU624187 EU624146 ND ND

Coelarthrum opuntia G0303 ND KU707867 KU707867 EU624188 DQ343671 KU722708 KU726720 89 (Endlicher) Borgesen 1 Cordylecladia erecta G0206 ND ND ND ND DQ343713 ND ND 30 (Greville) J. Agardh* Drouetia scutellata GWS032729 ND ND KU707846 ND KU722553 KU722688 KU726698 64 Filloramo & G.W. Saunders Drouetia sp. (South KZNb2258 ND ND ND ND DQ343676 ND ND 30 Africa) Drouetia viridescens GWS032624 KU722658 ND KU707881 KU722681 KU722609 KU722720 KU726732 93 Filloramo & G.W. Saunders Gloiosaccion brownii GWS001952 KU722645 KU707865 KU707865 KU722674 KU722588 KU722706 KU726719 100 Harvey* 1

238

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Gloiosaccion sp._4 GWS024525 KU722628 ND KU707849 KU722668 KU722557 ND KU726702 83 brownii

GWS022802 ND ND JQ907536 ND ND ND JQ907551 72

Halichrysis GWS002090 ND ND ND EU624189 DQ343672 ND ND concrescens (J. Agardh) DeToni Halichrysis micans GWS001065 KU722642 KU934262 ND EU624190 DQ343673 KU722701 JQ858278 100 (Hauptfleisch) P. Huvé & H. Huvé Halopeltis adnata GWS018249 ND ND JQ907538 ND JQ907563 ND JQ907552 53 (Okamura) G.W. Saunders & C.W. Schneider Halopeltis australis (J. G0253 ND ND HM033026 EU624198 EU624152 ND JQ858281 72 Agardh) G.W. Saunders* Halopeltis gracilis GWS002051 KU722657 KU934269 ND KU722679 ND KU722719 JQ907554 100 G.W. Saunders

GWS002049 ND ND ND ND HM033165 ND ND

Irvinea ardreana G0173 KU722637 KU934258 ND EU624191 DQ343714 KU722695 JQ907559 100 (Brodie & Guiry) Guiry* Leptosomia rosea G0147 KU722646 KU934264 ND ND ND KU722707 JQ907560 99 (Harvey) Womersley*

G0384 ND ND ND EU624192 EU624147 ND ND

239

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Maripelta rotata (E.Y. G0177 ND ND ND EU624193 EU624148 ND ND 49 Dawson) E.Y. Dawson* Microphyllum robustum GWS002047 ND ND ND KU722676 ND ND JQ858277 100 G.W. Saunders

GWS002057 ND ND ND ND GU997654 ND ND

GWS002046 KU722666 KU934274 ND ND ND KU722726 ND

Rhodymenia delicatula GWS001878 ND ND ND EU624195 EU624149 ND KU726696 72 P.J.L. Dang

GWS009237 ND ND KU707851 ND ND ND ND

Rhodymenia intricata OK136 ND ND ND EU624196 EU624150 ND ND 72 (Okamura) Okamura

GWS018541 ND ND ND ND ND ND KU726710

GWS018510 ND ND KU707844 ND ND ND ND

Rhodymenia RDW502 KU722649 KU934265 ND ND ND KU722712 KU726725 81 pseudopalmata (Lamouroux) P.C. GWS000261 ND ND ND ND GU997653 ND ND Silva*

Rhodymenia GWS001555 ND ND HM033152 EU624199 EU624153 ND KU726706 72 stenoglossa J. Agardh

240

% Taxonomy Voucher No. COB COI COI-5P EF2 LSU psbA rbcL complete Rhodymenia wilsonis GWS001517 ND ND HM033160 KU722680 HM033183 ND ND 56 (Sonder) G.W. Saunders Sparlingia pertusa GWS000581 KU722653 KU934267 ND EU624200 DQ343677 KU501337 JQ907561 100 (Postels & Ruprecht) G.W. Saunders, I.M. Strachan & Kraft* Rhodymeniaceae sp. GWS029498 ND ND KU707871 ND KU722600 KU722710 KU726722 64

241

Figure. S1. An alternative topology produced by neighbor-joining analysis with the

HKY model to assess the impact of the starting tree on SiteStripper analyses.

242

Appendix B

Table S1. List of species and voucher numbers used in this study. GenBank sequences in bold were generated for this study.

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS Champiaceae Neogastroclonium subarticulatum (Turner) L. Le Gall, Dalen & G.W. Saunders GWS000079 ABMMC2504-08 Piedras Blancas, California, USA GWS KM254679 ND ND ND GWS002196 ABMMC2514-08 Bear Cove Park, Port Hardy, CL, KU687738 ND ND ND Vancouver Island, British Columbia, GWS CAN GWS002821 ABMMC2521-08 Bamfield, Dixon I., British HK KU687768 ND ND ND Columbia, CAN GWS002940 ABMMC2073-08 Bamfield, Seppings I., British GWS KU687571 KU687838 ND ND Columbia, CAN GWS003918 ABMMC2536-08 Bamfield, Dixon I., British GWS, KU687577 ND ND ND Columbia, CAN BC, DM GWS006498 ABMMC2126-08 Stephenson Pt., Nanaimo, British DM, KU687773 ND ND ND Columbia, CAN BC, KR, SM GWS006614 ABMMC2127-08 Tahsis, Island #40 on Esperenza Inlet BC, KU687487 ND ND ND Chart, British Columbia, CAN DM, KR, SM GWS006762 ABMMC2131-08 Tahsis, Friendly Cove, British DM, KU687681 ND ND ND Columbia, CAN BC, KR, HK

243

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS008630 ABMMC2154-08 Point Holmes, Comox, British GWS, KU687694 ND ND ND Columbia, CAN BC, DM, KR GWS009614 ABMMC6107-10 Tahsis, Island #40 on Esperenza Inlet GWS, HM916702 ND ND ND Chart, British Columbia, CAN BC GWS012749 ABMMC4048-09 Burnaby Island near Saw Reef, GWS, HM915382 ND ND ND Gwaii Haanas, British Columbia, DM CAN GWS013331 ABMMC4366-09 Murchison Island Lagoon, Gwaii GWS, HM915634 ND ND ND Haanas, British Columbia, CAN DM GWS013552 ABMMC5466-09 Newberry Cove (east side of western GWS, HM916252 ND ND ND inlet), Gwaii Haanas, British DM Columbia, CAN GWS013700 ABMMC4484-09 Lawn Point, Haida Gwaii, British GWS, HM915699 ND ND ND Columbia, CAN DM GWS013701 ABMMC5619-09 Lawn Point, Haida Gwaii, British GWS, HM916378 ND ND ND Columbia, CAN DM GWS013704 ABMMC5562-09 Lawn Point, Haida Gwaii, British GWS, HM916333 ND ND ND Columbia, CAN DM GWS019968 ABMMC13220-10 Warden Station Huxley Island, GWS, HQ544690 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS020122 ABMMC13347-10 Saw Reef, Gwaii Haanas, British GWS, HQ544764 ND ND ND Columbia, CAN KD GWS020409 ABMMC13562-10 Hot Spring Island (east `back` side), GWS, HQ544898 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS020473 ABMMC13609-10 Hot Spring Island (east `back` side), GWS, HQ544931 ND ND ND Gwaii Haanas, British Columbia, KD CAN

244

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS020825 ABMMC13876-10 Telephone Point, Skidegate, Haida GWS, HQ545112 ND ND ND Gwaii, British Columbia, CAN KD GWS021183 ABMMC14190-10 Alder Island, Gwaii Haanas, British GWS, HQ919444 ND ND ND Columbia, CAN KD GWS021258 ABMMC11321-10 Pigeon Point Lighthouse, California, BC, KM254736 ND ND ND USA KH GWS021310 ABMMC11365-10 Pigeon Point Lighthouse, California, BC, KM254837 ND ND ND USA KH GWS021455 ABMMC11487-10 Sea Lion Point North (backside), BC, KM254416 ND ND ND Point Lobos State Reserve, KH California, USA GWS022035 ABMMC12209-10 Santa Cruz (Four Mile), California, BC, KM254743 ND ND ND USA KH, ST GWS022200 ABMMC12353-10 McAbee Beach, Monterey, BC, KM254521 ND ND ND California, USA KH, ST GWS022232 ABMMC12384-10 McAbee Beach, Monterey, BC, KM254655 ND ND ND California, USA KH, ST GWS022260 ABMMC12407-10 McAbee Beach, Monterey, BC, KM254812 ND ND ND California, USA KH, ST GWS035947 ABMMC18665-13 Woodruff Bay (NE beach), Gwaii GWS, KU687502 ND ND ND Haanas, British Columbia, CAN KD GWS036002 ABMMC18713-13 Beach W of Marco Island, Gwaii GWS, KU687671 ND ND ND Haanas, British Columbia, CAN KD Faucheaceae Faucheocolax attenuata Setchell B053a (on ABMMC2502-08 Bamfield, mouth of inlet, British JPD KU687720 ND ND ND B048)*U Columbia, CAN

245

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS FcolaxFRY n/a Mosquito Pass, San Juan Islands, n/a ND ND ND U30366 Washington, USA FcolaxLAC n/a Mosquito Pass, San Juan Islands, n/a ND ND ND U30365 Washington, USA GWS001423 (on ABMMC16295-11 Bamfield, Wizard I., British GWS KU687787 KU687886 ND KU687926 GWS001422)*U Columbia, CAN GWS004768A (on ABMMC16296-11 Browning Wall, north of Port Hardy, GWS KU687764 ND ND KU687924 GWS004768)*NU British Columbia, CAN GWS014315 (on ABMMC16033-11 Bamfield, Scotts Bay, British GWS, KU687657 ND ND KU687907 GWS014314)*NU Columbia, CAN KD GWS020832 (on ABMMC13883-10 Telephone Point, Skidegate, Haida GWS, HQ545118 KU687869 ND KU687917 GWS020831)*NU Gwaii, British Columbia, CAN KD Gloioladia fryeana (Setchell) N. Sánchez, Rodríguez-Prieto GWS001381 ABMMC10769-10 Pier near statue of diver, Sidney, GWS KR085174 KR085191 ND ND British Columbia, CAN GWS001421 ABMMC10774-10 Bamfield, Wizard I., British GWS N/A KR085187 ND ND Columbia, CAN GWS003010 ABMMC2529-08 Bamfield, Black Fish I., near Helby GWS, KU687654 ND ND ND I., British Columbia, CAN JM GWS008730 ABMMC6681-10 Bamfield, Wizard I., British GWS, KU687651 KU687855 ND KU687906 Columbia, CAN BC, DM, KR GWS010426 ABMMC6909-10 Bamfield, Seapool Rock, British BC, KU687733 ND ND ND Columbia, CAN ST GWS010433 ABMMC6912-10 Bamfield, Seapool Rock, British BC, KU687745 ND ND ND Columbia, CAN ST GWS012656 ABMMC3989-09 Mazarredo Islands, NW of Masset, GWS, HM915335 ND ND ND Haida Gwaii, British Columbia, CAN DM GWS014377 ABMMC16035-11 Bamfield, Bordelais Is. (North Bay), GWS, N/A ND KU687889 KU687927 British Columbia, CAN KD

246

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS019620 ABMMC12933-10 Bamfield, opening of Grappler Inlet, GWS, KU687587 ND ND ND British Columbia, CAN KD GWS019668 ABMMC12978-10 Bamfield, Scotts Bay, British GWS, KU687643 ND ND ND Columbia, CAN KD GWS027310 ABMMC15032-11 Beaver Pt., Salt Spring I., British GWS, KU687769 ND ND ND Columbia, CAN KH GWS027343 ABMMC15181-11 Beaver Pt., Salt Spring I., British GWS, KU687730 ND ND KU687918 Columbia, CAN KH GWS027370 ABMMC15190-11 Victoria Shoal, Salt Spring I., British GWS, KU687774 ND ND ND Columbia, CAN KH GWS027374 ABMMC15182-11 Victoria Shoal, Salt Spring I., British GWS, XXXXX ND ND ND Columbia, CAN KH GWS027557 ABMMC15183-11 Pier near statue of diver, Sidney, GWS, KU687789 ND ND ND British Columbia, CAN KH, BC GWS027566 ABMMC15218-11 Pier near statue of diver, Sidney, GWS, KU687517 ND ND ND British Columbia, CAN KH, BC GWS027918 ABMMC15683-11 Lost Islands, SE wall, Gwaii GWS, KU687798 ND ND ND Haanas, British Columbia, CAN KD GWS028347 ABMMC16084-11 Hoskins Islets (rocky knoll btw), GWS KU687677 ND ND ND Gwaii Haanas, British Columbia, CAN GWS028540 ABMMC16055-11 Kloo Rock, Gwaii Haanas, British GWS, KU687617 ND ND KU687904 Columbia, CAN KD GWS028555 ABMMC16056-11 Kloo Rock, Gwaii Haanas, British GWS, KU687549 ND KU687829 KU687896 Columbia, CAN KD GWS028557 ABMMC16057-11 Kloo Rock, Gwaii Haanas, British GWS, KU687762 KU687880 ND KU687923 Columbia, CAN KD GWS028558 ABMMC16058-11 Kloo Rock, Gwaii Haanas, British GWS, KU687663 ND KU687857 KU687908 Columbia, CAN KD

247

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS028563 ABMMC16060-11 Kloo Rock, Gwaii Haanas, British GWS, N/A KU687833 ND KU687897 Columbia, CAN KD GWS028564 ABMMC16061-11 Kloo Rock, Gwaii Haanas, British GWS, N/A ND KU687837 KU687899 Columbia, CAN KD GWS028570 ABMMC16062-11 Kloo Rock, Gwaii Haanas, British GWS, KU687673 ND ND ND Columbia, CAN KD PWG-841 n/a Canoe Island, San Juan Islands, PWG ND FJ713148 ND ND Washington, USA Gloiocladia media (Kylin) Filloramo & G.W. Saunders GlaciCA n/a Carmel, California, USA PWG ND FJ713149 ND ND GWS021443 ABMMC11475-10 Sea Lion Point North (backside), BC, KM254874 KU687877 ND KU687921 Point Lobos State Reserve, KH California, USA Gloiocladia laciniata (J. Agardh) N. Sánchez, Rodríguez-Prieto n/a n/a Ketchihan, Alaska, USA SL ND AY294355 ND ND B048GWS ABMMC16297-11 Bamfield, mouth of inlet, British JPD KR085170 KR085188 ND ND Columbia, CAN FHL14-014 n/a Salmon Bank, San Juan Islands, ND KM253824 ND ND Washington, USA FHL14-015 n/a Salmon Bank, San Juan Islands, ND KM253823 ND ND Washington, USA FHL14-016 n/a Salmon Bank, San Juan Islands, ND KM253834 ND ND Washington, USA GWS001134 ABMMC2508-08 Bamfield, Dixon I., British GWS KU687647 ND ND ND Columbia, CAN GWS001694 ABMMC2857-08 Shields Island Bay, Rennell Sound, GWS, KU687513 ND ND ND Queen Charlotte Islands, British CL Columbia, CAN GWS002182 ABMMC5671-09 Bear Cove Park, Port Hardy, GWS, HM916412 ND ND ND Vancouver Island, British Columbia, CL CAN

248

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS002183 ABMMC5683-09 Bear Cove Park, Port Hardy, GWS, HM916418 ND ND ND Vancouver Island, British Columbia, CL CAN GWS002197 ABMMC5695-09 Bear Cove Park, Port Hardy, CL, HM916424 ND ND ND Vancouver Island, British Columbia, GWS CAN GWS002211 ABMMC2629-08 Daphne Pt., Port Hardy, Vancouver CL, KU687650 ND ND ND Island, British Columbia, CAN GWS GWS002871 ABMMC9000-10 Bamfield, `Sparlingia Pt.`, Bradys GWS HM918523 ND ND ND Beach, British Columbia, CAN GWS002962 ABMMC9012-10 Bamfield, Scotts Bay, British GWS, HM918533 ND ND ND Columbia, CAN JM GWS002970 ABMMC2527-08 Bamfield, Scotts Bay, British GWS, KU687572 ND ND ND Columbia, CAN JM GWS003016 ABMMC2631-08 Bamfield, Black Fish I., near Helby JM, KU687562 ND ND ND I., British Columbia, CAN GWS GWS003080 ABMMC2861-08 Bamfield, Satellite Passage Reef, MS, KU687540 ND ND ND British Columbia, CAN GWS GWS003253 ABMMC9056-10 Bamfield, Dixon I., British GWS HM918556 ND ND ND Columbia, CAN GWS003255 ABMMC2076-08 Bamfield, Dixon I., British GWS HM915227 ND ND ND Columbia, CAN GWS004324 ABMMC9259-10 Bamfield, Dixon I., British GWS, HM918648 ND ND ND Columbia, CAN BC, DM GWS004325 ABMMC9260-10 Bamfield, Dixon I., British GWS, HM918649 ND ND ND Columbia, CAN BC, DM GWS004328 ABMMC9261-10 Bamfield, Dixon I., British GWS, HM918650 ND ND ND Columbia, CAN BC, DM

249

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS004522 ABMMC2086-08 Bamfield, Seapool Rock, British GWS, KU687594 ND ND ND Columbia, CAN BC, DM GWS004523 ABMMC2087-08 Bamfield, Seapool Rock, British GWS, KU687575 ND ND ND Columbia, CAN BC, DM GWS004582 ABMMC2545-08 Satellite Reef, Bamfield, British GWS, KU687686 ND ND ND Columbia, CAN BC, DM GWS004768 ABMMC9341-10 Browning Wall, north of Port Hardy, BC, HM918695 ND ND KU687909 British Columbia, CAN DM GWS006715 ABMMC6286-10 Tahsis, Islands at ocean end of BC, HM916821 ND ND ND McKay Passage, British Columbia, DM CAN GWS008674 ABMMC6670-10 Bamfield, Seapool Rock, British GWS, HM917070 ND ND ND Columbia, CAN BC, DM, KR GWS008702 ABMMC6678-10 Bamfield, Seapool Rock, British GWS, HM917076 ND ND ND Columbia, CAN BC, DM, KR GWS008731 ABMMC2162-08 Bamfield, Wizard I., British GWS, KU687568 ND ND ND Columbia, CAN BC, DM, KR GWS008975 ABMMC9642-10 Bamfield, Execution Rock, British GWS, HM918862 ND ND ND Columbia, CAN BC GWS010071 ABMMC6803-10 Tahsis, Island south of Clotchman I., GWS, HM917184 ND ND ND Spanish Pilot Group, Tahsis, British BC Columbia, CAN

250

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS010072 ABMMC6804-10 Tahsis, Island south of Clotchman I., GWS, HM917185 ND ND ND Spanish Pilot Group, Tahsis, British BC Columbia, CAN GWS010081 ABMMC6806-10 Tahsis, Island south of Clotchman I., GWS, HM917186 ND ND ND Spanish Pilot Group, Tahsis, British BC Columbia, CAN GWS010282 ABMMC6855-10 Tahsis, Bay on north end of Bodega GWS, HM917229 ND ND ND Island, British Columbia, CAN BC GWS010283 ABMMC6856-10 Tahsis, Bay on north end of Bodega GWS, HM917230 ND ND ND Island, British Columbia, CAN BC GWS010438 ABMMC6915-10 Bamfield, Seapool Rock, British BC, HM917279 ND ND ND Columbia, CAN ST GWS012576 ABMMC3932-09 Chaatl Island across from Newton GWS, KU687758 ND ND KU687922 Point, Haida Gwaii, British DM Columbia, CAN GWS013548 ABMMC5513-09 Newberry Cove (east side of western GWS, KU687527 ND ND ND inlet), Gwaii Haanas, British DM Columbia, CAN GWS013549 ABMMC5525-09 Newberry Cove (east side of western GWS, HM916299 ND ND ND inlet), Gwaii Haanas, British DM Columbia, CAN GWS014314 ABMMC16046-11 Bamfield, Scotts Bay, British GWS, N/A ND KU687842 KU687902 Columbia, CAN KD GWS014331 ABMMC16047-11 Bamfield, Black Fish I., near Helby GWS, KU687582 ND ND ND I., British Columbia, CAN KD GWS019348 ABMMC12674-10 Bamfield, Seapool Rock, British GWS, N/A ND KU687817 ND Columbia, CAN KD GWS019451 ABMMC12774-10 Bamfield, `Sparlingia Pt.`, Bradys GWS, HQ544402 ND ND ND Beach, British Columbia, CAN KD GWS020025 ABMMC13260-10 Saw Reef, Gwaii Haanas, British GWS, N/A ND KU687887 ND Columbia, CAN KD

251

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS020237 ABMMC13428-10 East Copper Island (easterly point), GWS, HQ544812 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS020495 ABMMC13622-10 Hot Spring Island (north side), Gwaii GWS, KR085172 KR085189 ND ND Haanas, British Columbia, CAN KD GWS020751 ABMMC13803-10 Kwuna Island, Haida Gwaii, British GWS, HQ919380 ND ND ND Columbia, CAN KD GWS020752 ABMMC13804-10 Kwuna Island, Haida Gwaii, British GWS, HQ545059 ND ND ND Columbia, CAN KD GWS020753 ABMMC13805-10 Kwuna Island, Haida Gwaii, British GWS, HQ545060 ND ND ND Columbia, CAN KD GWS020760 ABMMC13811-10 Kwuna Island, Haida Gwaii, British GWS, HQ545066 ND ND ND Columbia, CAN KD GWS020777 ABMMC13828-10 Kwuna Island, Haida Gwaii, British GWS, HQ545077 ND ND ND Columbia, CAN KD GWS020829 ABMMC13880-10 Telephone Point, Skidegate, Haida GWS, HQ545115 ND ND ND Gwaii, British Columbia, CAN KD GWS020831 ABMMC13882-10 Telephone Point, Skidegate, Haida GWS, HQ545117 KU687845 ND KU687903 Gwaii, British Columbia, CAN KD GWS020836 ABMMC13887-10 Telephone Point, Skidegate, Haida GWS, HQ545121 ND ND ND Gwaii, British Columbia, CAN KD GWS020837 ABMMC13888-10 Telephone Point, Skidegate, Haida GWS, HQ545122 ND ND ND Gwaii, British Columbia, CAN KD GWS020859 ABMMC13909-10 Indian Head, Skidegate, Haida GWS, HQ545138 ND ND ND Gwaii, British Columbia, CAN KD GWS020861 ABMMC13911-10 Indian Head, Skidegate, Haida GWS, N/A ND KU687826 KU687894 Gwaii, British Columbia, CAN KD GWS020880 ABMMC13930-10 Indian Head, Skidegate, Haida GWS, HQ545152 ND ND ND Gwaii, British Columbia, CAN KD GWS020907 ABMMC13956-10 Indian Head, Skidegate, Haida GWS, HQ545171 ND ND ND Gwaii, British Columbia, CAN KD

252

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS020912 ABMMC13961-10 Indian Head, Skidegate, Haida GWS, HQ545176 ND ND ND Gwaii, British Columbia, CAN KD GWS020935 ABMMC13978-10 Indian Head, Skidegate, Haida GWS, HQ545187 ND ND ND Gwaii, British Columbia, CAN KD GWS020937 ABMMC13980-10 Indian Head, Skidegate, Haida GWS, HQ545189 ND ND ND Gwaii, British Columbia, CAN KD GWS021073 ABMMC14101-10 West of Juskatla Narrows, Masset GWS, HQ545287 ND ND ND Inlet, Haida Gwaii, British KD Columbia, CAN GWS021074 ABMMC14102-10 West of Juskatla Narrows, Masset GWS, HQ545288 ND ND ND Inlet, Haida Gwaii, British KD Columbia, CAN GWS021081 ABMMC14108-10 West of Juskatla Narrows, Masset GWS, HQ545294 ND ND ND Inlet, Haida Gwaii, British KD Columbia, CAN GWS021106 ABMMC14125-10 Cowley Islands, Masset Inlet, Haida GWS, HQ919396 ND ND ND Gwaii, British Columbia, CAN KD GWS021107 ABMMC14126-10 Cowley Islands, Masset Inlet, Haida GWS, N/A ND KU687861 ND Gwaii, British Columbia, CAN KD GWS027306 ABMMC15185-11 Beaver Pt., Salt Spring I., British GWS, N/A ND KU687881 ND Columbia, CAN KH GWS027354 ABMMC15184-11 Beaver Pt., Salt Spring I., British GWS, KU687553 ND ND ND Columbia, CAN KH GWS027371 ABMMC15191-11 Victoria Shoal, Salt Spring I., British GWS, N/A ND KU687831 ND Columbia, CAN KH GWS027372 ABMMC15192-11 Victoria Shoal, Salt Spring I., British GWS, KU687497 ND ND ND Columbia, CAN KH GWS027373 ABMMC15193-11 Victoria Shoal, Salt Spring I., British GWS, N/A ND KU687836 ND Columbia, CAN KH GWS027375 ABMMC15194-11 Victoria Shoal, Salt Spring I., British GWS, N/A ND KU687824 ND Columbia, CAN KH

253

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS027405 ABMMC15195-11 Panther Pt., Wallace I., British GWS, N/A ND KU687888 ND Columbia, CAN KH GWS027406 ABMMC15196-11 Panther Pt., Wallace I., British GWS, N/A ND KU687821 ND Columbia, CAN KH GWS027407 ABMMC15201-11 Panther Pt., Wallace I., British GWS, N/A KU687867 ND ND Columbia, CAN KH GWS027408 ABMMC15197-11 Panther Pt., Wallace I., British GWS, N/A ND KU687827 ND Columbia, CAN KH GWS027915 ABMMC16036-11 Lost Islands, SE wall, Gwaii GWS, N/A ND KU687830 ND Haanas, British Columbia, CAN KD GWS027916 ABMMC16037-11 Lost Islands, SE wall, Gwaii GWS, KU687482 ND ND ND Haanas, British Columbia, CAN KD GWS027923 ABMMC16038-11 Lost Islands, SE wall, Gwaii GWS, KU687522 ND ND ND Haanas, British Columbia, CAN KD GWS027926 ABMMC16048-11 Lost Islands, SE wall, Gwaii GWS, KU687612 ND ND ND Haanas, British Columbia, CAN KD GWS028031 ABMMC16051-11 Lost Islands, NE wall, Gwaii GWS, KU687598 ND ND ND Haanas, British Columbia, CAN KD GWS028075 ABMMC16052-11 Channel btw Murchison et Faraday GWS, KU687578 KU687841 ND KU687901 Is., Gwaii Haanas, British Columbia, KD CAN GWS028098 ABMMC16053-11 Channel btw Murchison et Faraday GWS, N/A ND KU687879 ND Is., Gwaii Haanas, British Columbia, KD CAN GWS028122 ABMMC16039-11 Murchison Is., East End, Gwaii GWS, KU687636 ND ND ND Haanas, British Columbia, CAN KD GWS028140 ABMMC16054-11 Murchison Is., East End, Gwaii GWS, N/A ND KU687849 ND Haanas, British Columbia, CAN KD GWS028397 ABMMC16041-11 Scudder Point (SE coast), Gwaii GWS, N/A ND KU687854 ND Haanas, British Columbia, CAN KD GWS028444 ABMMC16042-11 Saw Reef (W side), Gwaii Haanas, GWS, KU687552 ND KU687832 ND British Columbia, CAN KD

254

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS028559 ABMMC16059-11 Kloo Rock, Gwaii Haanas, British GWS, KU687631 ND KU687851 ND Columbia, CAN KD GWS028587 ABMMC16043-11 Kul Rocks, Gwaii Haanas, British GWS, N/A ND KU687856 ND Columbia, CAN KD GWS028598 ABMMC16044-11 Kul Rocks, Gwaii Haanas, British GWS, N/A ND KU687859 ND Columbia, CAN KD GWS028646 ABMMC16045-11 Point S of Dodge Point, Gwaii GWS, N/A ND KU687818 ND Haanas, British Columbia, CAN KD GWS028665 ABMMC16063-11 East side of Powrivco Bay, Gwaii GWS, N/A ND KU687882 KU687925 Haanas, British Columbia, CAN KD GWS028666 ABMMC16064-11 East side of Powrivco Bay, Gwaii GWS, N/A ND KU687875 KU687920 Haanas, British Columbia, CAN KD GWS028676 ABMMC16066-11 East side of Powrivco Bay, Gwaii GWS, KU687710 ND ND ND Haanas, British Columbia, CAN KD GWS030943 ABMMC17579-12 Nakons Islet, Gwaii Haanas, British GWS, KU687584 ND ND ND Columbia, CAN KD GWS035872 ABMMC18601-13 Small Cove, Lousconne Inlet, Gwaii GWS, KU687533 ND ND ND Haanas, British Columbia, CAN KD GWS036754 ABMMC19686-14 Rosario Beach Point, Washington, GWS, KU687542 ND ND ND USA MB, GF GWS036842 ABMMC21197-15 near Belle Pt., Skidegate Ch., Haida GWS, KU687725 ND ND ND Gwaii, British Columbia, CAN MB, TB JD031 ABMMC2623-08 Bamfield, Seapool Rock, British GWS KU687639 ND ND ND Columbia, CAN Gloioderma australe J. Agardh

G0306 ABMMC10599-10 Seabird, Western Australia, Australia RC, JX969722 JX969786 ND ND MC Gloioderma halymenioides

255

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS000469 ABMMC4891-09 Vivonne Bay, Kangaroo I, South GTK, HQ919528 KR085192 ND ND Australia, Australia GWS Gloiocladia vigneaultii Filloramo & G.W. Saunders Ffryeana n/a Mosquito Pass, San Juan Islands, n/a ND ND ND U30363 Washington, USA GlaciPygma n/a Botanical Beach, Vancouver Island, MJW ND FJ713150 ND ND British Columbia, CAN GWS004638 ABMMC9315-10 Vivian I., Vancouver Island, British BC, HM918677 KU687823 ND ND Columbia, CAN DM GWS008457 ABMMC6619-10 Tuwanek, near Sechelt, British GWS, HM917033 ND ND ND Columbia, CAN BC, DM, KR GWS008496 ABMMC6632-10 Backeddy Resort, British Columbia, GWS, HM917042 ND ND KU687919 CAN BC, DM, KR GWS027428 ABMMC15198-11 Norway Rock, Thetis Is., British GWS, N/A ND KU687834 ND Columbia, CAN KH, BC GWS027563 ABMMC15199-11 Pier near statue of diver, Sidney, GWS, KU687520 ND ND KU687892 British Columbia, CAN KH, BC GWS027570 ABMMC15200-11 Pier near statue of diver, Sidney, GWS, KU687715 ND ND KU687915 British Columbia, CAN KH, BC GWS027946 ABMMC16049-11 Lost Islands, SE wall, Gwaii GWS, N/A ND KU687870 ND Haanas, British Columbia, CAN KD GWS030614 ABMMC17576-12 Islets west of Adam Rocks, Gwaii GWS, KU687679 ND ND ND Haanas, British Columbia, CAN KD GWS030615 ABMMC17577-12 Islets west of Adam Rocks, Gwaii GWS, KU687640 ND ND ND Haanas, British Columbia, CAN KD

256

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS030616 ABMMC17578-12 Islets west of Adam Rocks, Gwaii GWS, KU687644 ND ND ND Haanas, British Columbia, CAN KD GWS035388 ABMMC18314-13 Marble Island (NW reef below GWS, KU687637 ND ND ND `Killer-Whale-Fin Rock`), Haida KD Gwaii, British Columbia, CAN GWS035802 ABMMC18533-13 SGang Gwaay (rocks to NW), Gwaii GWS, KU687739 ND ND ND Haanas, British Columbia, CAN KD GWS036877 ABMMC21198-15 Cape Knox, Haida Gwaii, British GWS, KU687492 ND ND ND Columbia, CAN MB, TB GWS038431 ABMMC19816-14 Marble Island (NW reef below GWS, KU687751 ND ND ND `Killer-Whale-Fin Rock`), Haida MB, Gwaii, British Columbia, CAN KD, AS GWS038464 ABMMC19849-14 Opening to Kitgoro Inlet, Haida GWS, KU687797 ND ND ND Gwaii, British Columbia, CAN MB, KD, AS GWS039059 ABMMC20183-14 Nereocystis bed South of Yan, Haida GWS, KU687559 ND ND ND Gwaii, British Columbia, CAN MB Gloiocladia furcata J. Agardh Gfur n/a I. Formigues, Palamos, Spain CRP ND FJ713146 ND ND Gloiocladia iyoensis (Okamura) R.E. Norris GiyoGF2005 n/a Jervis Bay, New South Wales, AM, ND FJ7131421 ND ND Australia DH Gloiocladia japonica (Okamura) Yoshida G718 n/a n/a, n/a, South Korea n/a KF547029 KF547021 ND Gloiocladia microspora (Bornet ex Rodríguez y Femenías) N. Sánchez, Rodríguez- Prieto HGI-A 7457 n/a Llevant de Mallorca, Balearic Islands, NS ND FJ713145 ND ND Spain

257

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS Gloiocladia repens (C. Agardh) N. Sánchez, Rodríguez-Prieto GWS001639 ABMMC10788-10 Pace, Messina, Straits of Messina, AC KR140326 KR140335 ND ND Mediterranean Sea, Sicily, Italy Gloiocladia pelicana Gavio, Fredericq Gpeli2GF2005 n/a Gulf of Mexico, n/a, USA SF, ND FJ713144.1 ND ND BG, CG, JLB Gloiocladia sp. 1WA GWS024910 OZSEA1886-10 Cozy Corner (Knobby Pt.), Western GWS, XXXXX ND ND ND Australia, Australia KD Gloiocladia spinulosa (Okamura, Segawa) N. Sánchez, Rodríguez-Prieto G695 n/a n/a, n/a, South Korea n/a KF547031 KF547023 ND ND Gloiocladia tenuissima Gavio, Fredericq GtenGF2005 n/a Gulf of Mexico, n/a, USA SF, ND FJ713141 ND ND BG, CG, JLB Leptofauchea nitophylloides (J.Agardh) Kylin GWS032631 OZSEA3885-13 Split Solitary Island (E), Coffs GWS, KR085173 KR085190 ND ND Harbour, New South Wales, Australia KD Leptofauchea pacifica E.Y. Dawson GWS001708 ABMMC049-05 Gospel Reef, Rennell Sound, Queen GWS, AY970580 ND ND ND Charlotte Islands, British Columbia, CL CAN GWS001713 ABMMC050-05 Gospel Reef, Rennell Sound, Queen GWS, AY970579 ND ND ND Charlotte Islands, British Columbia, CL CAN GWS002226 ABMMC051-05 Browning Wall, north of Port Hardy, GWS, AY970581 ND ND ND British Columbia, CAN CL

258

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS003049 ABMMC2532-08 Bamfield, Seapool Rock, British GWS, KU687693 ND ND ND Columbia, CAN JM GWS003072 ABMMC2533-08 Satellite Reef, Bamfield, British GWS, KU687784 ND ND ND Columbia, CAN MS GWS003078 ABMMC2534-08 Satellite Reef, Bamfield, British GWS, KU687532 ND ND ND Columbia, CAN MS GWS004524 ABMMC2088-08 Bamfield, Seapool Rock, British GWS, KU687791 ND ND ND Columbia, CAN BC, DM GWS004525 ABMMC2089-08 Bamfield, Seapool Rock, British GWS, KU687634 ND ND ND Columbia, CAN BC, DM GWS004763 ABMMC2094-08 Browning Wall, north of Port Hardy, BC, KU687511 ND ND ND British Columbia, CAN DM GWS004773 ABMMC19596-14 Browning Wall, north of Port Hardy, BC, KU687737 ND ND ND British Columbia, CAN DM GWS004861 ABMMC2097-08 Tree Knob Islands, Prince Rupert, GWS, KU687491 ND ND ND British Columbia, CAN BC, DM GWS008684 ABMMC2161-08 Bamfield, Seapool Rock, British GWS, KU687695 ND ND ND Columbia, CAN BC, DM, KR GWS008946 ABMMC2164-08 Bamfield, Seapool Rock, British GWS, KU687481 ND ND ND Columbia, CAN BC GWS010139 ABMMC6814-10 Tahsis, Flower Islet, Esperanza GWS, HQ919569 ND ND ND Channel, British Columbia, CAN BC GWS010140 ABMMC5339-09 Tahsis, Flower Islet, Esperanza GWS, HM916176 KR085195 ND ND Channel, British Columbia, CAN BC GWS010141 ABMMC5351-09 Tahsis, Flower Islet, Esperanza GWS, HM916187 ND ND ND Channel, British Columbia, CAN BC

259

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS010160 ABMMC6828-10 Tahsis, Flower Islet, Esperanza GWS, HM917205 ND ND ND Channel, British Columbia, CAN BC GWS010307 ABMMC5258-09 Tahsis, Mozina Pt., British GWS, HM916104 ND ND ND Columbia, CAN BC GWS010314 ABMMC5271-09 Tahsis, Mozina Pt., British GWS, HM916117 ND ND ND Columbia, CAN BC GWS010436 ABMMC5295-09 Bamfield, Seapool Rock, British BC, KU687788 ND ND ND Columbia, CAN ST GWS010437 ABMMC5306-09 Bamfield, Seapool Rock, British BC, HM916148 ND ND ND Columbia, CAN ST GWS014348 ABMMC16095-11 Bamfield, Seapool Rock, British GWS, KU687629 ND ND ND Columbia, CAN KD GWS014356 ABMMC16069-11 Bamfield, Seapool Rock, British GWS, KU687676 ND ND ND Columbia, CAN KD GWS014357 ABMMC16070-11 Bamfield, Seapool Rock, British GWS, KU687505 ND ND ND Columbia, CAN KD GWS014414 ABMMC16117-11 Bamfield, Bordelais Is. (South Wall), GWS, KU687489 ND ND ND British Columbia, CAN KD GWS014415 ABMMC16118-11 Bamfield, Bordelais Is. (South Wall), GWS, KU687579 ND ND ND British Columbia, CAN KD GWS019356 ABMMC12682-10 Bamfield, Seapool Rock, British GWS, KU687603 ND ND ND Columbia, CAN KD GWS019373 ABMMC12698-10 Bamfield, Seapool Rock, British GWS, HQ544360 ND ND ND Columbia, CAN KD GWS022355 ABMMC12493-10 Monterey Bay (Shale Bed), BC, KM254731 ND ND ND California, USA KH, ST GWS027914 ABMMC16125-11 Lost Islands, SE wall, Gwaii GWS, KU687494 ND ND ND Haanas, British Columbia, CAN KD GWS027929 ABMMC16126-11 Lost Islands, SE wall, Gwaii GWS, KU687504 ND ND ND Haanas, British Columbia, CAN KD

260

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS027963 ABMMC16080-11 Lost Islands, SE wall, Gwaii GWS, KU687585 ND ND ND Haanas, British Columbia, CAN KD GWS035392 ABMMC18318-13 Marble Island (NW reef below GWS, KU687801 ND ND ND `Killer-Whale-Fin Rock`), Haida KD Gwaii, British Columbia, CAN GWS035701 ABMMC18436-13 Burnaby Narrows (S opening, in GWS, KU687580 ND ND ND Macrocystic bed), Gwaii Haanas, KD British Columbia, CAN GWS038910 ABMMC20034-14 Tartu Point (E corner), Rennell GWS, KU687776 ND ND ND Sound, Haida Gwaii, British MB Columbia, CAN JD032 ABMMC052-05 Bamfield, Seapool Rock, British GWS AY970566 ND ND ND Columbia, CAN Webervanbossea splachnoides (Harvey) De Toni GWS002435 ABMMC5128-09 Flinders Jetty, Victoria, Australia GWS HM916011 KR085186 ND ND Webervanbossea tasmanensis Womersley GWS000922 ABMMC4906-09 Queenscliff Jetty, Port Phillip Heads, GWS HM915887 KR085196 ND ND Victoria, Australia GWS029664 OZSEA2767-11 Cape Dombey Jetty, Robe, South GWS, KR085171 ND ND ND Australia, Australia KD Fryeellaceae Fryeella callophyllidoides (Hollenberg, I.A. Abbott) Filloramo & G.W. Saunders GWS014349 ABMMC16073-11 Bamfield, Seapool Rock, British GWS, KU687589 ND ND ND Columbia, CAN KD GWS022358 ABMMC12496-10 Monterey Bay (Shale Bed), BC, KM254971 ND ND ND California, USA KH, ST GWS022336 ABMMC12476-10 Monterey Bay (Shale Bed), BC, HQ544242 ND ND ND California, USA KH, ST Fryeella gardneri (Setchell) Kylin

261

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS001131 ABMMC773-06 Bamfield, Mud Cove, British RU JX969692 JX969776 ND ND Columbia, CAN GWS001149 ABMMC2509-08 Bamfield, reef in Satellite Passage, GWS KU687716 ND ND ND British Columbia, CAN GWS002214 ABMMC8908-10 Croker Rock, north of Port Hardy, GWS, HM918466 ND ND ND British Columbia, CAN CL GWS002219 ABMMC2515-08 Browning Wall, north of Port Hardy, GWS, KU687755 ND ND ND British Columbia, CAN CL GWS002961 ABMMC2525-08 Bamfield, Scotts Bay, British GWS, KU687621 ND ND ND Columbia, CAN JM GWS002966 ABMMC2526-08 Bamfield, Scotts Bay, British GWS, KU687746 ND ND ND Columbia, CAN JM GWS003081 ABMMC9034-10 Bamfield, Satellite Passage Reef, MS, HM918547 ND ND ND British Columbia, CAN GWS GWS004109 ABMMC2540-08 Bamfield, Scotts Bay, British GWS, KU687790 ND ND ND Columbia, CAN BC, DM GWS004232 ABMMC1306-07 Saxe Pt., Victoria, British Columbia, GWS, GQ497306 ND ND ND CAN BC, DM GWS004247 ABMMC2084-08 Saxe Pt., Victoria, British Columbia, GWS, KU687799 ND ND ND CAN BC, DM GWS004583 ABMMC2090-08 Satellite Reef, Bamfield, British GWS, KU687534 ND ND ND Columbia, CAN BC, DM GWS004623 ABMMC2633-08 Vivian I., Vancouver Island, British BC, KU687699 ND ND ND Columbia, CAN DM GWS004749 ABMMC2091-08 Browning Wall, north of Port Hardy, BC, KU687778 ND ND ND British Columbia, CAN DM GWS004761 ABMMC2093-08 Browning Wall, north of Port Hardy, BC, KU687538 ND ND ND British Columbia, CAN DM

262

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS004860 ABMMC2096-08 Tree Knob Islands, Prince Rupert, GWS, KU687619 ND ND ND British Columbia, CAN BC, DM GWS006714 ABMMC1594-07 Tahsis, British Columbia, CAN BC, KU687605 ND ND ND DM GWS006716 ABMMC1595-07 Tahsis, British Columbia, CAN BC, KU687696 ND ND ND DM GWS008175 ABMMC2142-08 Bamfield, Scotts Bay, British DM, KU687765 ND ND ND Columbia, CAN BC, KR, SM GWS008409 ABMMC2148-08 Martin Rd., Beaver I., near Sechelt, GWS, KU687649 ND ND ND Sunshine Coast, British Columbia, BC, CAN KR GWS008950 ABMMC2252-08 Bamfield, Seapool Rock, British GWS, KU687708 ND ND ND Columbia, CAN BC GWS010149 ABMMC6821-10 Tahsis, Flower Islet, Esperanza GWS, HM917198 ND ND ND Channel, British Columbia, CAN BC GWS010354 ABMMC5283-09 Palliser Rock, Comox, British BC, HM916127 ND ND ND Columbia, CAN DM, KH GWS010358 ABMMC6880-10 Palliser Rock, Comox, British BC, HM917251 ND ND ND Columbia, CAN DM, KH GWS010398 ABMMC6898-10 Savoie Rocks, Hornby Island, British BC, HM917266 ND ND ND Columbia, CAN DM, KH GWS010466 ABMMC6926-10 Bamfield, Execution Rock, British BC, HM917289 ND ND ND Columbia, CAN ST GWS012549 ABMMC4461-09 Tcenakun Point, Chaatl Island, GWS, HM915687 ND ND ND Haida Gwaii, British Columbia, CAN DM

263

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS012612 ABMMC3949-09 Mazarredo Islands, NW of Masset, GWS, HM915304 ND ND ND Haida Gwaii, British Columbia, CAN DM GWS014311 ABMMC16072-11 Bamfield, Scotts Bay, British GWS, KU687506 ND ND ND Columbia, CAN KD GWS014353 ABMMC16074-11 Bamfield, Seapool Rock, British GWS, KU687655 ND ND ND Columbia, CAN KD GWS014382 ABMMC16075-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687616 ND ND ND British Columbia, CAN KD GWS020972 ABMMC14012-10 Between Wiah Point et Cape GWS, HQ545215 ND ND ND Edensaw (#2), NW of Masset, Haida KD Gwaii, British Columbia, CAN GWS020976 ABMMC14016-10 Between Wiah Point et Cape GWS, HQ545219 ND ND ND Edensaw (#2), NW of Masset, Haida KD Gwaii, British Columbia, CAN GWS022293 ABMMC12438-10 Lover`s Point, Pacific Grove, BC, KM254803 ND ND ND California, USA ST GWS022351 ABMMC12489-10 Monterey Bay (Shale Bed), BC, KM254300 ND ND ND California, USA KH, ST GWS022391 ABMMC12529-10 Aquarium Reef, Monterey Bay, BC, KM254792 ND ND ND California, USA ST GWS022394 ABMMC12532-10 Aquarium Reef, Monterey Bay, BC, KM254950 ND ND ND California, USA ST GWS027012 ABMMC16077-11 Bamfield, opening of Grappler Inlet, GWS, KU687712 ND ND ND British Columbia, CAN KD GWS027326 ABMMC15202-11 Beaver Pt., Salt Spring I., British GWS, KU687661 ND ND ND Columbia, CAN KH GWS027329 ABMMC15186-11 Beaver Pt., Salt Spring I., British GWS, KU687484 ND ND ND Columbia, CAN KH GWS027398 ABMMC15204-11 Panther Pt., Wallace I., British GWS, KU687525 ND ND ND Columbia, CAN KH

264

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS027421 ABMMC15206-11 Panther Pt., Wallace I., British BC, KU687700 ND ND ND Columbia, CAN AL GWS027441 ABMMC15207-11 South Galiano Wall, Galiano Is., GWS, KU687528 ND ND ND British Columbia, CAN KH, BC GWS027558 ABMMC15216-11 Pier near statue of diver, Sidney, GWS, KU687771 ND ND ND British Columbia, CAN KH, BC GWS027559 ABMMC15217-11 Pier near statue of diver, Sidney, GWS, KU687524 ND ND ND British Columbia, CAN KH, BC GWS027560 ABMMC15208-11 Pier near statue of diver, Sidney, GWS, KU687625 ND ND ND British Columbia, CAN KH, BC GWS027561 ABMMC15209-11 Pier near statue of diver, Sidney, GWS, KU687759 ND ND ND British Columbia, CAN KH, BC GWS027902 ABMMC16078-11 Lost Islands, SE wall, Gwaii GWS, KU687581 ND ND ND Haanas, British Columbia, CAN KD GWS027913 ABMMC16079-11 Lost Islands, SE wall, Gwaii GWS, KU687713 ND ND ND Haanas, British Columbia, CAN KD GWS028132 ABMMC16081-11 Murchison Is., East End, Gwaii GWS, KU687483 ND ND ND Haanas, British Columbia, CAN KD GWS028178 ABMMC16082-11 Murchison Is., Northwest Beach, GWS, KU687766 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS028321 ABMMC16083-11 Hoskins Islets (rocky knoll btw), GWS, KU687757 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS028536 ABMMC16085-11 Kloo Rock, Gwaii Haanas, British GWS, KU687770 ND ND ND Columbia, CAN KD

2

65

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS028541 ABMMC15461-11 Kloo Rock, Gwaii Haanas, British GWS, KU687798 ND ND ND Columbia, CAN KD GWS028550 ABMMC16086-11 Kloo Rock, Gwaii Haanas, British GWS, KU873095 ND ND ND Columbia, CAN KD GWS030801 ABMMC17580-12 Macrocystis bed at entrance to GWS, KU687665 ND ND ND George Bay, Gwaii Haanas, British KD Columbia, CAN GWS030818 ABMMC17582-12 Macrocystis bed at entrance to GWS, KU687633 ND ND ND George Bay, Gwaii Haanas, British KD Columbia, CAN GWS034838 ABMMC18028-13 Halkett Wall, Gambier Isl., British GWS, KU687717 ND ND ND Columbia, CAN KD GWS035122 ABMMC18068-13 Lagoon Inlet Tidal Rapids (inside), GWS, KU687516 ND ND ND Haida Gwaii, British Columbia, CAN KD GWS035697 ABMMC18432-13 Burnaby Narrows (S opening, in GWS, KU687531 ND ND ND Macrocystic bed), Gwaii Haanas, KD British Columbia, CAN GWS035700 ABMMC18435-13 Burnaby Narrows (S opening, in GWS, KU687709 ND ND ND Macrocystic bed), Gwaii Haanas, KD British Columbia, CAN GWS036753 ABMMC19685-14 Rosario Beach Point, Washington, GWS, KU687785 ND ND ND USA MB, GF GWS036818 ABMMC21201-15 Smythe Passage East, British GWS, KU687731 ND ND ND Columbia, CAN MB, TB GWS038474 ABMMC19859-14 Opening to Kitgoro Inlet, Haida GWS, KU687586 ND ND ND Gwaii, British Columbia, CAN MB, KD, AS Hymenocladiopsis prolifera (Reinsch) M.J. Wynne CCMP455 ABMMC10503-10 n/a, n/a, Antarctica n/a KU934271 KU382071 ND ND

266

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS Minium parvum R.L. Moe GWS019350 ABMMC12676-10 Bamfield, Seapool Rock, British GWS, KU934255 KU687822 ND ND Columbia, CAN KD Pseudohalopeltis tasmanensis G.W. Saunders GWS001980 ABMMC772-06 Bicheno, in Governor Island GWS, KU934259 KU726712 ND ND Reserve, Tasmania, Australia RW Lomentariaceae Lomentaria hakodatensis Yendo GWS001643 ABMMC2510-08 Bamfield, Dixon I., British GWS KU687496 ND ND ND Columbia, CAN GWS002739 ABMMC2517-08 Bamfield, Dixon I., British GWS, KU687656 ND ND ND Columbia, CAN SC GWS002792 ABMMC2520-08 Bamfield, Dixon I., British GWS, KU687521 ND ND ND Columbia, CAN SC GWS003280 ABMMC9059-10 Bamfield, Seppings I., British GWS HM918559 ND ND ND Columbia, CAN GWS003495 ABMMC9099-10 Bamfield, Dixon I., British GWS, KU687599 ND ND ND Columbia, CAN BC, DM GWS003916 ABMMC9181-10 Bamfield, Dixon I., British GWS, KU687685 ND ND ND Columbia, CAN BC, DM GWS006479 ABMMC6228-10 Stephenson Pt., Nanaimo, British HK, HM916776 ND ND ND Columbia, CAN DM, BC, KR, SM GWS008182 ABMMC6569-10 Bamfield, Scotts Bay, British HK HM916995 ND ND ND Columbia, CAN GWS008412 ABMMC2569-08 Martin Rd., Beaver I., near Sechelt, GWS, KU687539 ND ND ND Sunshine Coast, British Columbia, BC, CAN KR

267

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS008638 ABMMC2156-08 Point Holmes, Comox, British GWS, KU687632 ND ND ND Columbia, CAN BC, DM, KR GWS009585 ABMMC6732-10 Posie Island, Sechelt, British GWS, HM917120 ND ND ND Columbia, CAN BC, DM, KH GWS010698 ABMMC6968-10 Bamfield, Dixon I., British GWS, HM917323 KU687860 ND ND Columbia, CAN BC GWS010728 ABMMC6973-10 Bamfield, Dixon I., British GWS, HM917328 ND ND ND Columbia, CAN BC GWS013333 ABMMC4477-09 Murchison Island Lagoon, Gwaii GWS, HM915692 ND ND ND Haanas, British Columbia, CAN DM GWS013643 ABMMC5522-09 Warden Station Huxley Island, GWS, HQ545349 ND ND ND Gwaii Haanas, British Columbia, DM CAN GWS019979 ABMMC13230-10 Warden Station Huxley Island, GWS, HQ544698 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS020588 ABMMC13699-10 Tanuu Island (Watchmen Station), GWS, HQ544999 ND ND ND Haida Gwaii, British Columbia, CAN KD GWS036028 ABMMC18737-13 Newberry Cove (west side of western GWS, KU687684 ND ND ND inlet), Gwaii Haanas, British KD Columbia, CAN GWS038530 ABMMC19909-14 Between Murchison and East KD, KU687530 ND ND ND Murchison Island, Gwaii Haanas, AS British Columbia, CAN GWS038641 ABMMC20020-14 Southern entrance of Louise Narrows, KD, KU687680 ND ND ND Haida Gwaii, British Columbia, CAN AS Rhodymeniaceae Botryocladia bermudana

268

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS BDA0028 Due south of Frick`s Beach, CWS, HQ933249 ND ND ND Tucker`s Town , outer reef, n/a, CL, Bermuda DM, TP Botryocladia ebriosa GWS032587 OZSEA3834-13 Korora Beach, Coffs Harbour, New GF KU873091 ND ND ND South Wales, Australia Botryocladia hawkesii Filloramo & G.W. Saunders GWS003006 ABMMC2528-08 Bamfield, Black Fish I., near Helby GWS, KU687620 ND ND ND I., British Columbia, CAN JM GWS003013 ABMMC2530-08 Bamfield, Black Fish I., near Helby JM, KU687683 ND ND ND I., British Columbia, CAN GWS GWS003120 ABMMC2074-08 Bamfield, Wizard I., British ML KU687687 ND ND ND Columbia, CAN GWS004849 ABMMC2095-08 Tree Knob Islands, Prince Rupert, GWS, KU687794 ND ND ND British Columbia, CAN BC, DM GWS004852 ABMMC2547-08 Tree Knob Islands, Prince Rupert, GWS, KU687583 ND ND ND British Columbia, CAN BC, DM GWS006709 ABMMC6284-10 Tahsis, Islands at ocean end of BC, HM916819 ND ND KU687911 McKay Passage, British Columbia, DM CAN GWS008484 ABMMC2153-08 Tuwanek, near Sechelt, British GWS, KU687638 ND ND ND Columbia, CAN BC, DM, KR GWS008485 ABMMC2570-08 Tuwanek, near Sechelt, British GWS, KU687543 ND ND ND Columbia, CAN BC, DM, KR

269

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS008821 ABMMC2065-08 Mosquito Pass, San Juan Co., PWG KU687546 KU687828 ND ND Washington, USA GWS008973 ABMMC2574-08 Bamfield, Execution Rock, British GWS, KU687493 ND ND ND Columbia, CAN BC GWS009023 ABMMC2172-08 Gilbert Island, Broken Group, British GWS, KU687615 ND ND ND Columbia, CAN BC GWS009081 ABMMC2182-08 Bamfield, Wizard I., British GWS, KU687486 ND ND ND Columbia, CAN BC GWS009082 ABMMC2183-08 Bamfield, Wizard I., British GWS, KU687604 ND ND ND Columbia, CAN BC GWS009083 ABMMC2576-08 Bamfield, Wizard I., British GWS, KU687608 ND ND ND Columbia, CAN BC GWS009461 ABMMC6700-10 Tuwanek, near Sechelt, British GWS, HM917094 ND ND KU687898 Columbia, CAN BC, DM, KH GWS009486 ABMMC6707-10 Pipers Point, Sechelt, British GWS, HM917099 ND ND KU687893 Columbia, CAN BC, DM, KH GWS009487 ABMMC6708-10 Pipers Point, Sechelt, British GWS, HM917100 ND ND KU687916 Columbia, CAN BC, DM, KH GWS010406 ABMMC6901-10 Savoie Rocks, Hornby Island, British BC, HM917269 KU687839 ND KU687900 Columbia, CAN DM, KH GWS027321 ABMMC15211-11 Beaver Pt., Salt Spring I., British GWS, KU687706 ND ND ND Columbia, CAN KH GWS027419 ABMMC15212-11 Panther Pt., Wallace I., British BC, KU687613 ND ND ND Columbia, CAN AL

270

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS028319 ABMMC16089-11 Hoskins Islets (rocky knoll btw), GWS, KU687741 KU687873 ND ND Gwaii Haanas, British Columbia, KD CAN GWS028320 ABMMC16090-11 Hoskins Islets (rocky knoll btw), GWS, N/A KU687872 ND ND Gwaii Haanas, British Columbia, KD CAN GWS034841 ABMMC18031-13 Halkett Wall, Gambier Isl., British GWS, KU687707 ND ND ND Columbia, CAN KD GWS034844 ABMMC18032-13 Channel marker 2 NW Bowen Isl., GWS, KU687607 ND ND ND British Columbia, CAN KD GWS034873 ABMMC18033-13 Worlcombe Isl. (east point), British GWS, KU687653 ND ND ND Columbia, CAN KD GWS034899 ABMMC18034-13 Dorman Bay, Bowen Isl., British GWS, KU687609 ND ND ND Columbia, CAN KD GWS035160 ABMMC18106-13 Sewell Rocks, Haida Gwaii, British GWS, KU687554 ND ND ND Columbia, CAN KD GWS035635 ABMMC18370-13 Lagoon Inlet Tidal Rapids (inside), GWS, KU687703 ND ND ND Haida Gwaii, British Columbia, CAN KD Botryocladia leptopoda GWS008828 ABMMC9584-10 Dancalan, Bulusan, Sorsogon, EC HM918826 KU726723 ND ND South Luzon, n/a, Philippines Botryocladia neushulii GWS008824 ABMMC2067-08 Baja region, n/a, Mexico MHH, KU873092 KU873090 ND ND JH Botrocladia occidentalis GWS008827 ABMMC9583-10 Onslow Bay, North Carolina, USA MD HM918825 ND ND ND Botryocladia pseudodichotoma (Farlow) Kylin GWS008822 ABMMC9580-10 Cannery Row, Monterey Co., PWG HM918822 ND ND ND California, USA GWS008823 ABMMC2066-08 Point Loma, San Diego Co., MHH, KM254544 KU687846 ND ND California, USA MV

271

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS020664 ABMMC16515-12 Salt Creek, California, USA CWS N/A KU687885 ND ND GWS022230 ABMMC12382-10 McAbee Beach, Monterey, BC, KM254747 KU687863 ND KU687913 California, USA KH, ST GWS022231 ABMMC12383-10 McAbee Beach, Monterey, BC, KM254663 ND ND KU687910 California, USA KH, ST GWS022262 ABMMC12408-10 McAbee Beach, Monterey, BC, KM254741 ND ND ND California, USA KH, ST GWS022279 ABMMC12424-10 Lover`s Point, Pacific Grove, BC, KM254254 ND ND KU687890 California, USA ST GWS022381 ABMMC12519-10 Aquarium Reef, Monterey Bay, BC, KM254591 KU687853 ND KU687905 California, USA ST Botryocladia sonderi GWS029469 OZSEA2578-11 Breakwater at harbour, Portland, GWS, KU873093 ND ND ND Victoria, Australia KD Chrysymenia ornata G0281 ABMMC4756-09 Kallymenia Flats' off Hole in the AM, KU382038 KU382073 ND ND Wall, Jervis Bay, New South Wales, PR Australia Chrysymenia wrightii GWS000306 ABMMC689-06 Muroran, HokkaidoJapan KK KU707857 ND ND ND MH004 ABMMC11204-10 Ouranohama, Yamada-wan, Iwate- MHH ND KU726707 ND ND Ken Pref., Japan Drouetia viridescens GWS032624 OZSEA3777-13 Split Solitary Island (NW), Coffs GWS, KU707881 KU726732 ND ND Harbour, New South Wales, Australia KD Irvinea ardreana G0173 ABMMC10568-10 SinesPortugal MDG, JQ907545 JQ907559 ND ND JAW

272

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS Minium parvum R.L. Moe GWS014360 ABMMC16088-11 Bamfield, Seapool Rock, British GWS, KU687569 ND ND ND Columbia, CAN KD GWS019350 ABMMC12676-10 Bamfield, Seapool Rock, British GWS, HQ544344 KU687822 ND ND Columbia, CAN KD GWS019354 ABMMC12680-10 Bamfield, Seapool Rock, British GWS, HQ544346 ND ND ND Columbia, CAN KD GWS019355 ABMMC12681-10 Bamfield, Seapool Rock, British GWS, HQ544347 ND ND ND Columbia, CAN KD GWS019580 ABMMC12899-10 Ucluelet, Sargison Bank, British GWS, HQ544505 ND ND ND Columbia, CAN KD GWS020962 ABMMC14002-10 Between Wiah Point et Cape GWS, HQ545206 ND ND ND Edensaw (#2), NW of Masset, Haida KD Gwaii, British Columbia, CAN Rhodymenia bamfieldensis Filloramo & G.W. Saunders GWS003038 ABMMC2531-08 Bamfield, Seapool Rock, British GWS, KU687743 ND ND ND Columbia, CAN JM GWS006347 ABMMC2124-08 Pier near statue of diver, Sidney, DM, KU687564 KU687835 ND ND British Columbia, CAN BC, KR, SM GWS014378 ABMMC16096-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687786 KU687884 ND ND British Columbia, CAN KD GWS014383 ABMMC16100-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687719 ND ND ND British Columbia, CAN KD GWS014388 ABMMC16104-11 Bamfield, Bordelais Is. (North Bay), GWS, N/A KU687876 ND ND British Columbia, CAN KD GWS014391 ABMMC16107-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687664 KU687858 ND ND British Columbia, CAN KD GWS036775 ABMMC19705-14 Keystone Breakwater, Whidbey I., GWS, KU687689 ND ND ND Washington, USA MB, GF

273

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS036776 ABMMC19706-14 Keystone Breakwater, Whidbey I., GWS, KU687675 ND ND ND Washington, USA MB, GF Rhodymenia californica Kylin GWS002262 ABMMC2516-08 Bamfield, Seppings I., British GWS KU687623 ND ND ND Columbia, CAN GWS002752 ABMMC2518-08 Bamfield, Dixon I., British GWS, KU687748 ND ND ND Columbia, CAN SC GWS002762 ABMMC2519-08 Bamfield, Dixon I., British GWS, KU687752 ND ND ND Columbia, CAN SC GWS003140 ABMMC2075-08 Bamfield, Execution Rock, British RW KU687509 ND ND ND Columbia, CAN GWS003192 ABMMC3650-09 Bamfield, Wizard I., British RW KU687627 ND ND ND Columbia, CAN GWS003239 ABMMC746-06 Bamfield, Bradys Beach, British GWS HM033085 ND ND ND Columbia, CAN GWS003279 ABMMC747-06 Bamfield, Seppings I., British GWS KU687750 ND ND ND Columbia, CAN GWS003496 ABMMC2535-08 Bamfield, Dixon I., British GWS, KU687601 ND ND ND Columbia, CAN BC, DM GWS006626 ABMMC4611-09 Tahsis, Island #40 on Esperenza Inlet BC, KU687781 ND ND ND Chart, British Columbia, CAN DM, KR, SM GWS006657 ABMMC2129-08 Tahsis, Island #40 on Esperenza Inlet BC, KU687500 ND ND ND Chart, British Columbia, CAN DM, KR, SM

274

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS006729 ABMMC2130-08 Tahsis, Friendly Cove, British DM, KU687592 ND ND ND Columbia, CAN BC, KR, HK GWS006872 ABMMC2133-08 Lands End? up the left side of BC, KU687570 ND ND ND Pachena Bay where it opens to the DM, ocean, Bamfield, British Columbia, KR, CAN HK GWS008974 ABMMC2166-08 Bamfield, Execution Rock, British GWS, KU687753 ND ND ND Columbia, CAN BC GWS009654 ABMMC2969-08 Tahsis, Island #40 on Esperenza Inlet GWS, KU687574 ND ND ND Chart, British Columbia, CAN BC GWS009983 ABMMC3126-08 Tahsis, Island #40 on Esperenza Inlet GWS, KU687480 ND ND ND Chart, British Columbia, CAN BC GWS010465 ABMMC5317-09 Bamfield, Execution Rock, British BC, HM916158 ND ND ND Columbia, CAN ST GWS010498 ABMMC5329-09 Bamfield, Edward King Island, BC HM916168 ND ND ND British Columbia, CAN GWS010499 ABMMC5341-09 Bamfield, Edward King Island, BC HM916178 ND ND ND British Columbia, CAN GWS010500 ABMMC5259-09 Bamfield, Edward King Island, BC HM916105 ND ND ND British Columbia, CAN GWS010501 ABMMC3145-08 Bamfield, Edward King Island, BC KU687597 ND ND ND British Columbia, CAN GWS010616 ABMMC2974-08 Bamfield, Scotts Bay, British BC, KU687622 ND ND ND Columbia, CAN ST GWS010617 ABMMC2975-08 Bamfield, Scotts Bay, British BC, KU687796 ND ND ND Columbia, CAN ST GWS010618 ABMMC3154-08 Bamfield, Scotts Bay, British BC, KU687508 ND ND ND Columbia, CAN ST GWS010677 ABMMC6067-10 Bamfield, Dixon I., British GWS, HM916672 KU687878 ND ND Columbia, CAN BC

275

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS010689 ABMMC6079-10 Bamfield, Dixon I., British GWS, HM916681 ND ND ND Columbia, CAN BC GWS010691 ABMMC3178-08 Bamfield, Dixon I., British GWS, KU687495 ND ND ND Columbia, CAN BC GWS010692 ABMMC3179-08 Bamfield, Dixon I., British GWS, KU687536 ND ND ND Columbia, CAN BC GWS010714 ABMMC3189-08 Bamfield, Dixon I., British GWS, KU687672 ND ND ND Columbia, CAN BC GWS010715 ABMMC3190-08 Bamfield, Dixon I., British GWS, KU687727 ND ND ND Columbia, CAN BC GWS010785 ABMMC6092-10 Bamfield, Seppings I., British GWS, HM916692 ND ND ND Columbia, CAN BC GWS010794 ABMMC3206-08 Bamfield, Seppings I., British GWS, KU687535 ND ND ND Columbia, CAN BC GWS010795 ABMMC3706-09 Bamfield, Seppings I., British GWS, KU687724 ND ND ND Columbia, CAN BC GWS010816 ABMMC3212-08 Bamfield, Seppings I., British GWS, KU687529 ND ND ND Columbia, CAN BC GWS010875 ABMMC3227-08 Bamfield, Blowhole at Bradys GWS, KU687618 ND ND ND Beach, British Columbia, CAN BC GWS011011 ABMMC3870-09 Bamfield, Seppings I., British RW HM915260 ND ND ND Columbia, CAN GWS013397 ABMMC4358-09 Burnaby (Dolomite) Narrows, Gwaii GWS, HM915627 ND ND ND Haanas, British Columbia, CAN DM GWS013533 ABMMC5500-09 Newberry Cove (east side of western GWS, HM916278 KU687816 ND ND inlet), Gwaii Haanas, British DM Columbia, CAN GWS013534 ABMMC5512-09 Newberry Cove (east side of western GWS, HM916289 ND ND ND inlet), Gwaii Haanas, British DM Columbia, CAN

276

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS013535 ABMMC5524-09 Newberry Cove (east side of western GWS, HM916298 ND ND ND inlet), Gwaii Haanas, British DM Columbia, CAN GWS013575 ABMMC5492-09 Newberry Cove (east side of western GWS, HM916273 ND ND ND inlet), Gwaii Haanas, British DM Columbia, CAN GWS014379 ABMMC16097-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687588 ND ND ND British Columbia, CAN KD GWS014380 ABMMC16098-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687490 ND ND ND British Columbia, CAN KD GWS014381 ABMMC16099-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687744 ND ND ND British Columbia, CAN KD GWS014386 ABMMC16103-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687728 ND ND ND British Columbia, CAN KD GWS014389 ABMMC16105-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687563 ND ND ND British Columbia, CAN KD GWS014390 ABMMC16106-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687565 ND ND ND British Columbia, CAN KD GWS014394 ABMMC16109-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687551 ND ND ND British Columbia, CAN KD GWS014395 ABMMC16110-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687648 ND ND ND British Columbia, CAN KD GWS014396 ABMMC16111-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687702 ND ND ND British Columbia, CAN KD GWS014409 ABMMC16112-11 Bamfield, Bordelais Is. (South Wall), GWS, KU687732 ND ND ND British Columbia, CAN KD GWS014410 ABMMC16113-11 Bamfield, Bordelais Is. (South Wall), GWS, KU687567 ND ND ND British Columbia, CAN KD GWS014411 ABMMC16114-11 Bamfield, Bordelais Is. (South Wall), GWS, KU687705 ND ND ND British Columbia, CAN KD GWS014412 ABMMC16115-11 Bamfield, Bordelais Is. (South Wall), GWS, KU687800 ND ND ND British Columbia, CAN KD

277

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS014413 ABMMC16116-11 Bamfield, Bordelais Is. (South Wall), GWS, KU687515 ND ND ND British Columbia, CAN KD GWS014434 ABMMC16119-11 Bamfield, Bordelais Is. (East Bay), GWS, KU687668 ND ND ND British Columbia, CAN KD GWS014435 ABMMC16120-11 Bamfield, Bordelais Is. (East Bay), GWS, KU687763 ND ND ND British Columbia, CAN KD GWS014436 ABMMC16121-11 Bamfield, Bordelais Is. (East Bay), GWS, KU687756 ND ND ND British Columbia, CAN KD GWS014471 ABMMC16122-11 Bamfield, Deadmans Cove (rocks GWS, KU687691 ND ND ND out towards Kia Bay), British KD Columbia, CAN GWS014472 ABMMC16123-11 Bamfield, Deadmans Cove (rocks GWS, KU687635 ND ND ND out towards Kia Bay), British KD Columbia, CAN GWS014473 ABMMC16124-11 Bamfield, Deadmans Cove (rocks GWS, N/A ND KU687874 ND out towards Kia Bay), British KD Columbia, CAN GWS019207 ABMMC12544-10 Bamfield, Seppings I., British GWS KU687793 ND ND ND Columbia, CAN GWS019265 ABMMC12600-10 Bamfield, Wizard I., British GWS, HQ544293 ND ND ND Columbia, CAN KD GWS019278 ABMMC12609-10 Bamfield, Blowhole at Bradys GWS, HQ544301 ND ND ND Beach, British Columbia, CAN KD GWS019398 ABMMC12722-10 Bamfield, Execution Rock, British GWS, HQ544372 ND ND ND Columbia, CAN KD GWS019403 ABMMC12727-10 Bamfield, Execution Rock, British GWS, HQ544375 ND ND ND Columbia, CAN KD GWS019448 ABMMC12771-10 Bamfield, `Sparlingia Pt.`, Bradys GWS, HQ544399 ND ND ND Beach, British Columbia, CAN KD GWS019452 ABMMC12775-10 Bamfield, `Sparlingia Pt.`, Bradys GWS, HQ544403 ND ND ND Beach, British Columbia, CAN KD

278

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS019453 ABMMC12776-10 Bamfield, `Sparlingia Pt.`, Bradys GWS, HQ544404 ND ND ND Beach, British Columbia, CAN KD GWS019454 ABMMC12777-10 Bamfield, `Sparlingia Pt.`, Bradys GWS, HQ544405 ND ND ND Beach, British Columbia, CAN KD GWS019455 ABMMC12778-10 Bamfield, `Sparlingia Pt.`, Bradys GWS, HQ544406 ND ND ND Beach, British Columbia, CAN KD GWS019541 ABMMC12862-10 Bamfield, Wizard I., British GWS, HQ544473 ND ND ND Columbia, CAN KD GWS019543 ABMMC12864-10 Bamfield, Wizard I., British GWS, HQ544475 ND ND ND Columbia, CAN KD GWS019680 ABMMC12989-10 Bamfield, Scotts Bay, British GWS, HQ544573 ND ND ND Columbia, CAN KD GWS019757 ABMMC13065-10 Alder Island, Gwaii Haanas, British GWS, HQ544604 ND ND ND Columbia, CAN KD GWS019814 ABMMC13113-10 Alder Island, Gwaii Haanas, British GWS, HQ544633 ND ND ND Columbia, CAN KD GWS019825 ABMMC13123-10 Alder Island, Gwaii Haanas, British GWS, HQ544640 ND ND ND Columbia, CAN KD GWS020021 ABMMC13256-10 Saw Reef, Gwaii Haanas, British GWS, KU687606 ND ND ND Columbia, CAN KD GWS020046 ABMMC13281-10 Saw Reef, Gwaii Haanas, British GWS, HQ544729 ND ND ND Columbia, CAN KD GWS020051 ABMMC13286-10 Saw Reef, Gwaii Haanas, British GWS, HQ544732 ND ND ND Columbia, CAN KD GWS020058 ABMMC13293-10 Saw Reef, Gwaii Haanas, British GWS, HQ544737 ND ND ND Columbia, CAN KD GWS020072 ABMMC13304-10 Saw Reef, Gwaii Haanas, British GWS, HQ544743 ND ND ND Columbia, CAN KD GWS020200 ABMMC13393-10 East Copper Island (easterly point), GWS, HQ544789 ND ND ND Gwaii Haanas, British Columbia, KD CAN

279

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS020226 ABMMC13417-10 East Copper Island (easterly point), GWS, HQ544804 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS020334 ABMMC13511-10 East Copper Island (easterly point), GWS, HQ544869 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS020573 ABMMC13685-10 Tanuu Island (Watchmen Station), GWS, HQ544987 ND ND ND Haida Gwaii, British Columbia, CAN KD GWS020574 ABMMC13686-10 Tanuu Island (Watchmen Station), GWS, HQ544988 ND ND ND Haida Gwaii, British Columbia, CAN KD GWS021545 ABMMC11551-10 Sea Lion Point North (frontside), BC, KM254401 ND ND ND Point Lobos State Reserve, KH California, USA GWS021546 ABMMC11552-10 Sea Lion Point North (frontside), BC, KM254354 ND ND ND Point Lobos State Reserve, KH California, USA GWS021547 ABMMC11553-10 Sea Lion Point North (frontside), BC, HQ544068 KU687847 ND ND Point Lobos State Reserve, KH California, USA GWS021548 ABMMC11554-10 Sea Lion Point North (frontside), BC, KM254783 ND ND ND Point Lobos State Reserve, KH California, USA GWS022198 ABMMC12351-10 McAbee Beach, Monterey, BC, KM254523 KU687844 ND ND California, USA KH, ST GWS022219 ABMMC12372-10 McAbee Beach, Monterey, BC, KM254976 ND ND ND California, USA KH, ST GWS022228 ABMMC12380-10 McAbee Beach, Monterey, BC, KM254260 KU687820 ND ND California, USA KH, ST

280

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS022284 ABMMC12429-10 Lover`s Point, Pacific Grove, BC, HQ544225 ND ND ND California, USA ST GWS028013 ABMMC16091-11 Lost Islands, NE Bay, Gwaii GWS, KU687697 KU687862 ND ND Haanas, British Columbia, CAN KD GWS028014 ABMMC16092-11 Lost Islands, NE Bay, Gwaii GWS, KU687760 ND ND ND Haanas, British Columbia, CAN KD GWS028038 ABMMC16093-11 Lost Islands, NE Bay, Gwaii GWS, KU687503 ND ND ND Haanas, British Columbia, CAN KD GWS030501 ABMMC17583-12 Gordon Islands (West), Gwaii GWS, KU687576 ND KU687840 ND Haanas, British Columbia, CAN KD GWS030743 ABMMC17595-12 East corner of entrance to Slim Inlet, GWS, KU687662 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS031231 ABMMC19576-14 Cascade Head Marine Reserve, TF KU687772 ND ND ND Oregon, USA GWS031232 ABMMC19577-14 Cascade Head Marine Reserve, TF KU687595 ND ND ND Oregon, USA GWS031412 ABMMC17584-12 Gordon Islands (West), Gwaii GWS, KU687548 ND ND ND Haanas, British Columbia, CAN KD GWS031413 ABMMC17585-12 Gordon Islands (West), Gwaii GWS, KU687519 ND ND ND Haanas, British Columbia, CAN KD GWS031437 ABMMC17586-12 Gordon Islands (West), Gwaii GWS, KU687566 ND ND ND Haanas, British Columbia, CAN KD GWS031487 ABMMC17587-12 Blackburn Point, Gwaii Haanas, GWS, KU687802 ND ND ND British Columbia, CAN KD GWS035395 ABMMC18321-13 Marble Island (NW reef below GWS, KU687514 ND ND ND `Killer-Whale-Fin Rock`), Haida KD Gwaii, British Columbia, CAN GWS035667 ABMMC18402-13 Burnaby Narrows (S opening, in GWS, KU687734 ND ND ND Macrocystic bed), Gwaii Haanas, KD British Columbia, CAN

281

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS035678 ABMMC18413-13 Burnaby Narrows (S opening, in GWS, KU687561 ND ND ND Macrocystic bed), Gwaii Haanas, KD British Columbia, CAN GWS035689 ABMMC18424-13 Burnaby Narrows (S opening, in GWS, KU687659 ND ND ND Macrocystic bed), Gwaii Haanas, KD British Columbia, CAN GWS035691 ABMMC18426-13 Burnaby Narrows (S opening, in GWS, KU687721 ND ND ND Macrocystic bed), Gwaii Haanas, KD British Columbia, CAN GWS035692 ABMMC18427-13 Burnaby Narrows (S opening, in GWS, KU687507 ND ND ND Macrocystic bed), Gwaii Haanas, KD British Columbia, CAN GWS035738 ABMMC18470-13 Louscoone Pt. (wall to the NE), GWS, KU687560 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS035780 ABMMC18512-13 SGang Gwaay (rocks to NW), Gwaii GWS, KU687736 ND ND ND Haanas, British Columbia, CAN KD GWS035782 ABMMC18514-13 SGang Gwaay (rocks to NW), Gwaii GWS, KU687573 ND ND ND Haanas, British Columbia, CAN KD GWS035783 ABMMC18515-13 SGang Gwaay (rocks to NW), Gwaii GWS, KU687624 ND ND ND Haanas, British Columbia, CAN KD GWS035784 ABMMC18516-13 SGang Gwaay (rocks to NW), Gwaii GWS, KU687682 ND ND ND Haanas, British Columbia, CAN KD GWS035785 ABMMC18517-13 SGang Gwaay (rocks to NW), Gwaii GWS, KU687722 ND ND ND Haanas, British Columbia, CAN KD GWS035796 ABMMC18528-13 SGang Gwaay (rocks to NW), Gwaii GWS, KU687726 ND ND ND Haanas, British Columbia, CAN KD GWS035804 ABMMC18535-13 SGang Gwaay (rocks to NW), Gwaii GWS, KU687742 ND ND ND Haanas, British Columbia, CAN KD GWS035824 ABMMC18555-13 Gordon Islands (across from access GWS, KU687761 ND ND ND beach), Gwaii Haanas, British KD Columbia, CAN

282

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS036006 ABMMC18717-13 Beach W of Marco Island, Gwaii GWS, KU687626 ND ND ND Haanas, British Columbia, CAN KD GWS036009 ABMMC18720-13 Beach W of Marco Island, Gwaii GWS, KU687674 ND ND ND Haanas, British Columbia, CAN KD GWS036022 ABMMC18731-13 Newberry Cove (east side of western GWS, KU687523 ND ND ND inlet), Gwaii Haanas, British KD Columbia, CAN GWS036026 ABMMC18735-13 Newberry Cove (east side of western GWS, KU687690 ND ND ND inlet), Gwaii Haanas, British KD Columbia, CAN GWS036027 ABMMC18736-13 Newberry Cove (east side of western GWS, KU687596 ND ND ND inlet), Gwaii Haanas, British KD Columbia, CAN GWS036044 ABMMC18750-13 East Limestone Island (N of Cabin GWS, KU687735 ND ND ND Cove), Haida Gwaii, British KD Columbia, CAN GWS036054 ABMMC18760-13 East Limestone Island (N of Cabin GWS, KU687669 ND ND ND Cove), Haida Gwaii, British KD Columbia, CAN GWS036896 ABMMC21202-15 Eisenia Pt. (south of Riley Beach), GWS, KU687714 ND ND ND Rennell Sound, Haida Gwaii, British MB, Columbia, CAN TB GWS037017 ABMMC21200-15 Marbel Island, Haida Gwaii, British GWS, KU687501 ND ND ND Columbia, CAN MB, TB GWS038549 ABMMC19928-14 Outer side of headland south of Gogit KD, KU687660 ND ND ND point, Lyell Island, Gwaii Haanas, AS British Columbia, CAN GWS038561 ABMMC19940-14 Outer side of headland south of Gogit KD, KU687666 ND ND ND point, Lyell Island, Gwaii Haanas, AS British Columbia, CAN

283

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS038563 ABMMC19942-14 Outer side of headland south of Gogit KD, KU687782 ND ND ND point, Lyell Island, Gwaii Haanas, AS British Columbia, CAN GWS038585 ABMMC19964-14 Alder Islet, Gwaii Haanas, British JB KU687688 ND ND ND Columbia, CAN GWS038588 ABMMC19967-14 Alder Islet, Gwaii Haanas, British JB KU687485 ND ND ND Columbia, CAN GWS038591 ABMMC19970-14 Rocks off the northeast corner of KD, KU687555 ND ND ND Murchison Island, Gwaii Haanas, AS British Columbia, CAN GWS038596 ABMMC19975-14 Rocks off the northeast corner of KD, KU687667 ND ND ND Murchison Island, Gwaii Haanas, AS British Columbia, CAN GWS038613 ABMMC19992-14 Rocks off the northeast corner of KD, KU687783 ND ND ND Murchison Island, Gwaii Haanas, AS British Columbia, CAN GWS038775 ABMMC21203-15 Gospel Pt., Haida Gwaii, British GWS, KU687692 ND ND ND Columbia, CAN MB, TB JD009 ABMMC2881-08 Bamfield, Dixon I., British JD HM033084 ND ND ND Columbia, CAN Rhodymenia compressa Filloramo & G.W. Saunders GWS032780 OZSEA3798-13 Mutton Bird Island (South), Coffs GWS, KT781926 ND ND ND Harbour, New South Wales, Australia KD Rhodymenia contortuplicata Filloramo & G.W. Saunders GWS001959 ABMMC754-06 Reef west of Verona Sands boat GWS, HM033088 ND ND ND launch, in mouth of Huon River, RW Tasmania, Australia Rhodymenia corallina (Bory) Greville GWS038159 ABMMC20722-14 Bahia de pumillahue, n/a, Chile MB KU565721 ND ND ND

284

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS Rhodymenia delicatula P.J.L. Dangeard GWS030103 ABMMC16734-12 n/a, Massachusetts, USA GWS KU565722 ND ND ND Rhodymenia gladiata Filloramo & G.W. Saunders GWS002467 ABMMC751-06 Breakwater at harbour, Portland, GWS HM033151 ND ND ND Victoria, Australia Rhodymenia holmesii Ardissone GWS000260 ABMMC1261-07 Cap Gris-Nez, n/a, France GWS HM033086 ND ND ND Rhodymenia insularis Filloramo & G.W. Saunders GWS001039 ABMMC752-06 Roach I., Admiralty Group, Lord GWS HM033148 ND ND ND Howe I., New South Wales, Australia Rhodymenia intricata (Okamura) Okamura GWS018541 ABMMC11892-10 Channel between Little, Big GWS, HQ544101 ND ND ND Munseom Islands, Jeju, South Korea HGC Rhodymenia leptophylla J. Agardh GWS002130 ABMMC755-06 Pahi, near AucklandNew Zealand GWS HM033149 ND ND ND Rhodymenia lociperonica Filloramo & G.W. Saunders GWS025150 OZSEA2091-10 Canal Rocks, Western Australia, GWS, KT781923 ND ND ND Australia KD Rhodymenia norfolkensis Filloramo & G.W. Saunders GWS032284 OZSEA3879-13 Simon`s Water Flats, Norfolk Island, GWS, KT781935 ND ND ND Australia KD Rhodymenia novahollandica G.W. Saunders GWS000925 ABMMC2856-08 Queenscliff Jetty, Port Phillip Heads, GWS HM033135 ND ND ND Victoria, Australia Rhodymenia novazelandica E.Y. Dawson

285

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS022832 OZSEA2080-10 Malabar Reef, Lord Howe Island, GWS, KT781924 ND ND ND New South Wales, Australia KD, RW Rhodymenia obtusa (Greville) Womersley G0200 ABMMC2494-08 Grossebucht, Luderitz, n/a, Namibia MHH HM033136 ND ND ND Rhodymenia pacifica Kylin GWS002948 ABMMC2523-08 Bamfield, Seppings I., British GWS KU687645 ND ND ND Columbia, CAN GWS002949 ABMMC2524-08 Bamfield, Seppings I., British GWS KU687591 ND ND ND Columbia, CAN GWS003221 ABMMC838-06 Bamfield, Bradys Beach, British GWS HM033137 ND ND ND Columbia, CAN GWS003957 ABMMC2537-08 Bamfield, Seppings I., British GWS, KU687600 ND ND ND Columbia, CAN BC, DM GWS008976 ABMMC2167-08 Bamfield, Execution Rock, British GWS, KU687723 ND ND ND Columbia, CAN BC GWS010502 ABMMC6102-10 Bamfield, Edward King Island, BC N/A KU687883 ND ND British Columbia, CAN GWS010503 ABMMC6113-10 Bamfield, Edward King Island, BC HM916707 ND ND ND British Columbia, CAN GWS014384 ABMMC16101-11 Bamfield, Bordelais Is. (North Bay), GWS, N/A ND KU687825 ND British Columbia, CAN KD GWS014385 ABMMC16102-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687526 ND ND ND British Columbia, CAN KD GWS014392 ABMMC16108-11 Bamfield, Bordelais Is. (North Bay), GWS, N/A ND KU687866 ND British Columbia, CAN KD GWS014397 ABMMC15977-11 Bamfield, Bordelais Is. (North Bay), GWS, KU687614 KU687848 ND ND British Columbia, CAN KD GWS021330 ABMMC11383-10 Pigeon Point Lighthouse, California, BC, KM254225 ND ND ND USA KH

286

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS021331 ABMMC11384-10 Pigeon Point Lighthouse, California, BC, HQ544050 ND ND ND USA KH GWS021495 ABMMC11516-10 Sea Lion Point North (frontside), BC, KM254517 KU687843 ND ND Point Lobos State Reserve, KH California, USA GWS022080 ABMMC12244-10 Stillwater Cove, Pebble Beach, BC, HQ544187 ND ND ND California, USA KH, ST GWS022173 ABMMC12327-10 Stillwater Cove, Pebble Beach, BC, KM254368 ND ND ND California, USA KH, ST GWS022211 ABMMC12364-10 McAbee Beach, Monterey, BC, HQ544217 KU687850 ND ND California, USA KH, ST GWS022292 ABMMC12437-10 Lover`s Point, Pacific Grove, BC, HQ544229 ND ND ND California, USA ST GWS035668 ABMMC18403-13 Burnaby Narrows, Gwaii Haanas, GWS, KU687611 ND ND ND British Columbia, CAN KD Rhodymenia prolificans Zanardini GWS002593 ABMMC3703-09 Blowhole, near Eaglehawk Nest, GWS HM033139 ND ND ND Tasmania, Australia Rhodymenia pseudopalmata RMAR2003 n/a Guimereux, Brittany, France JG, KJ961083 ND ND ND LF Rhodymenia rhizoides E.Y. Dawson GWS010797 ABMMC3207-08 Bamfield, Seppings I., British GWS, KM254819 ND ND ND Columbia, CAN BC GWS010798 ABMMC3208-08 Bamfield, Seppings I., British GWS, KM254294 ND ND ND Columbia, CAN BC GWS022144 ABMMC12305-10 Stillwater Cove, Pebble Beach, BC, KM254396 ND ND ND California, USA KH, ST

287

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS022378 ABMMC12516-10 Aquarium Reef, Monterey Bay, BC, KM254761 KU687865 ND ND California, USA ST GWS028614 ABMMC16127-11 Point S of Dodge Point, Gwaii GWS, KM254590 KU687852 ND ND Haanas, British Columbia, CAN KD GWS028615 ABMMC16128-11 Point S of Dodge Point, Gwaii GWS, KM254646 ND ND ND Haanas, British Columbia, CAN KD GWS028616 ABMMC16129-11 Point S of Dodge Point, Gwaii GWS, KM254501 ND ND ND Haanas, British Columbia, CAN KD GWS028617 ABMMC16130-11 Point S of Dodge Point, Gwaii GWS, KM254780 ND ND ND Haanas, British Columbia, CAN KD GWS028618 ABMMC16131-11 Point S of Dodge Point, Gwaii GWS, KM254265 ND ND ND Haanas, British Columbia, CAN KD GWS028621 ABMMC15975-11 Point S of Dodge Point, Gwaii GWS, KM254329 ND ND ND Haanas, British Columbia, CAN KD GWS028648 ABMMC16132-11 Point S of Dodge Point, Gwaii GWS, KM254841 KU687871 ND ND Haanas, British Columbia, CAN KD GWS030504 ABMMC17588-12 Gordon Islands (West), Gwaii GWS, KM254252 KU687819 ND ND Haanas, British Columbia, CAN KD GWS030726 ABMMC17589-12 East corner of entrance to Slim Inlet, GWS, KM254317 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS030733 ABMMC17593-12 East corner of entrance to Slim Inlet, GWS, KM254689 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS030734 ABMMC17594-12 East corner of entrance to Slim Inlet, GWS, KM254481 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS030742 ABMMC17590-12 East corner of entrance to Slim Inlet, GWS, KM254346 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS031438 ABMMC17591-12 Gordon Islands (West), Gwaii GWS, KU687610 ND ND ND Haanas, British Columbia, CAN KD

288

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS031439 ABMMC17592-12 Gordon Islands (West), Gwaii GWS, KM254759 KU687864 ND ND Haanas, British Columbia, CAN KD GWS035394 ABMMC18320-13 Marble Island (NW reef below GWS, KM254860 ND ND ND `Killer-Whale-Fin Rock`), Haida KD Gwaii, British Columbia, CAN GWS035803 ABMMC18534-13 SGang Gwaay (rocks to NW), Gwaii GWS, KM254493 ND ND ND Haanas, British Columbia, CAN KD GWS035863 ABMMC18592-13 SGang Gwaay (rocks to NW), Gwaii GWS, KM254619 ND ND ND Haanas, British Columbia, CAN KD GWS036043 ABMMC18749-13 East Limestone Island (N of Cabin GWS, KM254868 ND ND ND Cove), Haida Gwaii, British KD Columbia, CAN GWS036047 ABMMC18753-13 East Limestone Island (N of Cabin GWS, KM254772 ND ND ND Cove), Haida Gwaii, British KD Columbia, CAN GWS036052 ABMMC18758-13 East Limestone Island (N of Cabin GWS, KM254871 ND ND ND Cove), Haida Gwaii, British KD Columbia, CAN GWS037021 ABMMC21205-15 Marbel Island, Haida Gwaii, British GWS, KU687602 ND ND ND Columbia, CAN MB, TB GWS038460 ABMMC19845-14 Opening to Kitgoro Inlet, Haida GWS, KU687593 ND ND ND Gwaii, British Columbia, CAN MB, KD, AS GWS038772 ABMMC21204-15 Gospel Pt., Haida Gwaii, British GWS, KU687792 ND ND ND Columbia, CAN MB, TB Rhodymenia sp. ARS02432 n/a n/a, n/a, Hawaii n/a HQ423135 ND ND ND Rhodymenia stenoglossa J. Agardh GWS001555 ABMMC767-06 Warrnambool, Victoria, Australia GWS HM033152 KU726706 ND ND

289

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS Rhodymenia wilsonis (Sonder) G.W. Saunders GWS001496 ABMMC2040-08 Devils Hole, south of Eaglehawk GWS HM033162 ND ND ND Nest, Tasmania, Australia Rhodymenia womersleyi Filloramo & G.W. Saunders DV006 ABMMC2039-08 Smooth Pools, Eyre Peninsula, South GTK, HM033150 ND ND ND Australia, Australia KD Sparlingia pertusa (Postels, Ruprecht) G.W.Saunders, I.W.Strachan, Kraft GWS000581 ABMMC5637-09 Bamfield, Dixon I., British GWS HM916393 JQ907561 ND ND Columbia, CAN GWS002227 ABMMC5707-09 San Juan I., Cattle Pt., Washington, GWS HQ545351 ND ND ND USA GWS002876 ABMMC9001-10 Browning Wall, north of Port Hardy, GWS, HM918524 ND ND ND British Columbia, CAN CL GWS002886 ABMMC2522-08 Bamfield, `Sparlingia Pt.`, Bradys GWS KU687747 ND ND ND Beach, British Columbia, CAN GWS004009 ABMMC5688-09 Bamfield, `Sparlingia Pt.`, Bradys GWS KU687527 ND ND ND Beach, British Columbia, CAN GWS004010 ABMMC2539-08 Bamfield, `Sparlingia Pt.`, Bradys GWS, KU687754 ND ND ND Beach, British Columbia, CAN BC, DM GWS004150 ABMMC2541-08 Bamfield, `Sparlingia Pt.`, Bradys GWS, KU687512 ND ND ND Beach, British Columbia, CAN BC, DM GWS004156 ABMMC5653-09 Otter Point, near Sooke, Vancouver GWS, HM916402 ND ND ND Island, British Columbia, CAN BC, DM GWS004234 ABMMC5665-09 Otter Point, near Sooke, Vancouver GWS, HM916409 ND ND ND Island, British Columbia, CAN BC, DM GWS004370 ABMMC5689-09 Saxe Pt., Victoria, British Columbia, GWS, HM916420 ND ND ND CAN BC, DM

290

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS004400 ABMMC5701-09 Botanical Beach, Port Renfrew, GWS, HQ919542 ND ND ND Vancouver I., British Columbia, CAN BC, DM GWS004521 ABMMC2085-08 Botanical Beach, Port Renfrew, GWS, KU687498 ND ND ND Vancouver I., British Columbia, CAN BC, DM GWS004754 ABMMC2092-08 Bamfield, Seapool Rock, British GWS, KU687779 ND ND ND Columbia, CAN BC, DM GWS004769 ABMMC1464-07 Browning Wall, north of Port Hardy, BC, KU687510 ND ND ND British Columbia, CAN DM GWS004865 ABMMC2098-08 Browning Wall, north of Port Hardy, BC, KU687767 ND ND ND British Columbia, CAN DM GWS004929 ABMMC5655-09 Tree Knob Islands, Prince Rupert, GWS, HM916403 ND ND ND British Columbia, CAN BC, DM GWS005042 ABMMC2100-08 Stenhouse Reef, Prince Rupert, GWS, KU687780 ND ND ND British Columbia, CAN BC GWS005043 ABMMC5715-09 Ridley Island (south of coal GWS, HM916433 ND ND ND terrminal), Prince Rupert, British BC, Columbia, CAN DM GWS005045 ABMMC2101-08 Ridley Island (south of coal GWS, KU687652 ND ND ND terrminal), Prince Rupert, British BC, Columbia, CAN DM GWS005156 ABMMC5680-09 Ridley Island (south of coal GWS, HM916415 ND ND ND terrminal), Prince Rupert, British BC, Columbia, CAN DM GWS006340 ABMMC5704-09 Butze Rapids, Prince Rupert, British GWS, HM916428 ND ND ND Columbia, CAN BC, DM

291

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS006414 ABMMC2125-08 Pier near statue of diver, Sidney, DM, KU687704 ND ND ND British Columbia, CAN BC, KR, SM GWS008423 ABMMC2149-08 Spring Bay, British Columbia, CAN DM, KU687729 ND ND ND BC, KR, SM GWS008427 ABMMC2150-08 Martin Rd., Beaver I., near Sechelt, GWS, KU687749 ND ND ND Sunshine Coast, British Columbia, BC, CAN KR GWS008449 ABMMC2151-08 Martin Rd., Beaver I., near Sechelt, GWS, KU687488 ND ND ND Sunshine Coast, British Columbia, BC, CAN KR GWS008450 ABMMC6618-10 Tuwanek, near Sechelt, British GWS, HM917032 ND ND ND Columbia, CAN BC, DM, KR GWS008451 ABMMC2152-08 Tuwanek, near Sechelt, British GWS, KU687701 ND ND ND Columbia, CAN BC, DM, KR GWS008483 ABMMC1822-07 Tuwanek, near Sechelt, British GWS, KU687795 ND ND ND Columbia, CAN BC, DM, KR GWS008633 ABMMC2155-08 Tuwanek, near Sechelt, British GWS, KU687590 ND ND ND Columbia, CAN BC, DM, KR

292

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS008672 ABMMC2159-08 Point Holmes, Comox, British GWS, KU687550 ND ND ND Columbia, CAN BC, DM, KR GWS009458 ABMMC3033-08 Bamfield, Seapool Rock, British GWS, KU687630 ND ND ND Columbia, CAN BC, DM, KR GWS009459 ABMMC5662-09 Tuwanek, near Sechelt, British GWS, HM916408 ND ND ND Columbia, CAN BC, DM, KH GWS009476 ABMMC5698-09 Tuwanek, near Sechelt, British GWS, HQ919541 ND ND ND Columbia, CAN BC, DM, KH GWS009518 ABMMC6071-10 Tuwanek, near Sechelt, British GWS, HQ545359 ND ND ND Columbia, CAN BC, DM, KH GWS009542 ABMMC6083-10 Nine Mile Point, Sechelt, British GWS, HM916685 ND ND ND Columbia, CAN BC, DM, KH GWS010166 ABMMC6076-10 Nine Mile Point, Sechelt, British GWS, HM916679 ND ND ND Columbia, CAN BC, DM, KH GWS010431 ABMMC6101-10 Palliser Rock, Comox, British BC, HM916698 ND ND ND Columbia, CAN DM, KH

293

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS010504 ABMMC6031-10 Bamfield, Seapool Rock, British BC, HM916645 ND ND ND Columbia, CAN ST GWS010507 ABMMC6043-10 Bamfield, Edward King Island, BC HM916652 ND ND ND British Columbia, CAN GWS010799 ABMMC6104-10 Bamfield, Edward King Island, BC HM916700 ND ND ND British Columbia, CAN GWS012643 ABMMC3978-09 Bamfield, Seppings I., British GWS, HM915324 ND ND ND Columbia, CAN BC GWS012983 ABMMC4094-09 Scudder Point, Burnaby Island, GWS, N/A ND KU687868 ND Gwaii Haanas, British Columbia, DM CAN GWS013007 ABMMC4199-09 Mazarredo Islands, NW of Masset, GWS, HM915501 ND ND ND Haida Gwaii, British Columbia, CAN DM GWS013624 ABMMC5533-09 Scudder Point, Burnaby Island, GWS, HM916307 ND ND ND Gwaii Haanas, British Columbia, DM CAN GWS019484 ABMMC12806-10 Warden Station Huxley Island, GWS, HQ544425 ND ND ND Gwaii Haanas, British Columbia, DM CAN GWS019635 ABMMC12947-10 Bamfield, Taylor Islet, British GWS, HQ544545 ND ND ND Columbia, CAN KD GWS019824 ABMMC13122-10 Bamfield, opening of Grappler Inlet, GWS, HQ544639 ND ND ND British Columbia, CAN KD GWS020030 ABMMC13265-10 Alder Island, Gwaii Haanas, British GWS, HQ544720 ND ND ND Columbia, CAN KD GWS020201 ABMMC13394-10 Saw Reef, Gwaii Haanas, British GWS, HQ544790 ND ND ND Columbia, CAN KD GWS020235 ABMMC13426-10 East Copper Island (easterly point), GWS, HQ544810 ND ND ND Gwaii Haanas, British Columbia, KD CAN

294

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS020618 ABMMC13722-10 East Copper Island (easterly point), GWS, HQ545014 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS020721 ABMMC13773-10 North Beach (near Naval Station), GWS, HQ545040 ND ND ND Haida Gwaii, British Columbia, CAN KD GWS020763 ABMMC13814-10 Kwuna Island, Haida Gwaii, British GWS, HQ545069 ND ND ND Columbia, CAN KD GWS020845 ABMMC13895-10 Kwuna Island, Haida Gwaii, British GWS, HQ545126 ND ND KU687912 Columbia, CAN KD GWS020913 ABMMC13962-10 Indian Head, Skidegate, Haida GWS, KU687777 ND ND ND Gwaii, British Columbia, CAN KD GWS021051 ABMMC14080-10 Indian Head, Skidegate, Haida GWS, HQ545274 ND ND KU687895 Gwaii, British Columbia, CAN KD GWS021070 ABMMC14098-10 Juskatla Narrows, Masset Inlet, GWS, HQ545284 ND ND ND Haida Gwaii, British Columbia, CAN KD GWS021071 ABMMC14099-10 West of Juskatla Narrows, Masset GWS, HQ545285 ND ND ND Inlet, Haida Gwaii, British KD Columbia, CAN GWS021072 ABMMC14100-10 West of Juskatla Narrows, Masset GWS, HQ545286 ND ND KU687891 Inlet, Haida Gwaii, British KD Columbia, CAN GWS021084 ABMMC14111-10 West of Juskatla Narrows, Masset GWS, HQ545296 ND ND ND Inlet, Haida Gwaii, British KD Columbia, CAN GWS021127 ABMMC14146-10 West of Juskatla Narrows, Masset GWS, KU687698 ND ND ND Inlet, Haida Gwaii, British KD Columbia, CAN GWS027549 ABMMC15223-11 Cowley Islands, Masset Inlet, Haida GWS, KU687658 ND ND ND Gwaii, British Columbia, CAN KD GWS027905 ABMMC15990-11 Beddis Beach (left point et seagrass GWS, KU687628 ND ND ND bed), Salt Spring, British Columbia, KH, CAN BC

295

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS027937 ABMMC16134-11 Lost Islands, SE wall, Gwaii GWS, KU687711 ND ND KU687914 Haanas, British Columbia, CAN KD GWS028509 ABMMC15452-11 Lost Islands, SE wall, Gwaii GWS, KU687718 ND ND ND Haanas, British Columbia, CAN KD GWS028619 ABMMC16136-11 Ramsey Island (N Bay w anchorage), GWS, KU687646 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS028620 ABMMC16137-11 Point S of Dodge Point, Gwaii GWS, KU687556 ND ND ND Haanas, British Columbia, CAN KD GWS030590 ABMMC17596-12 Point S of Dodge Point, Gwaii GWS, KU687537 ND ND ND Haanas, British Columbia, CAN KD GWS030715 ABMMC17597-12 Louscoone Point, Gwaii Haanas, GWS, KU687547 ND ND ND British Columbia, CAN KD GWS030720 ABMMC16994-12 East corner of entrance to Slim Inlet, GWS, KU687775 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS030795 ABMMC17598-12 East corner of entrance to Slim Inlet, GWS, KU687544 ND ND ND Gwaii Haanas, British Columbia, KD CAN GWS030798 ABMMC17599-12 Macrocystis bed at entrance to GWS, KU687545 ND ND ND George Bay, Gwaii Haanas, British KD Columbia, CAN GWS034814 ABMMC18035-13 Macrocystis bed at entrance to GWS, KU687642 ND ND ND George Bay, Gwaii Haanas, British KD Columbia, CAN GWS034833 ABMMC18036-13 South Wall Bowyer Isl., British GWS, KU687518 ND ND ND Columbia, CAN KD GWS034885 ABMMC17997-13 Halkett Wall, Gambier Isl., British GWS, KU687541 ND ND ND Columbia, CAN KD GWS035389 ABMMC18315-13 Bird Islet, Horseshoe Bay, British GWS, KU687558 ND ND ND Columbia, CAN KD

296

Bold Accession Number Collection Site1 Coll.2 GenBank Accession Numbers3 COI-5P rbcL full rbcL-3P ITS GWS035391 ABMMC18317-13 Marble Island (NW reef below GWS, KU687670 ND ND ND `Killer-Whale-Fin Rock`), Haida KD Gwaii, British Columbia, CAN GWS035773 ABMMC18505-13 Marble Island (NW reef below GWS, KU687678 ND ND ND `Killer-Whale-Fin Rock`), Haida KD Gwaii, British Columbia, CAN GWS036718 ABMMC19651-14 SGang Gwaay (rocks to NW), Gwaii GWS, KU687740 ND ND ND Haanas, British Columbia, CAN KD GWS036752 ABMMC19684-14 Rosario Beach, Washington, USA GWS, KU687557 ND ND ND MB, GF GWS038909 ABMMC20033-14 Tartu Point (E corner), Rennell GWS, KU687499 ND ND ND Sound, Haida Gwaii, British MB Columbia, CAN GWS038969 ABMMC20093-14 Cape Knox, Haida Gwaii, British GWS, KU687641 ND ND ND Columbia, CAN MB 1 Country abbreviations: CAN= Canada; USA= United States of America.

2 Collectors’ initials: AC= A. Celona, AL= A. Lamb, AM= A. Millar, AS= Amanda Savoie, BC= Bridgette Clarkston, BG= B. Gavio, CG= C. Gurgel CL= Chris Lane, CRP= C. Rodriguez-Prieto, CWS= C.W. Schneider, DH= D. Hardin, DM= Dan McDevitt, EC= E. Coppejans, GF= Gina Filloramo, GTK= Gerry T. Kraft, GWS= G.W. Saunders, HGC= H-G Choi, HK= Hana Kucera, JAW= J.A. West, JB= J. Brown, JD= Jen Dalen, JG= J. Guillaudeau, JH= John Hughey, JLB= J. Lopez-Bautista, JM= J. Mortimer, JPD= J.P. Danko, KD= Kyatt Dixon, KH= Katy Hind, KK= K. Kogame, KR= Kat Roy, LF= L. Frotte, MB= Meghann Bruce, MC= M. Cowan, MD= Mike Deal, MDG= M.D. Guiry, MHH= M.H. Hommersand, MJW= M.J. Wynne, ML= M. Lloyd, MS= M. Spani, MV= M. Volovsek, NS= N. Sanchez, PR= P. Richards, PWG= P.W. Gabrielson, RC= R. Cowan, RU= R. Urquarte, RW= Rodney Withall, SC= Susan Clayden, SF= Suzanne Fredericq, SL= Sandra Lindstrom, SM= Sarah Hamsher, ST= S. Toews, TB= Trevor Bringloe, TF= T. Foley, TP= Thea Popolizio.

3 ND= data not generated.

297

Appendix C

Table S1. Collection details for samples included in molecular analyses. GenBank accessions in bold were determined for this

study.

BOLD PROCESS Species VOUCHER SITE COI-5P LSU rbcL ID1 Gloiocladia fryeana ABMMC10769-10 GWS001381 Sidney, British KR085174 EU624157 ND (Setchell) Sánchez & Columbia, Canada Rodríguez-Prieto ABMMC10774-10 GWS001421 Bamfield, Wizard ND ND KR085187 Island, British Columbia, Canada Gloiocladia ABMMC4891-09 GWS000469 Vivonne Bay, HQ919528 DQ873283 KR085192 halymenioides (Harvey) Kangaroo Island, R.E. Norris South Australia, Australia Gloiocladia laciniata ABMMC16297-11 B048GWS Bamfield, British KR0851702 EU624158 ND (J. Agardh) Sánchez & Columbia, Canada Rodríguez-Prieto ABMMC13622-10 GWS020495 Hot Spring Island, KR085172 ND KR085189 Gwaii Haanas, British Columbia, Canada Gloiocladia repens (C. ABMMC10603-10 G0332 Lachea Isle, Catania, ND AF419143 ND Agardh) Sánchez & Ionian Sea, Sicily, Rodríguez-Prieto Italy

298

BOLD PROCESS Species VOUCHER SITE COI-5P LSU rbcL ID1 ABMMC10788-10 GWS001639 Pace, Messina, Straits KR140326 ND KR140335 of Messina, Mediterranean Sea, Sicily, Italy Gloioderma australe J. ABMMC10599-10 G0306 Seabird, Western JX969722 DQ873282 JX969786 Agardh Australia, Australia Leptofauchea chiloensis ABMMC10662-10 GWS000503 Ancud Bay, Chiloé ND DQ873285 KR140338 Dalen & G.W. Island, Chile Saunders Leptofauchea cocosana ABMMC18915-13 GWS037755 Winter Wall, south of KR140330 KR140334 KR140340 Filloramo & G.W. Horsburgh Island, Saunders Cocos (Keeling) Islands, Australia Leptofauchea N/A OCD1501 Musclera Trencada, ND EU418774 ND coralligena Rodríguez- Llafranch, Spain Prieto & De Clerck Leptofauchea earleae N/A LAF-26-5-00- Offshore Louisiana, ND ND HQ400570 Gavio & Fredericq 1-1 Gulf of Mexico, USA Leptofauchea ABMMC11825-10 GWS018362 Gimnyeong, Jeju, KR1403292 KR140333 ND leptophylla (Segawa) South Korea Mas.Suzuki, Nozaki, R. Terada, Kitayama, Tetsu.Hashimoto & Yoshizaki ABMMC11826-10 GWS018363 Gimnyeong, Jeju, KR140328 ND ND South Korea

299

BOLD PROCESS Species VOUCHER SITE COI-5P LSU rbcL ID1 ABMMC11891-10 GWS018540 Channel between KR140327 ND KR140336 Little & Big Munseom Islands, Jeju, South Korea N/A TNS-AL- Yojiro, Kagoshima, ND AB677823 AB6931203 174121 Kagoshima Prefecture, Japan Leptofauchea ABMMC11883-10 GWS018532 Channel between HQ5440942 KR140331 KR140337 munseomica Filloramo Little & Big & G.W. Saunders Munseom Islands, Jeju, South Korea ABMMC11884-10 GWS018533 Channel between HQ544095 ND ND Little & Big Munseom Islands, Jeju, South Korea ABMMC11888-10 GWS018537 Channel between HQ544099 ND ND Little & Big Munseom Islands, Jeju, South Korea ABMMC11943-10 GWS018674 Channel between HQ544137 ND ND Little & Big Munseom Islands, Jeju, South Korea Leptofauchea OZSEA3885-13 GWS032631 Split Solitary Island KR085173 KR085179 KR085190 nitophylloides (E), Coffs Harbour, (J.Agardh) Kylin New South Wales, Australia

300

BOLD PROCESS Species VOUCHER SITE COI-5P LSU rbcL ID1 Leptofauchea pacifica ABMMC052-05 JD032 Bamfield, Seapool AY970566 DQ873286 ND E.Y. Dawson Rock, British Columbia, Canada ABMMC5339-09 GWS010140 Tahsis, Flower Islet, HM9161762 ND KR085195 Esperanza Channel, British Columbia, Canada Leptofauchea N/A TNS-AL- Yamada, Shimohei ND AB677824 AB383124 rhodymenioides W.R. 161637 County, Iwate Taylor4 Prefecture, Japan Leptofauchea sp._1WA ABMMC4807-09 G0400 NE of White Island, HM915831 DQ873287 KR085194 Easter Group, Abrolhos Island, Western Australia, Australia Webervanbossea ABMMC8154-10 GWS016349 Sanctuary Reef, HM918169 KR085175 KR085185 sp._1splachnoides Wynyard, Tasmania, Australia Webervanbossea ABMMC7495-10 GWS015310 Burying Ground HM917690 KR085182 KR085193 sp._1TAS Point, Tasmania, Australia Webervanbossea ABMMC5128-09 GWS002435 Flinders Jetty, HM916011 DQ873288 KR085186 splachnoides (Harvey) Victoria, Australia De Toni Webervanbossea ABMMC4906-09 GWS000922 Queenscliff Jetty, HM9158872 ND KR085196 tasmanensis Port Phillip Heads, Womersley Victoria, Australia

301

BOLD PROCESS Species VOUCHER SITE COI-5P LSU rbcL ID1 OZSEA2767-11 GWS029664 Cape Dombey Jetty, KR085171 KR085178 ND Robe, South Australia, Australia 1 Process id for the Barcode of Life Data System (BOLD).

2 Where more than one voucher was used for molecular data, the COI-5P sequence used in multigene is noted.

3 Chimeric sequence deposited in GenBank for L. leptophylla. Only the last ~730 bp were representative of L. leptophylla and used in

our analyses.

4 This collection is molecularly similar to and morphologically representative of L. leptophylla as described herein.

N/A Data acquired from GenBank without corresponding sample records in (BOLD).

ND Data not determined for this study.

302

Appendix D

Table S1. Collection data for specimens used in this study. All samples were collected from Australia unless otherwise

indicated.

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g Rhodymenia GWS032780 OZSEA3798-13 Mutton Bird G.W. KT781926 ND compressa Filloramo Island (South), Saunders & & G.W. Saunders Coffs Harbour, K. Dixon NSW GWS032782 OZSEA3800-13 Mutton Bird G.W. KT781957 ND Island (South), Saunders & Coffs Harbour, K. Dixon NSW Rhodymenia GWS001959 ABMMC754-06 Reef west of G.W. HM033088 ND contortuplicata Verona Sands Saunders & Filloramo & G.W. boat launch, in R. Withall Saunders mouth of Huon River, TAS GWS015201 ABMMC7404-10 Burying G.W. HM917629 ND Ground Point, Saunders & TAS K. Dixon GWS016020 ABMMC7919-10 Lucas Point G.W. HM917989 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS

303

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS016050 ABMMC7947-10 Lucas Point G.W. HM918010 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016060 ABMMC7954-10 Lucas Point G.W. KT782004 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016106 ABMMC7995-10 Lucas Point G.W. HM918044 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016121 ABMMC8005-10 Lucas Point G.W. HM918053 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS Rhodymenia corallina GWS038159 ABMMC20722- Chile M.R. Bruce KU565721 ND (Bory) Greville 14

Rhodymenia GWS030103 ABMMC16734- Massachusetts, G.W. KU565722 ND delicatula 12 United States Saunders P.J.L. Dangeard Rhodymenia gladiata GWS002467 ABMMC751-06 Breakwater at G.W. HM033151 ND Filloramo & G.W. harbour, Saunders Saunders Portland, VIC

304

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS002477 ABMMC136-06 Williamstown, G.W. KT781958 ND Port Phillip Saunders Bay, VIC GWS015611 ABMMC13015- Barrel Rock, F. Scott KT781999 KT782014 10 TAS GWS015830 ABMMC7777-10 Tinderbox G.W. HM917892 KT782011 (Fiona’s Saunders & Point), TAS K. Dixon GWS016372 ABMMC8172-10 Sanctuary G.W. HM918176 KT782009 Reef, Saunders & Wynyard, TAS K. Dixon GWS016541 ABMMC8301-10 Stanley G.W. HM918241 ND Breakwater, Saunders, TAS L. Kraft & K. Dixon GWS029450 OZSEA2560-11 Breakwater at G.W. KT781973 ND harbour, Saunders & Portland, VIC K. Dixon GWS029465 OZSEA2574-11 Breakwater at G.W. KT782003 KT782015 harbour, Saunders & Portland, VIC K. Dixon GWS029466 OZSEA2575-11 Breakwater at G.W. KT781934 KT782005 harbour, Saunders & Portland, VIC K. Dixon

305

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS029510 OZSEA2619-11 Nuns Beach G.W. KT781955 KT782008 under Saunders & Lighthouse, K. Dixon Portland, VIC GWS029511 OZSEA2620-11 Nuns Beach G.W. KT781986 KT782012 under Saunders & Lighthouse, K. Dixon Portland, VIC GWS032615 OZSEA3884-13 Split Solitary G.W. KT781956 KT782010 Island Saunders & (Northwest), K. Dixon Coffs Harbour, NSW GWS032639 OZSEA3795-13 Split Solitary G.W. KT781988 KT782013 Island (East), Saunders & Coffs Harbour, K. Dixon NSW GWS032660 OZSEA3796-13 South Solitary G.W. KT781946 KT782006 Island (SE), Saunders & Coffs Harbour, K. Dixon NSW GWS032783 OZSEA3886-13 Mutton Bird G.W. KT781968 ND Island (South), Saunders & Coffs Harbour, K. Dixon NSW

306

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS032784 OZSEA3887-13 Mutton Bird G.W. KT781953 KT782007 Island (South), Saunders & Coffs Harbour, K. Dixon NSW GWS032785 OZSEA3888-13 Mutton Bird G.W. KT781985 ND Island (South), Saunders & Coffs Harbour, K. Dixon NSW Rhodymenia insularis GWS001039 ABMMC752-06 Roach I., G.W. HM033148 ND Filloramo & G.W. Admiralty Saunders Saunders Group, Lord Howe I., NSW GWS002043 ABMMC753-06 Islands off G.W. HM033147 ND Balls Pyramid, Saunders Lord Howe I., NSW GWS023535 OZSEA2084-10 Algae Hole G.W. KT781961 ND North, Lord Saunders, Howe, NSW K. Dixon & R. Withall GWS023557 OZSEA2085-10 Algae Hole G.W. KT781942 ND North, Lord Saunders, Howe, NSW K. Dixon & R. Withall

307

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS023563 OZSEA2086-10 Algae Hole G.W. KT781993 ND North, Lord Saunders, Howe, NSW K. Dixon & R. Withall GWS023681 OZSEA2088-10 Split Rock, G.W. KT781970 ND Lord Howe, Saunders & NSW K. Dixon GWS023711 OZSEA2098-10 Split Rock, G.W. KT781951 ND Lord Howe, Saunders & NSW K. Dixon GWS023750 OZSEA2089-10 Split Rock, G.W. KT781971 ND Lord Howe, Saunders & NSW K. Dixon GWS023751 OZSEA2090-10 Split Rock, G.W. KT781983 ND Lord Howe, Saunders & NSW K. Dixon Rhodymenia intricata GWS018541 ABMMC11892- Jeju, South G.W. HQ544101 ND (Okamura) Okamura 10 Korea Saunders & H-G. Choi

Rhodymenia GWS002130 ABMMC755-06 Pahi, near G.W. HM033149 ND leptophylla J. Agardh Auckland, Saunders New Zealand

308

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g Rhodymenia GWS025150 OZSEA2091-10 Canal Rocks, G.W. KT781923 ND lociperonica WA Saunders & Filloramo & G.W. K. Dixon Saunders GWS025411 OZSEA2093-10 Pt. Peron, WA G.W. KT781960 ND Saunders & K. Dixon GWS025412 OZSEA2077-10 Pt. Peron, WA G.W. KT781938 ND Saunders & K. Dixon GWS025430 OZSEA2094-10 Pt. Peron, WA G.W. KT781980 ND Saunders & K. Dixon Rhodymenia GWS032284 OZSEA3879-13 Simon`s Water G.W. KT781935 ND norfolkensis Flats, NI Saunders & Filloramo & G.W. K. Dixon Saunders GWS032285 OZSEA3880-13 Simon`s Water G.W. KT781921 ND Flats, NI Saunders & K. Dixon GWS032489 OZSEA3883-13 Little Organ, G.W. KT782000 ND NI Saunders & K. Dixon GWS034321 OZSEA3889-13 Little Organ, G.W. KT781982 ND NI Saunders & K. Dixon

309

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS034326 OZSEA3890-13 Little Organ, G.W. KT781979 ND NI Saunders & K. Dixon GWS034329 OZSEA3891-13 Little Organ, G.W. KT781952 ND NI Saunders & K. Dixon GWS034333 OZSEA3892-13 Little Organ, G.W. KT781950 ND NI Saunders & K. Dixon GWS034352 OZSEA3893-13 Little Organ, G.W. KT781929 ND NI Saunders & K. Dixon GWS034372 OZSEA3894-13 Duncombe G.W. KT781974 ND Bay, NI Saunders & K. Dixon GWS034515 OZSEA3897-13 Fig Valley, NI G.W. KT781995 ND Saunders & K. Dixon Rhodymenia GWS000925 ABMMC2856-08 Queenscliff G.W. HM033135 ND novahollandica G.W. Jetty, Port Saunders Saunders Phillip Heads, VIC GWS000929 ABMMC4910-09 Queenscliff G.W. HM915888 ND Jetty, Port Saunders Phillip Heads, VIC

310

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS000930 ABMMC710-06 Queenscliff G.W. HM033130 ND Jetty, Port Saunders Phillip Heads, VIC GWS000933 ABMMC4911-09 Queenscliff G.W. KT781984 ND Jetty, Port Saunders Phillip Heads, VIC GWS000950 ABMMC711-06 Queenscliff G.W. HM033128 ND Jetty, Port Saunders Phillip Heads, VIC GWS001278 ABMMC712-06 Northwest tip J. Huisman HM033126 ND of Carnac Island, WA GWS001507 ABMMC2042-08 Arch Rock, G.W. HM033112 JN021154, south of Saunders JN021173d Hobart, TAS GWS001567 ABMMC2044-08 Queenscliff G.T. Kraft HM033109 ND Jetty, Port Phillip Heads, VIC

311

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS001575 ABMMC713-06 Glaneuse Reef G.W. HM033124 ND near Point Saunders Lonsdale Lighthouse Reef, VIC GWS001576 ABMMC2046-08 Glaneuse Reef G.W. HM033106 ND near Point Saunders Lonsdale Lighthouse Reef, VIC GWS001577 ABMMC714-06 Glaneuse Reef G.W. HM033122 ND near Point Saunders Lonsdale Lighthouse Reef, VIC GWS001579 ABMMC2047-08 Glaneuse Reef G.W. HM033104 ND near Point Saunders Lonsdale Lighthouse Reef, VIC GWS001600 ABMMC2048-08 Queenscliff G.W. HM033102 ND Jetty, Port Saunders Phillip Heads, VIC

312

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS001601 ABMMC715-06 Queenscliff G.W. HM033120 ND Jetty, Port Saunders Phillip Heads, VIC GWS001602 ABMMC2049-08 Queenscliff G.W. HM033100 ND Jetty, Port Saunders Phillip Heads, VIC GWS001603 ABMMC2050-08 Queenscliff G.W. HM033098 ND Jetty, Port Saunders Phillip Heads, VIC GWS001604 ABMMC716-06 Queenscliff G.W. HM033117 ND Jetty, Port Saunders Phillip Heads, VIC GWS001605 ABMMC2051-08 Queenscliff G.W. HM033096 ND Jetty, Port Saunders Phillip Heads, VIC GWS001606 ABMMC717-06 Queenscliff G.W. HM033116 ND Jetty, Port Saunders Phillip Heads, VIC

313

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS001607 ABMMC718-06 Queenscliff G.W. HM033115 ND Jetty, Port Saunders Phillip Heads, VIC GWS001608 ABMMC719-06 Queenscliff G.W. HM033114 ND Jetty, Port Saunders Phillip Heads, VIC GWS001905 ABMMC1275-07 Nine Pin Point, G.W. HM033094 JN021153, TAS Saunders & JN021172d R. Withall GWS002380 ABMMC720-06 Beach access G.W. HM033113 ND west of Bay of Saunders Islands, Great Ocean Road, VIC GWS002418 ABMMC721-06 Glaneuse Reef G.W. HM033111 ND near Point Saunders Lonsdale Lighthouse Reef, VIC GWS002419 ABMMC722-06 Glaneuse Reef G.W. HM033110 ND near Point Saunders Lonsdale Lighthouse Reef, VIC

314

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS002420 ABMMC723-06 Glaneuse Reef G.W. HM033108 ND near Point Saunders Lonsdale Lighthouse Reef, VIC GWS002421 ABMMC724-06 Glaneuse Reef G.W. HM033107 ND near Point Saunders Lonsdale Lighthouse Reef, VIC GWS002439 ABMMC725-06 Queenscliff G.W. HM033105 ND Jetty, Port Saunders Phillip Heads, VIC GWS002440 ABMMC726-06 Queenscliff G.W. HM033103 ND Jetty, Port Saunders Phillip Heads, VIC GWS002441 ABMMC727-06 Queenscliff G.W. HM033101 ND Jetty, Port Saunders Phillip Heads, VIC GWS002442 ABMMC728-06 Queenscliff G.W. HM033099 ND Jetty, Port Saunders Phillip Heads, VIC

315

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS002444 ABMMC729-06 Queenscliff G.W. HM033097 ND Jetty, Port Saunders Phillip Heads, VIC GWS002445 ABMMC730-06 Queenscliff G.W. HM033095 ND Jetty, Port Saunders Phillip Heads, VIC GWS002446 ABMMC731-06 Queenscliff G.W. HM033093 ND Jetty, Port Saunders Phillip Heads, VIC GWS002447 ABMMC732-06 Queenscliff G.W. HM033092 ND Jetty, Port Saunders Phillip Heads, VIC GWS002448 ABMMC733-06 Queenscliff G.W. HM033091 ND Jetty, Port Saunders Phillip Heads, VIC GWS002449 ABMMC734-06 Queenscliff G.W. HM033090 ND Jetty, Port Saunders Phillip Heads, VIC

316

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS002450 ABMMC735-06 Queenscliff G.W. HM033089 ND Jetty, Port Saunders Phillip Heads, VIC GWS002469 ABMMC736-06 Breakwater at G.W. HM033134 ND harbour, Saunders Portland, VIC GWS002470 ABMMC737-06 Breakwater at G.W. HM033133 ND harbour, Saunders Portland, VIC GWS002471 ABMMC2498-08 Breakwater at G.W. HM033118 ND harbour, Saunders Portland, VIC GWS002472 ABMMC738-06 Breakwater at G.W. HM033132 ND harbour, Saunders Portland, VIC GWS002473 ABMMC739-06 Breakwater at G.W. HM033131 ND harbour, Saunders Portland, VIC GWS002536 ABMMC740-06 Glaneuse Reef G.W. HM033129 ND near Point Saunders Lonsdale Lighthouse Reef, VIC

317

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS002540 ABMMC5144-09 Glaneuse Reef G.W. HM916021 ND near Point Saunders Lonsdale Lighthouse Reef, VIC GWS002563 ABMMC5150-09 Queenscliff G.W. HM916024 ND Jetty, Port Saunders Phillip Heads, VIC GWS002564 ABMMC5151-09 Queenscliff G.W. HM916025 ND Jetty, Port Saunders Phillip Heads, VIC GWS002566 ABMMC5153-09 Queenscliff G.W. HM916026 JN021152, Jetty, Port Saunders JN021171d Phillip Heads, VIC GWS002567 ABMMC741-06 Queenscliff G.W. HM033127 ND Jetty, Port Saunders Phillip Heads, VIC GWS002568 ABMMC742-06 Queenscliff G.W. HM033125 JN021170e Jetty, Port Saunders Phillip Heads, VIC

318

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS002569 ABMMC743-06 Queenscliff G.W. HM033123 ND Jetty, Port Saunders Phillip Heads, VIC GWS002570 ABMMC744-06 Queenscliff G.W. HM033121 ND Jetty, Port Saunders Phillip Heads, VIC GWS002571 ABMMC745-06 Queenscliff G.W. HM033119 ND Jetty, Port Saunders Phillip Heads, VIC GWS014803 ABMMC7080-10 Point Lonsdale G.W. HM917410 ND Lighthouse Saunders & Reef, VIC G.T. Kraft GWS015288 ABMMC7479-10 Burying G.W. HM917680 ND Ground Point, Saunders & TAS K. Dixon GWS015389 ABMMC7566-10 Reef west of G.W. HM917742 ND Verona Sands Saunders & boat launch, in K. Dixon mouth of Huon River, TAS

319

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS016044 ABMMC7941-10 Lucas Point G.W. HM918004 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016045 ABMMC7942-10 Lucas Point G.W. HM918005 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016047 ABMMC7944-10 Lucas Point G.W. HM918007 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016070 ABMMC7962-10 Lucas Point G.W. HM918019 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016094 ABMMC7983-10 Lucas Point G.W. HM918039 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016116 ABMMC8002-10 Lucas Point G.W. HM918051 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS

320

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS016502 ABMMC8275-10 Stanley G.W. HM918229 ND Breakwater, Saunders, TAS L. Kraft & K. Dixon GWS016568 ABMMC8322-10 Stanley G.W. HM918252 ND Breakwater, Saunders, TAS L. Kraft & K. Dixon GWS016607 ABMMC8348-10 The Springs, G.W. KT781941 ND Point Saunders, Lonsdale, TAS L. Kraft & K. Dixon GWS016608 ABMMC8349-10 The Springs, G.W. HM918269 ND Point Saunders, Lonsdale, TAS L. Kraft & K. Dixon GWS024630 OZSEA2072-10 Windy G.W. KT781933 ND Harbour, WA Saunders & K. Dixon GWS024649 OZSEA2073-10 Windy G.W. KT781947 ND Harbour, WA Saunders & K. Dixon GWS025295 OZSEA2092-10 Pt. Peron, WA G.W. KT781954 ND Saunders & K. Dixon

321

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS025315 OZSEA1634-10 Canal Rocks, G.W. KT782001 ND WA Saunders & K. Dixon GWS025392 OZSEA2075-10 Pt. Peron, WA G.W. KT781937 ND Saunders & K. Dixon GWS025393 OZSEA2076-10 Pt. Peron, WA G.W. KT781972 ND Saunders & K. Dixon GWS025427 OZSEA2078-10 Pt. Peron, WA G.W. KT781998 ND Saunders & K. Dixon GWS025586 OZSEA2079-10 Ricey Beach, G.W. KT781928 ND Rottnest I., Saunders & WA K. Dixon GWS025629 OZSEA2095-10 Ricey Beach, G.W. KT781975 ND Rottnest I., Saunders & WA K. Dixon GWS028880 OZSEA2197-11 Gravel Bay, K. Dixon KT781978 ND Yorke Peninsula, SA GWS029126 OZSEA2911-11 Brighton Pier, K. Dixon KT781930 ND Holdfast Bay, SA

322

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS029127 OZSEA2912-11 Brighton Pier, K. Dixon KT781969 ND Holdfast Bay, SA GWS029300 OZSEA2419-11 Glaneuse Reef G.W. KT781949 ND near Point Saunders Lonsdale Lighthouse Reef, VIC GWS029342 OZSEA2460-11 Warrnambool G.W. KT781963 ND Reef, VIC Saunders & K. Dixon GWS029451 OZSEA2561-11 Breakwater at G.W. KT781939 ND harbour, Saunders & Portland, VIC K. Dixon GWS029454 OZSEA2564-11 Breakwater at G.W. KT781976 ND harbour, Saunders & Portland, VIC K. Dixon GWS029459 OZSEA2569-11 Breakwater at G.W. KT781945 ND harbour, Saunders & Portland, VIC K. Dixon GWS029467 OZSEA2576-11 Breakwater at G.W. KT781994 ND harbour, Saunders & Portland, VIC K. Dixon

323

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS029532 OZSEA2641-11 Nuns Beach G.W. KT781931 ND under Saunders & Lighthouse, K. Dixon Portland, VIC GWS029564 OZSEA2671-11 Port G.W. KT781989 ND MacDonnell, Saunders & SA K. Dixon GWS029565 OZSEA2672-11 Port G.W. KT781990 ND MacDonnell, Saunders & SA K. Dixon GWS029678 OZSEA2780-11 Cape Dombey G.W. KT781948 ND Jetty, Robe, Saunders & SA K. Dixon GWS029692 OZSEA2794-11 Post Office G.W. KT781962 ND Rock (Salmon Saunders & Hole side), K. Dixon Beachport, SA GWS029882 OZSEA2881-11 Rapid Bay, SA G.W. KT781987 ND Saunders & K. Dixon GWS029884 OZSEA2883-11 Rapid Bay, SA G.W. KT781997 ND Saunders & K. Dixon GWS034905 OZSEA4229-13 Speeds Point, K. Dixon KT781991 ND Eyre & R. Dixon Peninsula, SA

324

Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g Rhodymenia GWS022832 OZSEA2080-10 Malabar Reef, G.W. KT781924 ND novazelandica E.Y. Lord Howe I., Saunders, Dawson NSW K. Dixon & R. Withall GWS022833 OZSEA2081-10 Malabar Reef, G.W. KT781925 ND Lord Howe I., Saunders, NSW K. Dixon & R. Withall GWS022858 OZSEA2082-10 Malabar Reef, G.W. KT781967 ND Lord Howe I., Saunders, NSW K. Dixon & R. Withall GWS022889 OZSEA2083-10 Malabar Reef, G.W. KT781992 ND Lord Howe I., Saunders, NSW K. Dixon & R. Withall Rhodymenia GWS002593 ABMMC3703-09 Blowhole, near G.W. HM033139 ND prolificans Zanardini Eaglehawk Saunders Nest, TAS GWS002594 ABMMC3704-09 Blowhole, near G.W. HM033138 ND Eaglehawk Saunders Nest, TAS

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Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS002595 ABMMC5307-09 Blowhole, near G.W. HM916149 NAg Eaglehawk Saunders Nest, TAS GWS002628 ABMMC8946-10 Bicheno, G.W. HM918489 NA Rocky front Saunders past Govenor`s Reserve, TAS GWS002629 ABMMC310-06 Bicheno, G.W. HM033144 NA Rocky front Saunders past, TAS GWS002630 ABMMC311-06 Bicheno, G.W. HM033143 ND Rocky front Saunders past, TAS GWS002631 ABMMC312-06 Bicheno, G.W. HM033142 ND Rocky front Saunders past, TAS GWS002633 ABMMC313-06 Bicheno, G.W. HM033141 NA Rocky front Saunders past, TAS GWS002634 ABMMC8948-10 Bicheno, G.W. HM918490 NA Rocky front Saunders past Govenor`s Reserve, TAS GWS002635 ABMMC314-06 Bicheno, L. LeGall HM033140 ND Rocky front past, TAS

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Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS002642 ABMMC8950-10 Bicheno, G.W. KT781922 NA Rocks to left of Saunders beach, TAS Rhodymenia GWS001555 ABMMC767-06 Warrnambool, G.W. HM033152 ND stenoglossa J. Agardh VIC Saunders Rhodymenia wilsonis GWS001496 ABMMC2040-08 Devils Hole, G.W. HM033162 JN021158, (Sonder) G.W. south of Saunders JN021184d Saunders Eaglehawk Nest, TAS GWS001517 ABMMC756-06 Arch Rock, C. HM033160 ND south of Sanderson Hobart, TAS GWS001556 ABMMC2043-08 Warrnambool, G.W. HM033161 JN021157, VIC Saunders JN021183d GWS001922 ABMMC757-06 Tinderbox G.W. HM033159 JN021182e Reserve, South Saunders & of Hobart, R. Withall TAS GWS001969 ABMMC758-06 Bicheno, in G. W. HM033158 JN021156, Governor Saunders & JN021181d Island Reserve, R. Withall TAS GWS002528 ABMMC761-06 Warrnambool, G.W. HM033155 JN021180 e VIC Saunders GWS002529 ABMMC762-06 Warrnambool, G.W. HM033154 JN021179 e VIC Saunders

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Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS002530 ABMMC763-06 Warrnambool, G.W. HM033153 JN021178 e VIC Saunders GWS002640 ABMMC759-06 Bicheno, G.W. HM033157 JN021155, Rocks to left of Saunders JN021177d beach, TAS GWS002641 ABMMC760-06 Bicheno, G.W. HM033156 JN021176 e Rocks to left of Saunders beach, TAS GWS015187 ABMMC7393-10 Burying G.W. KT782002 ND Ground Point, Saunders & TAS K. Dixon GWS015202 ABMMC7405-10 Burying G.W. HM917630 ND Ground Point, Saunders & TAS K. Dixon GWS015309 ABMMC7494-10 Burying G.W. KT781927 ND Ground Point, Saunders & TAS K. Dixon GWS015314 ABMMC7499-10 Burying G.W. HM917693 ND Ground Point, Saunders & TAS K. Dixon GWS015548 ABMMC7711-10 Nine Pin Point, G.T. Kraft KT781996 ND TAS & L. Kraft GWS015936 ABMMC7867-10 Tinderbox G.T. Kraft HM917954 ND (Fiona’s & L. Kraft Point), TAS

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Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS016018 ABMMC7918-10 Lucas Point G.W. KT781944 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016078 ABMMC7970-10 Lucas Point G.W. HM918027 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016087 ABMMC7977-10 Lucas Point G.W. HM918034 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016115 ABMMC8001-10 Lucas Point G.W. HM918050 ND (past Hells Saunders & Gates) Strahan, K. Dixon TAS GWS016352 ABMMC8156-10 Sanctuary G.W. KT781940 ND Reef, Saunders & Wynyard, TAS K. Dixon GWS029679 OZSEA2781-11 Cape Dombey G.W. KT781959 ND Jetty, Robe, Saunders & SA K. Dixon Rhodymenia DV006 ABMMC2039-08 Smooth Pools, G.T. Kraft HM033150 ND womersleyi Filloramo Eyre & K. & G.W. Saunders Peninsula, SA Dixon

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Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS032474 OZSEA3881-13 Bronte Beach, G.W. KT781932 NA Sydney, NSW Saunders & K. Dixon GWS032475 OZSEA3882-13 Bronte Beach, G.W. KT781965 ND Sydney, NSW Saunders & K. Dixon GWS032646 OZSEA3862-13 Sawtell G. KT781977 NA (Bonville Head Filloramo Lookout), & L. Kraft NSW GWS032741 OZSEA3864-13 Mutton Bird G.W. KT781943 NA Island (North), Saunders & Coffs Harbour, K. Dixon NSW GWS032742 OZSEA3865-13 Mutton Bird G.W. KT781964 ND Island (North), Saunders & Coffs Harbour, K. Dixon NSW GWS032769 OZSEA3866-13 Mutton Bird G.W. KT781966 ND Island (North), Saunders & Coffs Harbour, K. Dixon NSW GWS032794 OZSEA3763-13 Mutton Bird G.W. KT781981 ND Island (South), Saunders & Coffs Harbour, K. Dixon NSW

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Species Voucher BOLD Collection Collector COI-5P ITS GenBank Number Accessiona Siteb GenBank Accession Accessionc c,d,e,f,g GWS032795 OZSEA3764-13 Mutton Bird G.W. KT781936 NA Island (South), Saunders & Coffs Harbour, K. Dixon NSW

a “BOLD Accession” refers to the sample accession for the Barcode of Life Data Systems (BOLD). b Abbreviations for Australian states and territories: NI = Norfolk Island; NSW = New South Wales; SA = South Australia; TAS = Tasmania; VIC = Victoria; and WA = Western Australia. c GenBank numbers in bold were generated for this study. d Where two GenBank accessions are provided, the first refers to the ITS1 and the second, the ITS2.

e Denotes samples for which only the ITS2 was generated. f “ND”, data not determined for this study. g “NA”, denotes samples for which ITS data were generated; however, that data were too messy to deposit in BOLD or GenBank.

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Appendix E Multigene analyses resolve early diverging lineages in the

Rhodymeniophycidae (Florideophyceae, Rhodophyta)

Citation for published manuscript: Saunders, G.W., Filloramo, G., Dixon, K., Le Gall,

L., Maggs, C.A., & Kraft. G.T. (2016). Multigene analyses resolve early diverging lineages in the Rhodymeniophycidae (Florideophyceae, Rhodophyta). Journal of

Phycology, DOI: 10.1111/jpy.12426.

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Abstract

Multigene phylogenetic analyses were directed at resolving the earliest divergences in the red algal subclass Rhodymeniophycidae. The inclusion of key taxa (new to science and/or previously lacking molecular data), additional sequence data (SSU, LSU, EF2, rbcL, COI-5P), and phylogenetic analyses removing the most variable sites (site stripping) have provided resolution for the first time at these deep nodes. The earliest diverging lineage within the subclass was the enigmatic Catenellopsis oligarthra from

New Zealand (Catenellopsidaceae), which is here placed in the Catenellopsidales ord. nov. In our analyses Atractophora hypnoides was not allied with the other included

Bonnemaisoniales, but resolved as sister to the Peyssonneliales, and is here assigned to

Atractophoraceae fam. nov. in the Atractophorales ord. nov. Inclusion of Acrothesaurum gemellifilum gen. et sp. nov. from Tasmania has greatly improved our understanding of the Acrosymphytales, to which we assign three families, the Acrosymphytaceae,

Acrothesauraceae fam. nov. and Schimmelmanniaceae fam. nov.

Keyword index words: Acrosymphytales, Acrothesauraceae, Acrothesaurum,

Atractophoraceae, Atractophorales, Catenellopsidales, Schimmelmanniaceae

Abbreviations: COI-5P, 5’ region of the mitochondrial cytochrome oxidase subunit 1 gene; EF2, nuclear elongation factor 2 gene; LSU, nuclear large subunit ribosomal DNA; rbcL, plastid ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene; SSU, nuclear small subunit ribosomal DNA

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Introduction

As with most lineages of living organisms, molecular data have come to play an essential role in reshaping our understanding of organismal relationships and providing new evolutionary perspectives for red algae (see Saunders and Hommersand 2004, Yoon et al.

2006, Verbruggen et al. 2010). Among the five or six classes comprising the phylum

Rhodophyta (Saunders and Hommersand 2004, Yoon et al. 2006), the Florideophyceae is by far the most species-rich, containing upwards of 95% of the currently reported species

(Guiry and Guiry 2015). The Florideophyceae consists of multicellular marine and freshwater species currently assigned to five subclasses on the basis of molecular and morphological analyses (Saunders and Hommersand 2004, Le Gall and Saunders 2007).

The subclass Rhodymeniophycidae contains some 75% of the species currently assigned to the Florideophyceae (Guiry and Guiry 2015) including many that are well known to non-specialists, e.g., Irish moss (Chondrus crispus Stackhouse) and dulse [Palmaria palmata (Linnaeus) F.Weber & D.Mohr].

The Rhodymeniophycidae was established by Saunders and Hommersand (2004) on the basis of molecular data available at that time (e.g., Saunders and Bailey 1997,

1999, Harper and Saunders 2001), as well as the key ultrastructural synapomorphy of pit plugs that are covered by a cap membrane at their cytoplasmic faces (Pueschel and Cole

1982). Saunders et al. (2004) completed a relatively comprehensive molecular phylogenetic assessment of this subclass and established that all orders except the

Gigartinales were largely monophyletic, however, relationships among most orders were unresolved. Saunders et al. (2004) included data for only the SSU, for which varied rates of change in divergent lineages likely confounded phylogenetic determinations (Le

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Gall and Saunders 2007). Subsequent research included additional taxa and used the

LSU and SSU in combination. As a result more orders were recognized (e.g., Withall and Saunders 2006), but the relationships among most of them remained equivocal.

Indeed, of the 11 orders recognized in Withall and Saunders (2006), only the positioning of the Halymeniales as sister to the Sebdeniales and Rhodymeniales was consistently resolved. Studies using the rbcL have similarly failed to resolve interordinal relationships (e.g., Gavio et al. 2005, Krayesky et al. 2009).

Attempts to resolve ordinal relationships among florideophycean subclasses then took two divergent approaches. Le Gall and Saunders (2007) attempted to improve resolution by adding taxa and generating sequence data for an additional nuclear marker,

EF2, whereas Verbruggen et al. (2010) used a data-mining approach to prepare a supermatrix for phylogenetic analyses. Although support for monophyly of some orders was improved and the subclass Corallinophycidae was recognized as distinct from the

Nemaliophycidae (Le Gall and Saunders 2007), relationships among orders of the

Rhodymeniophycidae remained poorly resolved. Verbruggen et al. (2010) identified ordinal relationships among Rhodymeniophycidae as one of five poorly supported regions in the red algal tree of life that were in need of further study. They noted that

“data availability for (this subclass) is meager to poor”, but provided compelling evidence that resolution would be possible with the addition of more data (Verbruggen et al. 2010, fig. 3). In the most recent effort, Yang et al. (2015) analyzed mitochondrial genomes for 21 Rhodymeniophycidae. Again few novel interordinal relationships were resolved with meaningful support except for an early divergence of the

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Bonnemaisoniales, Gigartinales and Peyssonneliales relative to the remaining orders

(94% support in maximum likelihood analyses, Yang et al. 2015, fig. 1).

To improve the resolution of ordinal relationships within the

Rhodymeniophycidae we have generated data from more taxa, including some not previously included in phylogenetic analyses, e.g., Catenellopsis oligarthra (J.Agardh)

V.J.Chapman, and more genes combining the five markers SSU, LSU, EF2, rbcL, and

COI-5P. We additionally completed analyses on alignments of progressively more conservative characters (site stripping) in an effort to reduce the effects of saturation and thus improve phylogenetic signal (Verbruggen 2012).

Materials and Methods

Molecular methods

Samples for molecular investigation (Table E.S1) were processed and DNA extracted following Saunders and McDevit (2012). Sequence data were generated for the SSU,

LSU, EF2, rbcL and CO1-5P following Saunders and Moore (2013). Sequences were aligned using the ClustalW plugin for Geneious R7 version 7.1.5

(http://www.geneious.com; Kearse et al. 2012) and included data from GenBank (Table

E.S1). We generated five individual gene alignments: SSU (68 taxa, 1702 of 1895 sites included in analyses; 93% complete); LSU (72 taxa, 2605 of 3434 sites included in analyses; 99% complete); EF2 (67 taxa, 1681 sites; 92% complete); rbcL (69 taxa, 1358 sites; 95% complete) and CO1-5P (65 taxa, 664 sites; 89% complete). In addition, a concatenated alignment for all taxa and regions (73 taxa, 8010 aligned sites) was generated. Most taxa were at least 75% complete except for Acrosymphyton

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purpuriferum (J.Agardh) G.Sjöstedt (54% complete, data only for SSU and LSU) and

Pihiella liagoraciphila Huisman, A.R.Sherwood & I.A.Abbott (21% complete, data only for SSU), which together accounted for ~26% of the missing data.

Single-gene alignments were analyzed with a GTR+I+G model, with partitioning by codon for the three protein-coding genes, in the web-server program RAxML

(Stamatakis 2014) and robustness determined with 500 bootstrap replicates. There were no strong inconsistencies noted among the single-gene trees and the five genes were combined for phylogenetic analyses. Bayesian analysis was performed on the full dataset using the MrBayes plugin for Geneious R7 version 7.1.5 under a GTR+I+G model with parameter settings unlinked and the rates prior set to allow rate differentiation across partitions (by gene and then by codon for protein-coding genes = full partitioning scheme). This analysis was run twice for 1,000,000 generations with sampling every

1000 generations. Plotting the overall likelihood against the number of generations identified the stationary phase to determine the burn-in for each run. Maximum likelihood analysis was performed under a GTR+I+G model with the data fully partitioned using the RAxML plugin for Geneious R7 version 7.1.5 with 1000 bootstrap replicates.

Owing to the paucity of data for Pihiella (SSU only) the combined Bayesian analyses were repeated after removing this taxon. There were no significant changes in topology and only minor variations in posterior probability support indicating that its inclusion was not negatively impacting our phylogenetic results.

To assess phylogenetic inference problems due to substitution saturation, quickly evolving sites were calculated and systematically removed from the full alignment.

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Site-specific rates were determined using the “substitution rates” analysis tool in HyPhy

(Pond et al. 2005) under the JC69 model with a Bayes phylogram as a guide tree. To generate a series of progressively more conservative alignments the program SiteStripper

(Verbruggen 2012) was used to order the sites by rate then remove the more rapidly evolving sites in increments of 5%. Maximum likelihood analysis (ML) was performed on each “stripped” alignment (fully partitioned) using RAxML version 7.3.5 with a command line script available through SiteStripper under a GTR+I+G model and 1000 bootstrap replicates.

To assess how partitions might impact phylogenetic inference, the original alignment and “stripped” alignments were analyzed by running PartitionFinder (Lanfear et al. 2012) using the “greedy” algorithm under the BIC model selection method with linked branch-length estimation. The best partitioning scheme and most appropriate model of evolution as determined by PartitionFinder were subsequently used to reanalyze the alignments with RAxML again with 1000 bootstrap replicates.

Anatomical methods

For anatomical observations, whole-mounts of gelatinous species were made from liquid- preserved thallus fragments, and cross-sections of more robust species were prepared from rehydrated or formalin-fixed samples by hand-sectioning or in a cryostat (CM1850,

Leica). Tissues were stained with 1% aniline blue and mounted in 40-50% corn syrup.

Observations were made with a light microscope and documented with digital photography.

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Morphological development of Atractophora hypnoides P.Crouan & H.Crouan was followed in cultured material fixed in formalin-seawater (4%) and stained with aniline blue acidified in 1% HCl. Cell nuclei were visualized by staining formalin-fixed material in a drop of Hoechst 33258 solution (10 µg mL-1) and examined with an epifluorescence microscope (Leitz Dialux), or by staining with aceto-iron-haematoxylin- chloral hydrate (Wittmann 1965) and photographed using Nomarski interference, as described by Maggs (1989). Photographs were taken using Technical Pan film developed in Kodak HC110 liquid developer.

Culture studies

Atractophora hypnoides was isolated from tetraspores released by Rhododiscus tetrasporophytes collected at Finavarra, Co. Clare, Ireland, and Cloghy Rocks, Strangford

Lough, N. Ireland (multiple cultures were isolated from 1982-1986, several of which were maintained long-term; see Maggs 1988). Cultures were grown in half-strength modified von Stosch medium (Guiry and Cunningham 1984), at 15°C, in a regime of

16:8 h light: dark (LD), under a photon irradiance of c. 20 µmol photons m-2 s-1, and subject to changes in photoperiod as described in the text.

Results

Molecular phylogenetic analyses

Topologies were congruent for all four analyses of the full combined alignment (Bayes and maximum likelihood under full and PartitionFinder partitioning) and the Bayesian result with partitioning by gene and codon is presented (Fig. E.1; with support

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values for all analyses summarized in Table E.1). Tree scores and branch support were typically slightly better for the fully partitioned analyses, i.e., not using partitions identified by PartitionFinder (Table E.1). To assess the effects of substitution saturation a series of progressively more conservative alignments were analyzed with maximum likelihood, again fully partitioned and with the schemes determined by PartitionFinder.

Consistent with the full combined alignment, tree scores and overall support were typically better for analyses in which the data were fully partitioned and only those values are presented (Table E.1).

A neighbor-joining tree constructed with the HKY model was generated in

Geneious R7 (Supplementary Fig. E.S1) to determine if the starting tree employed by

HyPhy to determine individual site rates had biased downstream analyses. These NJ- based site rates were used by SiteStripper to generate a series of subalignments, which were analyzed in RAxML. The results of a Shimodaira-Hasegawa test indicated that the neighbor-joining tree (likelihood -144264.31) was significantly different (p<0.01) from the Bayesian Inference tree (likelihood -144682.75) used in the initial site-stripping analyses; however, use of the neighbor-joining topology for calculating sites rates did not impact downstream site-stripping analyses (data not shown).

In general terms support was moderate to strong at many key ordinal and interordinal nodes, with some deeper nodes seeing enhanced support values at 10% site removal (Table E.1). Neither the Bonnemaisoniales nor Gigartinales was monophyletic

(Fig. E.1, Table E.1). Catenellopsis oligarthra, not previously included in phylogenetic analyses, resolved as an independent lineage sister to the remainder of the

Rhodymeniophycidae and was not associated with other taxa assigned to the

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Gigartinales (Fig. E.1). The next diverging lineage was the fully supported

Bonnemaisoniales sensu stricto (Fig. E.1), i.e., excluding Atractophora hypnoides, which was resolved as sister to the strongly supported Peyssonneliales in a lineage with the remaining Gigartinales (Fig. E.1, Table E.1). The remaining orders were resolved as a large clade subdivided into two well-supported groups. The first of these consisted of

Acrosymphytales + Ceramiales + Schmitzia (Calosiphoniaceae). The novel Australian taxon Acrothesaurum gemellifilum resolved within a fully supported lineage encompassing species of the genera Acrosymphyton and Schimmelmannia, both currently assigned to a single family in the Acrosymphytales (Fig. E.1, Table E.1). This expanded

Acrosymphytales was sister to the Ceramiales, which included with moderate support the genus Inkyuleea (Fig. E.1). Relationships among the Gelidiales, Gracilariales,

Nemastomatales, Plocamiales and the Halymeniales+Rhodymeniales+Sebdeniales lineage remained largely unresolved, although some support for an alliance of the

Rhodymeniales+Sebdeniales was recognized (Fig. E.1, Table E.1). Finally, moderate support was acquired for the continued inclusion of the Sarcodiaceae in the Plocamiales

(Fig. E.1, Table E.1).

Taxonomic changes

Catenellopsidales K.R.Dixon, Filloramo & G.W.Saunders, ord. nov.

Description: Thalli develop from triaxial apices. Gonimoblasts numerous, arising from an extensive conjugated reticulum with associated nutritive tissue. Tetrasporangia cruciate or decussate, terminal, embedded in outer cortical tissue.

Type and only family: Catenellopsidaceae Robins 1990, p. 698.

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Atractophorales Maggs, L.Le Gall, Filloramo & G.W.Saunders, ord. nov.

Description: Gametangial thallus consisting of erect axes arising from a basal disc; lubricous with erect branches spirally arranged; uniaxial with four periaxial cells per whorl. Monoecious; carpogonial branches 3-celled; procarpic, supporting cell functioning as auxiliary cell, fusing with the fertilized carpogonium, from which the gonimoblast arises. Gonimoblast a diffuse system of loose descending filaments, forming a covering around one or more cells of the main axis; pericarp absent. Mature cystocarps spindle-shaped. Spermatangia in superficial clusters. Tetrasporangial thallus crustose; tetrasporangia regularly cruciate, terminal.

Type and only family: Atractophoraceae Maggs, L.Le Gall & G.W.Saunders, fam. nov.

Atractophoraceae Maggs, L.Le Gall & G.W.Saunders, fam. nov.

Description: as for Atractophorales.

Type genus: Atractophora P.Crouan & H.Crouan 1848: 371.

Additional genus: Liagorothamnion Huisman, D.L.Ballantine & M.J.Wynne, 2000: 507,

508 (discussed below).

Lectotypification: Atractophora hypnoides was provisionally lectotypified by Dixon and

Irvine (1977) in CO. The collection of the brothers Crouan contains five specimens of

Atractophora hypnoides, as well as an illustration (IC BOT/Herb. CO/0001) with a fragment of a plant (CO00287). None of the specimens accord with the protologue, which mentioned a specimen dredged at 8-10 m depth on August 20th 1848 in the Rade de Brest and growing on Melobesia polymorpha (Linnaeus) Harvey and on

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Ceramium rubrum C.Agardh. Among the five specimens, one lacks collection information (CO00289), one is from Noirmoutier (CO00291), and the remainder are from the Rade de Brest. Among the last, one has no collection date (CO00290) and the other two were collected at Baie Sainte Anne in 1847. Specimen CO00288 was growing on

Ceramium, a host mentioned in the protologue. We therefore designate specimen

CO00288 as the lectotype of Atractophora hypnoides (Fig. E.2).

Gametophyte observations in culture: Atractophora hypnoides tetraspores form small multicellular loosely coherent discs that give rise centrally to an erect axis. Erect axes consist initially of a single filament produced by transverse divisions of a more or less isodiametric apical cell. Beginning at about the sixth cell from the apex, each axial cell cuts off a periaxial cell from a lateral protuberance. Alternate axial cells give rise to two periaxial cells/whorl branch initials at 180° to each other, initially forming a distichous arrangement of branchlets (Fig. E.3a). As axes develop further, periaxial cells are also cut off at 90° to the first branchlets, resulting in whorls of four branchlets of limited growth in a cruciate arrangement (Fig. E.3b). Axial cells enlarge greatly in length and diameter, mainly below the insertion of the whorl, so that the whorl is eventually positioned around the distal part of the axial cell, all enclosed in a thin (<10 µm) mucilaginous sheath (Fig. E.3b, d). Whorl branchlets consist of inflated cells 10 µm in diameter, tapering to cylindrical/conical apical cells that often bear hairs up to 100 µm long (Fig. E.3c). Occasional whorl branchlets are replaced by axes of indeterminate growth, of the same construction as the primary axis, but generally forming the cruciate arrangement of whorl branchlets within the apical 10-12 axial cells (Fig. E.4a).

When about one month old, thalli start to produce a distichous arrangement

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of lateral ramuli from the whorl branchlets, and axes develop a filamentous cortication formed by down-growing rhizoidal filaments that originate from the basal cell of every whorl branchlet (Fig. E.3d). All vegetative cell types are uninucleate and contain irregular ribbon-like to reticulate chloroplasts; neither secondary pit connections nor cell fusions are formed.

At about 1.5 months old, thalli form spermatangia and carpogonial branches just below the apices of axes (Figs E.3e-j, E.4a-e). Spermatangia develop in dense clusters all around axes, arising from modified whorl branchlets, each cell of which cuts off small spermatangial mother cells in all directions, singly or in chains. Spermatangial mother cells are rectangular/pyriform and by oblique divisions cut off 2-3 uninucleate spermatangia 1.5-2 µm long (Figs E.3e, E.4b). Released spermatia are spherical, uninucleate and 2.5-3 µm in diameter (Fig. E.4c).

Carpogonial branches usually develop from the basal cell of a modified whorl branch, which is thus the supporting cell (Figs E.3f-j, E.4d-e). The carpogonial branch is

3-celled, the carpogonium and hypogynous cell lying at right angles to the first branch cell, which brings the carpogonium close to the supporting cell (Fig. E.4d, e). The supporting cell bears a 1-celled and a 2-celled lateral branch, and the first cell of the carpogonial branch also bears a lateral cell, forming together an 8-celled structure (Figs

E.3j, E.4e). The carpogonium is triangular as one side lies along the hypogynous cell, and another along the first carpogonial branch cell. The trichogyne develops from the third side, towards the axis at first, and then bending outwards and growing to about 250

µm in length (Figs E.3f-i, E.4d, e). The cytoplasm of the trichogyne is constricted near the carpogonium and then expands to about 2 µm wide, surrounded by a mucilage

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sheath 2 µm thick. Numerous spermatia are observed on hairs and trichogynes, forming cytoplasmic continuity with the trichogynes (Figs E.3k, E.4c).

Following fertilization, the carpogonium and supporting cell fuse to form a dumb- bell shaped cell in some cases, with the hypogynous cell remaining separate (Fig. E.4h).

In other examples the carpogonium and hypogynous cell appear to fuse before joining with the supporting cell. It appears that the first carpogonial branch cell sometimes becomes part of the fusion cell (Fig. E.4f, g). An additional fusion can occur between two of the lateral cells, but this fusion cell is separate from the one involving the carpogonium (Fig. E.4f, g). Early post-fertilization development is apparently quite variable but is obscured by the production of dense clusters of small “nutritive” cells by the first carpogonial branch cell, its lateral cell, and the hypogynous cell (Figs E.3j, E.4f- h). These persist as a small group of cells attached to the fusion cell(s). The fusion cell formed from the carpogonium and the supporting cell cuts off a gonimoblast initial from the carpogonium end (Figs E.3l, E.4f, g). The gonimoblast initial quickly gives rise to several non-pigmented branched gonimoblast filaments, which surround the axis, weaving amongst the whorl branchlets and giving rise to radiating filaments (Fig. E.4i).

Deeply pigmented carposporangia 12-13 µm in diameter are borne terminally on these outward-growing filaments (Figs E.3m, E.4i). Approximately 1.5 months after the first appearance of gametangia, globular mature cystocarps about 250 µm in diameter are present, often arranged in series on an axis due to its continued growth and formation of carpogonial branches.

Tetrasporophyte observations: Carpospores of about 20 µm diameter released in culture and grown under the same conditions as field-collected tetraspores (i.e., 15°C;

345

16:8h LD) germinate in the same manner as the tetraspores to produce cohesive discs

(Fig. E.5a). Basal layer filaments of crusts branch pseudodichotomously (Fig. E.5c), forming a polyflabellate pattern due to the cessation of growth of most filaments soon after branching, causing considerable variation in cell dimensions. This pattern is also seen in field material of Rhododiscus pulcherrimus P.Crouan & H.Crouan (Fig. E.5f), and results in a lobed or irregular margin. No cell fusions or secondary pit connections are formed; calcification is absent. Three months after germination, crusts are up to 2 mm in diameter and 30 µm thick, including a thick mucilage layer on the upper and lower surfaces. They consist of basal layer cells, each bearing one or two 5-6 celled erect filaments 8-14 µm in diameter (Fig. E.5d). The cell cut off behind the apical cell of a basal layer filament immediately divides periclinally (Fig. E.5b) to form crust margins two cells thick. No erect axes developed from these crusts. Crusts transferred to 15°C;

8:16h LD and then to 10°C; 8:16h LD formed tetrasporangia across the surface, developing from the darkly pigmented apical cells of the erect filaments (Figs E.5d, e).

Tetrasporangia are regularly cruciately divided (Fig. E.5d), c. 16-20 µm in length and diameter, smaller and rounder than in field-collected material (Fig. E.5f), in which they are 16-37 x 10-20 µm. In culture, tetrasporangia release tetraspores about a month after formation.

Acrosymphytales Withall & G.W.Saunders 2006, p. 389-390.

Type family: Acrosymphytaceae S.C.Lindstrom 1987, p. 52.

Type genus: Acrosymphyton G.Sjöstedt 1926, 8-9.

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Acrothesauraceae G.W.Saunders & Kraft, fam. nov.

Description: Thalli uniaxial, apical cell division transverse, the central-axial cells each bearing nodal whorls of sub-/pseudodichotomous determinate laterals. Carpogonial and auxiliary-cell filaments both simple, occurring singly, in pairs or in clusters.

Diploidization of the auxiliary cells effected by direct fusion with a presumably fertilized carpogonium, or its derivative cell, in the same (procarpic) or a separate (nonprocarpic) branch system. Tetrasporophytes unknown.

Type genus: Acrothesaurum Kraft & G.W.Saunders, gen. nov.

Additional genus: Peleophycus I.A.Abbott 1984, 325-327 (discussed below).

Acrothesaurum Kraft & G.W.Saunders, gen. nov.

Description: Thalli flaccid, lubricous; central-axial cells each bearing nodal whorls of sub-/pseudo-dichotomous determinate laterals; mid and lower axes corticated between nodal whorls by branched, basipetally directed rhizoids. Thalli monoecious; spermatangia borne directly on terminal and subterminal cells of whorl branchlets; carpogonial and auxiliary-cell filaments both unbranched, occurring singly, in pairs or in clusters on periaxial and one or two distal cells of whorl branchlets, directed basipetally, the auxiliary cells terminal. Diploidization of auxiliary cells effected by direct fusion with presumably fertilized carpogonia, the diploidized auxiliary cell either borne on the same supporting cell as the donor carpogonium (procarpic) or on one of several adjacent supporting cells (nonprocarpic); gonimoblast initials single, carposporophytes composed of up to three synchronously maturing gonimolobes consisting entirely of carposporangia. Proximal portions of diploidized auxiliary cells frequently emitting

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one or two stout, basally directed filaments that fuse apically to central-axial cells and/or adjacent lower whorl-branchlet cells. Tetrasporophytes unknown.

Etymology: from “acro”, referring to objects at an extremity, and “thesaurum”, for a treasury or treasure chamber, in reference to the terminal auxiliary cells that receive and house the “precious” zygote nucleus that initiates the embryonic carposporophyte.

Type and only species: Acrothesaurum gemellifilum Kraft & G.W.Saunders, sp. nov.

Acrothesaurum gemellifilum Kraft & G.W.Saunders, sp. nov. Figs E.6-E.8

Description: portion of thallus on holotype slide (Fig. E.6a) banded, 14 mm in height,

17.8 mm in width, the whole specimen (Fig. E.6b) lubricous, 60 mm in height, 73 mm in greatest breadth, erect from a holdfast pad of consolidated rhizoidal filaments; axes terete

(Fig. E.6c, j), irregularly radially branched to four orders, indeterminate lateral initials scattered (Fig. E.6c), arising on epi-periaxial cells of determinate whorl-laterals (Fig.

E.6d); proximal axes to 1600 µm in diameter, 550-650 µm in lower first-order laterals, narrowing to 10-20 µm at gradually tapered tips (Fig. E.6c). Cells of central-axial filaments 60-90 µm long, 18-25 µm wide (Fig. E.6d), each with (3-)4 periaxial cells at distal poles, the periaxial cells subtending a determinate subdichotomous whorl branchlet with domed or lacrimiform (Fig. E.6c, d), sometimes hair-terminated (Fig. E.6e), apical cells, the nodal appearance of fronds accentuated by the regular spacing of adjacent whorls (Fig. E.6a, d). Rhizoidal filaments basipetal, 2-4 µm in diameter, initially simple

(Fig. E.6f), later branched (Fig. E.6g), mostly arising from periaxial cells, also frequently from apical and subapical cells of apparently non-functioning carpogonial and auxiliary- cell branches, in mature axes issuing adventitious filaments perpendicularly to fully

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corticate the central-axial cells between adjacent whorl-branch nodes (Fig. 6h).

Spermatangia spherical, 2.0-2.5 µm in diameter, borne singly or usually in pairs or threes

(occasionally fours) mostly on terminal cells of whorl-branchlets (Fig. E.6i, j), less frequently also singly or in pairs on subterminal cells. Carpogonial and auxiliary-cell branches basipetally directed (Figs E.6f, i, E.7a-c), borne individually and intermixed on periaxial or epi-periaxial supporting cells (Fig. E.7b), the auxiliary-cell branches usually three-celled, predominant (Fig. E.7a, b), the carpogonial branches scarcer, normally four- celled (Fig. E.7a-c), rarely five-celled (Fig. E.7d), the carpogonia campanulate and with straight (Fig. E.7c, d) or sinuous (Fig. E.7e) trichogynes, the carpogonia usually borne on an inflated, subspherical to ovoid hypogynous cell c. 7.5-8 µm x 6 µm (Fig. E.7b-e); carpogonia frequently non-functional, at various stages of breaking down (Fig. E.7b, e), carpogonial branches then resembling three-celled auxiliary-cell branches because hypogynous cells are the size and shape of auxiliary cells (Fig. E.7b, e). Auxiliary cells terminal, usually ovoid (Fig. E.7a-c), 6-15 x 6-9 µm in diameter; each functional carpogonial branch invariably associated with an adjacent auxiliary-cell branch on the same or an adjoining supporting cell (Fig. E.7a-c). Diploidization of auxiliary cells effected by direct fusion of the presumably fertilized carpogonium (Fig. E.7f, g), the auxiliary cell enlarging, becoming eccentrically swollen (Fig. E.8a, b) and cutting off a single terminal gonimoblast initial (Figs E.7g, E.8a). Gonimolobes compact, the auxiliary cell elongating, thickening distally (Fig. E.8b-d), darkly staining (Fig. E.8a-e), frequently initiating two stout single-celled arms of undetermined function proximal to the carposporophyte (Figs E.7h, E.8d, e), the arms ultimately fusing apically with central- axial cells (Fig. E.8b, d). Carposporophytes globular (Figs E.7h, E.8c, e), at

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maturity 250-450 µm in diameter and composed of three compact synchronously maturing gonimolobes of carposporangia (Fig. E.8f), the gonimolobes consisting of tightly folded filaments of pit-connected subspherical to angular carposporangia 25-50

µm in diameter.

Etymology: from “gemellus” (paired or twinned), and “filum” (filament), in reference to the adjacency of carpogonial and auxiliary-cell filaments that connect after fertilization either procarpically or nonprocarpically.

Holotype: GWS016355, slide A (Fig. E.6a). The holotype slide and six duplicate slides

(GWS016355B-G) permanently housed at UNB. Habit of the entire type specimen was photographed before it was dried in silica as a voucher (Fig. E.6b).

Type locality: Wynyard, Tasmania (40o 58’ 48.7” S; 145o 45’ 04” E), -12 m on shell at

Sanctuary Reef (G.W. Saunders & K.R. Dixon, 28 Jan. 2010).

Distribution: known only from the single holotype specimen.

Schimmelmanniaceae G.W.Saunders & Kraft, fam. nov.

Description: Thalli uniaxial, apical cell division transverse, the central-axial cells each bearing nodal whorls of sub-/pseudodichotomous determinate laterals. Procarpic; carpogonial and auxiliary-cell branches in pairs on supporting cells. Diploidization of auxiliary cells effected by direct fusion with presumably fertilized carpogonia, typically following division of the latter. Tetrasporophytes crustose; tetrasporangial division cruciate.

Type genus: Schimmelmannia Schousboe ex Kützing, 1849: 722.

Additional genus: Gloeophycus I.K.Lee & S.A.Yoo 1979, p. 347 (discussed below).

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Discussion

The combination of more taxa, notably some key lineages previously poorly studied or newly discovered, additional sequence data and exploration of phylogenetic analyses that account for site saturation have resulted in increased resolution among the deep-diverging lineages of Rhodymeniophycidae. This problematic portion of the red algal tree of life

(Withall and Saunders 2006) was considered solvable in the analyses of Verbruggen et al.

(2010), which was consistent with the results here. Phylogenetic reconstruction can be difficult when sequences become saturated and when deep evolutionary events have occurred in relatively close succession such that the available signal is masked by noise in an alignment. Site stripping as performed here can enhance the signal to noise ratio improving phylogenetic inference (Verbruggen 2012). Logically, a node of interest in evolutionary time will be impacted such that resolution of deeper nodes could see greater improvement with more site removal relative to more recent nodes. However, as more and more sites are removed signal will also be removed and support across the phylogeny will degrade. Although stochastic events can complicate matters, the previous patterns were generally observed in our analyses (Fig. E.1, Table E.1). As with many studies

Bayesian posterior probabilities were typically higher in support of various relationships than were the corresponding ML bootstrap percentages (Table E.1). Although the former values are typically considered to overestimate support (see Wróbel 2008), our analyses of progressively more conservative alignments showed enhanced ML bootstrap support for relationships with high posterior probability support in our original analyses of the full alignment (Table E.1). For the current alignment and model at least, higher

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posterior probabilities in analyses of the full alignment appeared to be indicative of phylogenetic signal for the resolved relationships (e.g., Table E.1 nodes E and H), i.e.,

Bayesian posterior probabilities may have been more indicative of evolutionary relationships when the full alignment was analyzed than were the ML bootstrap values.

It should also be noted that the latter values are typically considered as conservative estimators of support (Wróbel 2008). Although the general applicability of site stripping for enhancing phylogenetic resolution awaits more study, the novel phylogenetic insights generated here have necessitated a suite of taxonomic changes at the familial and ordinal levels to represent the full diversity of the lineages under study and to adhere to the principle of monophyly.

Agardh (1876) originally described Catenellopsis oligarthra as a species of

Catenella, at the time placed in the Solieriaceae. Kylin (1932) transferred it to

Nemastoma based on tetrasporangial anatomy, but Chapman (1979) later described the new genus Catenellopsis (Gymnophloeaceae/‘Nemastomataceae’) because the carposporangia were formed strictly in constricted regions of the saccate thalli. Later, when erecting the monospecific family Catenellopsidaceae, Robins (1990) compared the reproductive anatomy of C. oligarthra to several other families. Although he did not observe carpogonia or early post-fertilization development, Robins (1990) considered the post-fertilization anatomy of C. oligarthra to be so distinct that even its ordinal position was uncertain. Nevertheless Robins (1990) provisionally retained Catenellopsis and the

Catenellopsidaceae in the Gigartinales. Our molecular data, the first published for this species, resolved Catenellopsis as an isolated lineage sister to the remainder of the

Rhodymeniophycidae necessitating the recognition of this taxon at the ordinal level.

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Members of the Bonnemaisoniaceae (represented by Asparagopsis and Delisea) and two members of the Naccariaceae (Naccaria and Reticulocaulis) group together

(although the Bonnemaisoniaceae is paraphyletic and further familial level study is needed in this order), but Atractophora, previously assigned to the Naccariaceae, does not join this clade (Fig. E.1). The systematic position of Atractophora has long been debated. In describing A. hypnoides Crouan and Crouan (1848) posited an alliance with

Dudresnaya. Agardh (1863) transferred this species to Naccaria, which he placed in his family Wrangelieae (although given the rank of 'ordo' Agardh's name was equivalent to a family), while Zerlang (1889) again recognized Atractophora as a distinct genus.

Schmitz and Hauptfleisch (1897) allied Atractophora, Naccaria and Wrangelia in the

Wrangelieae of the family Gelidiaceae, with Oltmanns (1904) soon after recognizing the family Wrangeliaceae for these three genera. Kylin (1928) erected the Naccariaceae to include Naccaria and Atractophora. He discussed the relationships of the Naccariaceae and suggested, based on post-fertilization development, that the family was allied to the

Bonnemaisoniaceae, which at that time was included in the Nemaliales (as

Nemalionales). Feldmann and Feldmann (1942) separated the Bonnemaisoniaceae from the Nemaliales owing to the heteromorphic life cycle of Asparagopsis and

Bonnemaisonia and proposed the Bonnemaisoniales. Kylin (1956) did not recognize the

Bonnemaisoniales and included the Naccariaceae (including Atractophora, Naccaria and

Neoardissonia) and Bonnemaisoniaceae (including Asparagopsis, Bonnemaisonia,

Delisea, Leptophyllis and Ptilonia) at the end of his treatment of the Nemaliales (as

Nemalionales).

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Fan (1961), discussing relationships of the Gelidiales, stressed a major difference between Atractophora and Naccaria in that the nutritive filaments associated with carpogonial branches were produced by the supporting cell versus the hypogynous cell, respectively. Fan considered that both genera displayed direct development of the gonimoblast from the carpogonium like the Gelidiales, and felt that the

Bonnemaisoniaceae should be recognized at the ordinal level, as proposed by Feldmann and Feldmann (1942). However, Papenfuss (1966) and Dixon and Irvine (1977) continued to place the Bonnemaisoniaceae in the Nemalionales.

Pueschel and Cole (1982) showed that both Atractophora hypnoides and

Bonnemaisonia hamifera Hariot have pit plugs characterized by a membrane only and lacking plug caps, whereas bona fide Nemaliales have two-layered plug caps. They considered this as strong evidence in support of the Bonnemaisoniales as distinct from the Nemaliales and continued to include the Bonnemaisoniaceae and Naccariaceae in the former order. However, it is important to note that this pit-plug type is shared by virtually all Rhodymeniophycidae and provides no evidence on the relationships between these two families and the many orders of this subclass (Saunders and Hommersand

2004). Womersley (1996) speculated that the Naccariaceae may not be related to the

Bonnemaisoniaceae owing to significant differences in the reproductive structures including the diffuse rather than compact gonimoblast and the complete absence of a pericarp.

Separation of Atractophora from the rest of the Naccariaceae is not entirely unexpected despite similarities of the uniaxial mucilaginous erect gametophytes and mature carposporophytes composed of diploid tissue tightly surrounding the

354

primary axis, intermixed with sterile filaments, and lacking a consolidated pericarp.

There are several potentially significant vegetative differences, such as the transverse apical cell division in Atractophora (Fig. E.3c) compared to the oblique division in

Naccaria (Kylin 1928, fig. 7A) and Reticulocaulis (Schils et al. 2003, fig. 23), the number of periaxial cells cut off each axial cell (four in Atractophora versus two in members of the Bonnemaisoniales sensu stricto), and the absence of secondary pit connections in Atractophora (Figs E.3d, E.5c) versus their presence in Naccaria and

Reticulocaulis (Schils et al. 2003). There are many similarities in female development between Atractophora and Naccaria, such as the presence of nutritive-cell clusters on the carpogonial branch (Kylin 1928, Chihara and Yoshizaki 1972, Hommersand and

Fredericq 1990), which was the basis of their association with the Bonnemaisoniaceae.

However, there are also significant differences between the early post-fertilization development of Atractophora and the other Naccariaceae, the most important of which is that whereas in Atractophora the supporting cell (= auxiliary cell) fuses with the fertilized carpogonium (Fig. E.4h), in Naccaria and Reticulocaulis it remains discrete

(Kylin 1928, Schils et al. 2003). As reported by Kylin (1928), in Atractophora the gonimoblast develops from the carpogonial element of the fusion cell (Fig. E.4g), not from the auxiliary cell. We have also observed fusion between the carpogonium and hypogynous cell (Fig. E.4g), and among some of the lateral (nutritive) cells, which was not reported by Kylin (1928), but resembles that in Reticulocaulis in which the fertilized carpogonium fuses directly with the hypogynous cell via the expansion or breakdown of the pit connection (Schils et al. 2003).

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Atractophora hypnoides resolved distant from the included Bonnemaisoniales and as sister to the Peyssonneliales (Fig. E.1). The sister relationship observed between

Atractophora and the Peyssonneliales was unexpected and intriguing, particularly considering their contrasting morphologies and the anatomical similarities that

Atractophora shares with members of the Naccariaceae (Bonnemaisoniales), to which it was previously attributed. However, there are some significant morphological links between Atractophora and the Peyssonneliales. The tetrasporophyte of Atractophora hypnoides, described by Crouan and Crouan (1859) as Rhododiscus pulcherrimus, was placed provisionally in the Squamariaceae (= Peyssonneliaceae) by Denizot (1968). The

Rhododiscus phase of Atractophora consists of a compact disc with a heterotrichous construction, in which prostrate filaments growing from a multiaxial margin give rise to erect filaments (Fig. E.5a-f). Tetrasporangia are formed terminally on the erect filaments, large, and regularly cruciately divided (Fig. E.5d-f), resembling those of Peyssonnelia species (Maggs and Irvine 1983). Furthermore, during development of male reproductive structures, members of the Peyssonneliaceae often exhibit a uniaxial filamentous construction with whorls of periaxial filaments around each “axial” cell (Kylin 1956, fig.

118B, Maggs and Irvine 1983, fig. 30). Post-fertilization development in Peyssonnelia species reportedly involves the fusion of the carpogonium with the hypogynous and subhypogynous cells of the carpogonial branch, but the auxiliary cell is in a separate filament, i.e., nonprocarpic, and gonimoblasts arise either from a diploidized auxiliary cell or directly from connecting filaments that form expansive networks (Maggs and

Irvine 1983, Dixon and Saunders 2013).

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The question of whether the similarities between Atractophora and the

Peyssonneliales are sufficient to justify expanding the Peyssonneliales to include

Atractophora is a difficult one. Clearly, there are marked contrasts between

Atractophora and the Peyssonneliales, such as procarpic versus nonprocarpic reproduction. The Peyssonneliales exhibits a consistent vegetative and reproductive architecture such that we find it impossible to reconcile the assignment of Atractophora to this order. We therefore propose placing the genus Atractophora in the

Atractophoraceae fam. nov., Atractophorales ord. nov.

Schils et al. (2003) noted similarities between the Naccariaceae and the monotypic genus Liagorothamnion, described as an atypical member of the Ceramiaceae

(Huisman et al. 2000). These similarities include the formation of sterile cell groups on the supporting cell and carpogonial branch cells. However, many key features of its vegetative and post-fertilization development are closer to those of Atractophora than to the rest of the Naccariaceae. In particular, the vegetative axis consists of a narrow axial filament lacking the expanded “jacket” cells observed in the Naccariaceae (Huisman et al. 2000, Schils et al. 2003). The formation in Liagorothamnion of gonimoblasts from the fertilized carpogonium following fusion with the supporting cell also contrasts with

Naccaria and the rest of the Naccariaceae, in which the carpogonium first fuses with the hypogynous cell (Kylin 1928, Hommersand and Fredericq 1990). Based on this anatomical evidence we propose that Liagorothamnion (for Liagorothamnion mucoides

Huisman, D.L.Ballantine & M.J.Wynne) be placed in the Atractophoraceae.

Lindstrom (1987) erected the family Acrosymphytaceae based mainly on the terminal rather than intercalary position of the auxiliary cell for species of

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Acrosymphyton versus the Dumontiaceae sensu stricto. Lindstrom (1987) commented on similarities to the Calosiphoniaceae or Naccariaceae, but argued for a separate family because members of the Calosiphoniaceae have intercalary auxiliary cells while those of the Naccariaceae were considered procarpic in contrast to the terminal auxiliary cells and nonprocarpy of the Acrosymphytaceae.

Tai et al. (2001) provided molecular evidence in support of the

Acrosymphytaceae as distinct from the Dumontiaceae and further suggested that this family might not even be a member of the Gigartinales. Saunders et al. (2004) expanded on that study and uncovered a strong association of the genus Schimmelmannia, at that time assigned to the Gloiosiphoniaceae (Gigartinales), with the Acrosymphytaceae. This was an interesting discovery because species of Schimmelmannia, despite being procarpic, produce a terminal auxiliary cell as in the Acrosymphytaceae and unlike the generitype of the Gloiosiphoniaceae (Kylin 1930). Saunders et al. (2004) transferred

Schimmelmannia to the Acrosymphytaceae despite the respective procarpic versus nonprocarpic post-fertilization patterns, placing taxonomic significance on the terminal auxiliary cells. The Acrosymphytaceae (including Schimmelmannia) weakly resolved in a larger clade including the Ceramiales (members of which also produce terminal auxiliary cells) and the Calosiphoniaceae. Despite the previous molecular indications,

Saunders et al. (2004) retained the Acrosymphytaceae and Calosiphoniaceae in the

Gigartinales arguing that formal taxonomic proposals were premature. Subsequent molecular analyses by Withall and Saunders (2006) were sufficiently robust to recognize a new order for Acrosymphyton and Schimmelmannia, Acrosymphytales, solidly resolved as sister to the Ceramiales with the Calosiphoniaceae as sister to the previous two

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orders. The Calosiphoniaceae were considered incertae sedis pending study of the generitype Calosiphonia (Withall and Saunders 2006). The Calosiphoniaceae remain a distinct lineage in our analyses (Fig. E.1); however, taxonomic proposals remain premature as we lack molecular data for both the type of Schmitzia and, more importantly, Calosiphonia.

The resolution of our new Tasmanian species Acrothesaurum gemellifilum within the Acrosymphytales prompted us to consider family-level taxonomy. Acrosymphyton is highly distinctive from Acrothesaurum and Schimmelmannia as it is characterized by carpogonial branches bearing pinnate laterals, production of primary connecting filaments on presumed fertilization that first fuse with cells of the carpogonial-branch laterals, which in turn issue lengthy septate secondary connecting filaments that seek out distant (i.e., nonprocarpic) auxiliary cells terminating short, unbranched “adventitious” filaments, diploidize them, and then continue on to effect large numbers of further diploidizations (Sjöstedt 1926, Kraft 1981, fig. 1.1, Millar and Kraft 1984, figs 7-9, 15).

Schimmelmannia, on the other hand, is procarpic with the supporting cell bearing both the carpogonial and auxiliary-cell branches, the former simple and not pinnate as in

Acrosymphyton (Kylin 1930). Following fertilization in Schimmelmannia the carpogonium typically undergoes one or two divisions (with one exception; Ballantine et al. 2003), with one of the resulting cells fusing directly to the auxiliary cell (Kylin 1930), again in stark contrast to Acrosymphyton. Our new Tasmanian genus, Acrothesaurum, differs from both Acrosymphyton and Schimmelmannia in that following fertilization the carpogonium fuses directly with an auxiliary cell without intervening connecting filaments or connecting cells, respectively. It is further unusual in blurring the lines

359

between procarpy and nonprocarpy in that auxiliary cells are diploidized both in the same and in separate branch systems by post-fertilization carpogonia. To avoid paraphyly (Fig.

E.1, Table E.1), and in consideration of the significant anatomical differences for

Acrosymphyton relative to Acrothesaurum and Schimmelmannia, we have recognized the latter two at the family level in the Acrosymphytales.

The blurring of the procarpic and nonprocarpic conditions in this family may represent a transitional state from the procarpic Schimmelmanniaceae to the elaborate nonprocarpy characteristic of the Acrosymphytaceae (Fig. E.1). The sister relationship of the Acrosymphytales to the procarpic Ceramiales, combined with the early divergence of the procarpic Schimmelmanniaceae, render it parsimonious to conclude that procarpy is ancestral to nonprocarpy in the Acrosymphytales. For this lineage at least, this reverses the long-standing paradigm that nonprocarpy is an ancestral condition to procarpy and that the Ceramiales are the apogee of red algal evolution (Kylin 1956).

Acrothesaurum gemellifilum is another novel addition to our knowledge of

Tasmanian algal biodiversity as it displays vegetative characters seemingly typical of the

Gloiosiphoniaceae (Gigartinales), a family heretofore unknown in Australia (Womersley

1994). Molecular analyses, however, revealed an unexpected alliance with the

Acrosymphytales, a small order including species that classical morphologists would not have been likely to classify correctly. Recognition of the Acrosymphytales as presented here emphasizes the importance of the terminal auxiliary cell as a diacritical marker among “gloiosiphonioid” taxa (Yeh and Yeh 2008, p. 337). The affinities of the genera

Gloeophycus and Peleophycus need consideration as both are atypical members of the

Gloiosiphoniaceae in being characterized by terminal auxiliary cells.

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Gloeophycus is lubricous in habit and lacks the pinnate carpogonial branch of the

Acrosymphytaceae (Lee and Yoo 1979). It is procarpic and in this regard more reminiscent of the Acrothesauraceae and Schimmelmanniaceae, although more akin to the latter (Kaneko et al. 1980). Pending much needed insights of molecular data this genus is provisionally placed in the Schimmelmanniaceae, Acrosymphytales.

Peleophycus is a Hawaiian endemic monotypic genus (Abbott 1984). Limited

LSU data (664 bp) in GenBank (HQ421875) for Peleophycus multiprocarpius I.A.Abbott solidly ally this genus to the Acrothesauraceae (not shown). Like Acrothesaurum, P. multiprocarpius is lubricous, has similarly structured laterals (Abbott 1984, figs 2, 7, 8), spermatangia (Abbott 1984, fig. 9), and carpogonial/auxiliary-cell branches (Abbott

1984, figs 5, 6). It differs in the relative simplicity of its rhizoidal cortication (Abbott

1984, fig. 4), which apparently does not produce perpendicular corticating filaments, the division of presumably fertilized carpogonia and the diploidization of auxiliary cells by a connecting cell produced by a derivative cell of the divided carpogonium, and an apparent lack of the stout tubular gonimoblasts that arise from proximal auxiliary-cell surfaces to connect to subtending central-axial cells. Whereas Peleophycus was reported as strictly procarpic (Abbott 1984, p. 330), the possibility that carpogonia can diploidize auxiliary cells in separate branch systems as noted here for Acrothesaurum should be explored.

Acknowledgements All of the collectors (listed with the accessions for the COI-5P data in GenBank) are thanked for their contributions to this study, as is T. Moore for generating most of the

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sequence data. This research was supported through funding to GWS from the Canadian

Barcode of Life Network from Genome Canada through the Ontario Genomics Institute,

Natural Sciences and Engineering Research Council of Canada and other sponsors listed at www.BOLNET.ca. Additional support to GWS was provided by the Canada Research

Chair Program, the Canada Foundation for Innovation and the New Brunswick

Innovation Foundation, as well as NSF through the RedToL project (DEB 0937975).

References

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Table E.1 Bayesian posterior probabilities and RAxML bootstrap values corresponding to the nodes in Figure E.1. Results are included for the original alignment without PartitionFinder partitioning schemes (noPF; i.e., fully partitioned) and with PartitionFinder (PF), as well as maximum likelihood bootstrap values for the “stripped” alignments without PartitionFinder partition schemes. Dashes (-) indicate nodal support below 50%. n/a indicates a node that was not resolved.

Full alignment Bayes ML Percentage of stripping for ML no PF analyses Nodes no PF no PF 5% 10% 15% 20% 25% PF PF A 1 0.97 100 100 100 100 100 100 100 B 0.99 0.97 93 92 90 94 90 68 55 C 0.94 0.90 66 66 65 81 50 - n/a D 0.99 0.97 96 97 97 96 71 61 n/a E 1 0.95 89 91 97 79 n/a 54 n/a F 0.99 0.90 52 49 54 76 73 80 - G 0.99 1 65 66 67 76 71 - - H 0.99 0.99 77 77 75 92 87 61 55 I 0.99 0.97 89 89 87 90 80 80 - J 0.99 0.97 97 96 96 99 95 86 55 K 0.51 n/a n/a n/a n/a n/a n/a 60 - L 0.89 0.78 - - - - 53 51 n/a M 0.99 0.97 100 100 100 100 n/a 100 100 N 0.98 0.95 66 66 76 71 n/a - n/a a 0.99 0.99 96 98 98 97 99 100 98 b 0.99 1 100 100 100 100 100 100 100 c 0.99 1 80 81 85 81 80 50 53 d 0.95 0.94 68 69 75 81 83 68 58 e 1 1 100 100 100 100 100 100 89 f 1 1 100 100 100 100 100 100 100 g 1 1 100 100 100 100 100 100 100 h 0.99 1 100 100 100 100 100 100 99 i 0.99 1 100 100 100 100 100 100 100 j 1 1 100 100 100 100 100 100 100 k 0.99 0.98 100 100 100 100 100 100 100 m 0.99 0.97 64 64 77 70 55 67 61

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Figure 1. Bayesian phylogeny for multigene alignment analyzed with full partitioning. Letters at nodes refer to respective support values in Table 1. New taxa in bold type. The outgroup

Corallinophycidae have been cropped from the figure to facilitate presentation. Scale indicates substitutions per site.

Figure E.1 Bayesian phylogeny for multigene alignment analyzed with full partitioning. Letters at nodes refer to respective support values in Table E.1. New taxa in bold type. The outgroup Corallinophycidae have been cropped from the 368 figure to facilitate presentation. Scale indicates substitutions per site.

Figure E.2 Specimen CO00288 from the brothers Crouan herbarium housed at the

Muséum National d'Histoire Naturelle - Marinarium de Concarneau (CO) is here designated as the lectotype of Atractophora hypnoides.

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Figure E.3 Vegetative and reproductive structures of Atractophora hypnoides gametophytes in culture. The culture was isolated from tetraspores of field- collected Rhododiscus pulcherrimus. [Abbreviations: ax, axial cell; b, first (basal) carpogonial branch cell; c, carpogonium; cs, carposporangium; f, fusion cell; g, gonimoblast cell; gi, gonimoblast initial; h/hy, hypogynous cell; l, l1-l4, lateral cells of carpogonial branch; s, spermatangium; sm, spermatangial mother cell; sp, spermatium; su, supporting cell; tr, trichogyne.] a) Uniaxial erect axis of A. hypnoides 18 d after inoculation of tetraspores, showing distichous branching pattern. b) Erect axes after 29 d in culture, developing cruciate arrangement of whorl branchlets. c) Apex of thallus after 46 d, showing transverse apical cell division, repositioning of whorls around axial cells by elongation of axial cells,

370

and terminal hair (arrow). d) Cortication of axis (at 90 d) by downgrowing rhizoidal filaments produced by basal cells of whorl branchlets (shaded). e) Tufts of spermatangial mother cells bearing spermatangia after 46 d. f-i) Stages of development of carpogonial branch. j) Diagram of structure of mature carpogonial branch showing tufts of small "nutritive" cells on the hypogynous cell, first carpogonial branch cell, and fourth lateral cell. k) Carpogonial branch after fertilization, with spermatium attached to trichogyne. Carpogonium and hypogynous cell appear to have fused; protuberance has developed from supporting cell, to which lateral cell (l) is attached. l) First carpogonial branch cell has apparently been included in fusion structure formed by carpogonium, hypogynous cell and supporting cell, which has produced branching gonimoblast filaments. m)

Gonimoblast filaments of mature carposporophyte, bearing terminal carposporangia, after 95 d in culture. Scale bars represent: a, b, 50 µm; c, d, i, 20

µm; e-h, j-m, 25 µm.

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Figure E.4 Reproductive structures of A. hypnoides gametophytes in culture.

[Abbreviations as for Fig. E.3.] a) Thallus apex bearing tufts of spermatangial branches (arrows). b) Spermatangial mother cells giving rise to spermatangia. c)

Thallus with spermatangial tufts and mature carpogonial branch (arrow) with long trichogyne and numerous attached and unattached spherical spermatia. d) Young carpogonial branch with developing trichogyne. e) Mature carpogonial branch showing 8-celled structure. f, g) Post-fertilization development, with interpretation of complex structure drawn in multiple focal planes. Carpogonium has fused with supporting cell, still attached to axial cell (large arrow) to form fusion cell (shaded); gonimoblast initial and gonimoblast filaments produced from carpogonial end of

372

fusion cell (shown in f). Second fusion cell consisting of hypogynous cell and first carpogonial branch cell (lies over carpogonium); third fusion cell formed by lateral cells 2 and 4; three groups of small nutritive cells (nut; small arrow) attached to these fusion cells. h) Fusion cell (large arrow) with group of small nutritive cells

(small arrow indicates one cell). i) Branching gonimoblast filaments with terminal cells developing into carposporangia. Scale bars represent: a, 100 µm; b, d, 5 µm; c,

20 µm; e-i, 10 µm.

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Figure E.5 Vegetative and reproductive structures of Atractophora hypnoides tetrasporophytes in culture. a) Poorly attached spore (left) produced long filament that by lateral branching formed a disc after 31 d. b) Radial vertical section of crust

(at 102 d), showing origin of erect filaments from basal layer cells, and elongated apical cells. c) Stained crust from below. Polyflabellate pattern formed by cessation of growth of most filaments soon after branching. d) Radial vertical sections of crust showing origin of two erect filaments from long basal layer cells, and pigmented apical cells (shaded) that develop into tetrasporangia (t) following transfer to short daylengths (8 h). e) Surface view of crust with mature tetrasporangia; patchy appearance results from discharge of spores. f) Vertical section through field- collected tetrasporophyte (as Rhododiscus pulcherrimus) with developing and mature tetrasporangia. Scale bars represent: a, 50 µm; b, d, 20 µm; c, e, f, 25 µm.

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Figure E.6 a-j) Acrothesaurum gemellifilum vegetative and spermatangial features of holotype (GWS016355). Slide-mounted fragment serving as holotype. Habit of entire specimen from which holotype slide was prepared. Gradually tapering tips of indeterminate laterals, with higher-order laterals (arrows) arising from epi- periaxial cells of whorl branchlets.Whorl laterals borne on periaxial cells ringing distal poles of central-axial cells; higher-order lateral (arrowhead) growing from epi-periaxial cell (arrow). Apical cells of whorl-laterals that project into vegetative hairs (arrows). Rhizoids initiated basipetally from periaxial cells that also bear carpogonial (arrow) and auxiliary-cell (arrowhead) branches. Basipetally growing rhizoids (arrowheads) producing lateral branches. Complete internodal cover of central-axial cells by perpendicular determinate laterals borne on rhizoidal filaments. Whorl lateral of monoecious gametophyte with spermatangia borne singly (arrowheads) or in multiples of two or three (double arrowheads) formed terminally or subterminally, with an auxiliary-cell branch (arrow) directed basipetally from a periaxial cell. Cross-section of a gametophyte axis with terminal spermatangia on whorl laterals borne on the four periaxial cells (arrowheads) surrounding the central-axial filament.

377

378

Figure E.7 a-h). Acrothesaurum gemellifilum features of pre- and presumably post- fertilization events (GWS016355). (Designations “a”, “b”, “ac” are basal, epibasal and auxiliary cells, respectively; designations “1”, “2”, “3”, “cp” are basal, epibasal, hypogynous cells and carpogonia, respectively. “sc” = supporting cell of carpogonial and/or auxiliary-cell filaments.) Periaxial and epi-periaxial (double arrowheads) cells bearing numerous auxiliary-cell branches (arrowheads) and an immature carpogonial branch with rudimentary trichogyne (arrow). Periaxial and epi-periaxial cells bearing auxiliary-cell (arrowheads) and carpogonial branches, trichogyne of one (arrow) apparently breaking down and leaving hypogynous cell the size and position of an auxiliary cell. Terminal auxiliary cells (ac) and three carpogonial branches with early trichogynes (arrows).An anomalous five-celled carpogonial branch, carpogonium bearing a long sinuous trichogyne (arrow).

Carpogonial branch with adjacent auxiliary cells (ac), trichogyne (arrow) extending to whorl surface and apparently bearing attached spermatium (arrowhead).

Attachment of carpogonium to auxiliary cell (arrowhead) of sibling filament on supporting cell (sc) and cutting off of single gonimoblast initial (1’gbl). Three-celled stage of early gonimoblast (gbl) following procarpic fusion of carpogonium and auxiliary cell (arrow). As first gonimolobe matures, two basally directed arms of unknown function grow from extended auxiliary cell (ac).

379

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Figure E.8 Acrothesarum gemellifilum gonimoblast and carposporophyte features

(GWS016355). (Photo annotations as in Fig. E.7) a) Fused carpogonia (arrow) and auxiliary cell (ac), latter viewed end-on and stoutly connected to gonimoblast initial

(gi), which subtends early first gonimolobe. b, c) Diploidized auxiliary cells seen side-on (ac), cells eccentrically swollen and bearing early (5b) and mid (5c) primary gonimolobes. d) Terminal fusion of two auxiliary-cell arms (arrows) to adjacent central-axial cells (arrowheads). e) Mature primary gonimolobe on auxiliary cell

(arrow) that has issued two inwardly growing arms (arrowheads) that are yet to fuse with central-axial cells. f) Mature carposporophyte consisting of three gonimolobes (gl-1, -2, -3) of successively maturing crops of carposporangia.

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Table E.S1 A list of the taxa used in this study with the corresponding GenBank accessions for the five genes used in phylogenetic analyses.

Taxon Voucher SSU LSU EF2 rbcL COI-5P

Ahnfeltiophycidae

Ahnfeltiales

Ahnfeltiaceae

Ahnfeltia plicata

(Hudson) Fries GWS000352 AF419105

GWS001287 EF033537 JX969802 JN113201

Z141391

Pihiellales

Pihiellaceae

Pihiella

liagoraciphila

Huisman, A.R.

Sherwood & I.A.

Abbott AY3019921

Corallinophycidae

Corallinales

Corallinaceae

Corallina

officinalis Linnaeus GWS000343 AF419116

GWS002371 KC130168 KC134323 JQ615683

L261841

382

Jania sagittata

(Lamouroux)

Blainville GWS016470 KC157580 KC157591 KC130175 KC134331 HM918212

Mastophoropsis canaliculata

(Harvey)

Woelkerling GWS015547 KC157582 KC157592 KC130176 KC134335 HM917843

Hapalidiales

Hapalidiaceae

Lithothamnion glaciale Kjellman GWS007312 KC157593 KC130177 KC134336 HM918805

U607381

Mesophyllum vancouveriense

(Foslie) Steneck &

R.T. Paine GWS010090 KC157577 KC157589 KC130171 KC134326 HM918925

Neopolyporolithon reclinatum (Foslie)

W.H. Adey & H.W.

Johansen GWS008332 KC157587 KC130169 KC134324 KC130140

U612521

Synarthrophyton patena (J.D.

Hooker & Harvey)

R.A. Townsend KRD833 KC157578 KC134328 HM916443

LLG0081 EF033600 EF033531

383

Rhodogorgonales

Rhodogorgonacea e

Renouxia sp. HV508A EF033584 EF033601 EF033534 KC134345 KC130151

Rhodogorgon ramosissima J.N.

Norris & Bucher G0302 AF006089

HV434 EF033602 EF033535 KC134341 KC130149

Acrosymphytales

Acrosymphytaceae

Acrosymphyton caribaeum (J.

Agardh) G. Sjostedt CL020501 DQ343661 DQ343684 EF033539 KU382054

Acrosymphyton purpuriferum (J.

Agardh) G. Sjostedt AcroSym01 AF317091 DQ343683

Acrothesauraceae

Acrothesaurum gemellifilum Kraft

& G.W. Saunders GWS016355 KU382042 KU501317 KU382086 KU382064 HM918171

Schimmelmanniac eae

Schimmelmannia plumosa (Setchell)

Abbott GWS021743 KU501315 KU382085 KU382061 KM254739

384

Schimmelmannia schousboei (J.

Agardh) J. Agardh G0152 AY437681 AF419130 EF033540 KU382066

Atractophorales

Atractophoraceae

Atractophora hypnoides P.

Crouan & H.

Crouan GWS005200 KU382041 GQ497323 GQ497322 KU382063 GQ497303

Bonnemaisoniales

Bonnemaisoniacea e

Asparagopsis taxiformis (Delile)

Trevisan GWS000292 AY772724 KU501318 KU382088 HM915860

GQ337069

1

Delisea hypneoides

Harvey GWS001961 EF033585 EF033603 EF033541 JX969778 HM915956

Naccariaceae

Naccaria wiggii

(Turner) Endlicher GWS000398 AY772729 AF419111 KU382052

GWS001859 GQ497312

Reticulocaulis mucosissimus

Abbott GWS001345 DQ343656 DQ343680 EF033542 JX969777

385

Catenellopsidales

Catenellopsidacea e

Catenellopsis oligarthra (J.

Agardh) Chapman KRD824 KU382040 KU501312 KU382082 KU382053 HM916097

Ceramiales

Ceramiaceae

Centroceras clavulatum (C.

Agardh) Montagne GWS000193 DQ343657 AF419113 EF033543

GWS015026 HM917544

DQ458918

1

Ceramium virgatum

Roth GWS000203 AF236793

GWS002366 EF033604 EF033544 KT250235

AF439290

1

Dasyaceae

Heterosiphonia plumosa (Ellis)

Batters CH380 AF488396 EF033606 EF033546

GWS014655 KU382031

AF259494

1

386

Delesseriaceae

Grinnellia americana (C.

Agardh) Harvey GWS001880 EF033587 EF033607 EF033547

GWS005729 JX110919 HM916640

Sorella repens

(Okamura)

Hollenberg CH005 AF488406 EF033608 EF033548

Inkyuleeaceae

Inkyuleea mariana

(Harvey) H.G.

Choi, Kraft & G.W.

Saunders GWS000215 AF236792 DQ343681

GWS002521 KU382055 KU382034

Wrangeliaceae

Ptilota serrata

Kützing G0349 AF203884

Ptilota tenuis Kylin GWS002173 EF033605 EF033545 AY970640

GQ252574

1

Gelidiales

Pterocladiaceae

Pterocladiella capillacea (S.G.

Gmelin) Santelices

& Hommersand GWS001558 DQ343660 DQ343682 EF033549 KU382033

387

GU731222

1

Gigartinales

Cystocloniaceae

Cystoclonium purpureum

(Hudson) Batters G0421 AY437672 KC130242 KU382083 KC130217

GWS002294 HM918475

Dumontiaceae

Dilsea carnosa

(Schmidel) Kuntze GWS000746 AF317096 EF033609 EF033551 JN403065 AY971151

Endocladiaceae

Gloiopeltis furcata

(Postels &

Ruprecht) J.G.

Agardh G0163 U33130

GWS002264 EF033612 EF033553 JX969801

GWS013649 HM916317

Gigartinaceae

Chondrus crispus

Stackhouse CSM006A EF033554

GWS001841 GQ338090

GWS013811 KF026483 HM916371

Z141401

388

Gloiosiphoniaceae

Gloiosiphonia capillaris (Hudson)

Carmichael in

Berkeley GWS000374 AY437680 GQ406352

GWS013313 KU382056 HM915532

Kallymeniaceae

Euthora cristata (C.

Agardh) J. Agardh GWS000026 AY437684 AF419124 EF033555 GU140142

GWS013734 JX969805

Mychodeaceae

Mychodea acanthymenia Kraft GWS000960 EF033589 EF033614 EF033557 JX969795 HM915892

Solieriaceae

Solieria robusta

(Greville) Kylin G0136 AY437657

GWS001590 GQ406360 KC130210 HM915934

Gracilariales

Gracilariaceae

Gracilaria salicornia (C.

Agardh) Dawson GWS002001 EF033615 EF033559 KU382062 FJ499537

AY2041421

Gracilariopsis andersonii

(Grunow) Dawson GWS002276 KU382039 KU501311 KU382081

GWS028503 KU382057 KU382035

389

Melanthalia obtusata

(Labillardiere) J.

Agardh GWS001620 L26215 DQ343692 EF033561 KU382044 FJ499661

Halymeniales

Halymeniaceae

Cryptonemia undulata Sonder G0081 U33125 AF419133 GQ471902

GWS014849 KU382069 HM917444

Halymenia floresii

(Clemente) C.

Agardh GWS011961 GQ471912 GQ471904 GQ862071

AY772019

1

Isabbottia ovalifolia (Kylin)

Balakrishnan GWS001334 EF033590 EF033616 EF033563 KU382070 HM915678

Pachymenia carnosa (J. Agardh)

J. Agardh GWS000347 AF515289 DQ343695 EF033564 KU382032

AF385640

1

Prionitis sternbergii (C.

Agardh) J. Agardh GWS001168 EF033591 EF033617 EF033565 HM915707

GWS021741 JX969790

390

Zymurgia chondriopsidea (J.

Agardh) Lewis &

Kraft G0287 AF515304

GWS000966 DQ343698 EF033566 KU382065 HM916232

Nemastomatales

Nemastomataceae

Predaea kraftiana

Millar & Guiry GWS000132 AF515297 EF033618 EF033567 KP733872

Schizymeniaceae

Platoma cyclocolpum

(Montagne)

Schmitz GWS000133 AF515292 DQ343706 EF033568 JN659917

FJ8788571

Schizymenia pacifica (Kylin)

Kylin GWS000584 AF419129

GWS001682 EF033592 EF033569 JX969726

GWS012735 JX969800

Peyssonneliales

Peyssonneliaceae

Peyssonnelia atropurpurea P.L.

Crouan & H.M.

Crouan CM04 KU501313 JX969810 JX969782 JX969703

AB2313211

391

Peyssonnelia dubyi

P.L. Crouan &

H.M. Crouan CM03 KU501316 JX969812 JX969785 JX969715

AB2313221

Ramicrusta textilis

C.M. Pueschel &

G.W. Saunders GWS001755 FJ848977 FJ848970 JX969821 KC130226 JX969749

Sonderophycus sp. G0418 AY437688 AF419125 KU382087 KC130225 KC130203

Plocamiales

Plocamiaceae

Plocamium maggsiae G.W.

Saunders &

Lehmkuhl G0167 AY437708 AF419141 EF033570

GWS001754 JX969788 JF271602

Sarcodiaceae

Sarcodia ciliata

Zanardini GWS001027 DQ343666 DQ343708 EF033572 JX969793 HM915894

Rhodymeniales

Champiaceae

Chylocladia sp._1Ire G0210 AF085263 DQ343712 EF033573 KU382046 KU382030

Coelothrix irregularis

(Harvey) Borgesen BDA0233 HQ933315

CL020502 EU624160 EU624165 KU382050

EU0864581

392

Faucheaceae

Gloiocladia repens

(C. Agardh)

Sanchez &

Rodriguez-Prieto G0332 AF085267 AF419143

GWS001639 KU382080 KR140335 KR140326

Leptofauchea pacifica Dawson GWS010140 KR085195

JD032 DQ873286 EU624174 AY970566

Fryeellaceae

Fryeella gardneri

(Setchell) Kylin G0335 AF085273 EF033622

GWS001131 EF033578 JX969776 JX969692

Hymenocladiopsis prolifera (Reinsch)

M.J. Wynne CCMP455 EU624144 EU624203 KU382071 HQ919249

G0197 AF085274

Hymenocladiaceae

Asteromenia anastomosans

(Weber Bosse)

G.W. Saunders,

C.E. Lane, C.W.

Schneider & Kraft GWS001059 DQ068299 KU382059 HM915899

GWS001079 EU624182

GWS001080 DQ343652

393

Hymenocladia conspersa (Harvey)

J. Agardh GWS000423 AF117128 DQ068294 EU624202

GWS015238 KU382045 HM917652

Lomentariaceae

Lomentaria orcadensis

(Harvey) Collins ex

W.R. Taylor GWS001885 DQ873292 EU624179 KU382058 GQ497311

Stirnia prolifera 14092000-

M.J. Wynne 07-09MW DQ873294 EU624181 KU382072 KU382037

Rhodymeniaceae

Chrysymenia ornata (J. Agardh)

Kylin G0281 AF085257 DQ343670 EU624186 KU382073 KU382038

Sparlingia pertusa

(Postels &

Ruprecht) G.W.

Saunders, I.M.

Strachan & Kraft G0331 AF085261

GWS000581 DQ343677 EU624200 JQ907561 HM916393

Sebdeniales

Sebdeniaceae

Crassitegula imitans G.W.

Saunders & G.T.

Kraft GWS001076 AY437707 DQ343700 JN641180

394

GWS002089 EF033581

GWS022917 KU382068

Crassitegula

walsinghamii C.W.

Schneider, C.E.

Lane & G.W.

Saunders GWS001245 AY964057 EF033623 EF033580 JN641185

Sebdeniaceae

sp._1unknown GWS002074 JN602191 JN602197 EF033579

GWS023442 KU382048 JN641193

Incertae sedis

Calosiphoniaceae

Schmitzia sp._1WA G0266 AY437660 DQ343685 EF033538 KU382067 KU382036

Schmitzia sp._2WA G0397 AY437659 AF419131 KU382084 KU382060

1Data were mined from GenBank and thus lack voucher numbers.

395

Figure E.S1. An alternative topology generated by neighbor-joining with the

HKY model to assess the effect of the starting tree on our SiteStripper analyses. 396

Appendix F Key Kamchatkan collections provide new taxonomic and

distributional insights for reportedly pan-North Pacific species of

Rhodymeniophycidae (Rhodophyta)

Citation for published manuscript: Filloramo, G.V., Savoie, A.M., Selivanova, O.N.,

Wynne, M.J. & Saunders, G.W. Kamchatkan collections provide new taxonomic and distributional insights for reportedly pan-North Pacific species of Rhodymeniophycidae

(Rhodophyta). Phycologia, accepted May 2016.

397

Abstract

Northwestern Pacific specimens of Callophyllis radula, Callophyllis rhynchocarpa,

Pterosiphonia bipinnata and Sparlingia pertusa, all with type localities in Far East

Russia, were genetically compared to reportedly conspecific populations in the Northeast

Pacific. Analyses resolved genetically distinct species with a biogeographical split between the Northwest and Northeast Pacific for C. radula, C. rhynchocarpa and P. bipinnata. Callophyllis variforma sp. nov. was established to accommodate Northeast

Pacific plants previously attributed to C. radula, while synonymy of the Northeast Pacific

C. flabellulata with the Northwest C. rhynochocarpa was refuted. To accommodate the species in the Northeast Pacific previously attributed to P. bipinnata, the epithet P. robusta was resurrected. Comparison of Northwest to Northeast Pacific S. pertusa revealed COI-5P variation consistent with incipient speciation between these two regions; however, subsequent analysis of the ITS2 provided evidence for recognition of a single species with a pan-North Pacific distribution. Our results indicated greater floristic division between the Northwest and Northeast Pacific than is currently recognized.

Key Words: Callophyllis; COI-5P; cryptic diversity; Far East Russia; Kamchatka;

Northeast Pacific; Pterosiphonia; rbcL; Sparlingia

The contemporary application of molecular tools (e.g., DNA barcoding) to algal taxonomy has provided more accurate knowledge of species diversity and distribution

(Saunders 2005, 2014; Robba et al. 2006; Le Gall & Saunders 2010a). Of note here, molecular tools have routinely resolved reportedly cosmopolitan or broadly

398

distributed algal species as highly divergent and diverse complexes of species with more limited geographical ranges (e.g., Saunders & Lehmkuhl 2005; Saunders et al. 2006; Le

Gall & Saunders 2010b; Saunders & McDevit 2013).

There are a number of cases for which there is taxonomic debate related to populations of seaweed species in the Northwest and Northeast Pacific. For example, the kelp species Saccharina bongardiana (Postels & Ruprecht) Selivanova, Zhigadlova &

G.I. Hansen was described from Kamchatka in Far East Russia but was also considered to be present in the Northeast Pacific by various authors at various times (e.g., Saunders

1901, Lindstrom 1977; Lüning & tom Dieck 1990; Gabrielson et al. 2000; Mondragon &

Mondragon 2003). Genetic data for S. bongardiana from near the type locality, however, resolved the Northeast populations as a distinct species, which was assigned to S. groenlandica (Rosenvinge) C.E. Lane, C. Mayes, Druehl & G.W. Saunders [McDevit &

Saunders 2010; = Saccharina nigripes (J. Agardh) Longtin & G.W. Saunders (Longtin &

Saunders 2015)]. The previous study and others like it (e.g., Adey et al. 2015), highlight the importance of including collections from near the type locality in taxonomic surveys.

Callophyllis radula Perestenko, Callophyllis rhyncocharpa Ruprecht,

Pterosiphonia bipinnata (Postels & Ruprecht) Falkenberg, and Sparlingia pertusa

(Postels & Ruprecht) G.W. Saunders, I.M. Strachan & Kraft all have type localities in Far

East Russia. These names have also been variously applied to populations in the

Northeast Pacific. Recent collections of the previous four taxa from Far East Russia (Fig.

F.1) have provided the necessary genetic data to assess their respective distributions in the North Pacific and assess conspecificity. In most cases, genetic species groups were determined using the mitochondrial COI-5P DNA barcode (664 bp) as a marker

399

(Saunders & McDevit 2012a) (Table F.S1). When COI-5P failed to amplify, we assessed the plastid rbcL gene (1363 bp; Saunders & Moore 2013) (Table F.S1). Owing to the low levels of variation uncovered for Sparlingia pertusa with the previous markers, ITS2 data were also generated (Saunders & McDevit 2012b) (Table F.S1). A single representative for each of the four Callophyllis spp. include in this study were combined in an alignment with additional COI-5P Callophyllis data acquired from GenBank (GenBank numbers included with accessions in the figure). That alignment was assessed in Geneious R7

(http://www.geneious.com; Kearse et al. 2012) by RAxML analysis under a GTR+I+G model with partitioning by codon and support determined by 1000 rounds of bootstrap resampling was performed on a COI-5P alignment. Our molecular analyses have provided clarification of the respective distributions of C. radula, C. rhyncocharpa, P. bipinnata and S. pertusa in the North Pacific and here we present our taxonomic clarifications and revisions.

The species Callophyllis radula was described from Kamchatka by Perestenko

(1996) and was first reported in British Columbia, Canada by Clarkston & Saunders

(2013). At the time of that study, Clarkston & Saunders (2013) lacked topotype material for comparison to individuals from British Columbia; however, anatomical observations

(excepting blade thickness, which was less in the British Columbia plants) were largely consistent with Perestenko’s description. In their phylogram (Fig. 1, p. 30) based on COI-

5P data, collections from British Columbia assigned to C. radula were closely related to a species described from Washington, C. heanophylla Setchell (Clarkston & Saunders

2013); however, the COI-5P data for our single representative of C. radula from

Kamchatka (type locality; Fig. F.1) was most closely allied to another Russian

400

species, C. rhynchocarpa and only distantly related (6.53% interspecific divergence) to the plants assigned to C. radula from British Columbia (n= 35; Table F.S1; Fig. F.2).

Recognition of a novel species for the British Columbian entity was warranted. That species may be distinguished from C. radula based on morphology (see below) and biogeography.

Callophyllis variforma Filloramo, Selivanova & G.W. Saunders sp. nov. Figs F.3-F.5

DESCRIPTION: Plants superficially representative of C. radula. Plants collected as clumped thalli, rarely found as single blades. Thalli 3-6 cm tall, regularly to irregularly dichotomously divided with 3-5 orders of branching (Fig. F.3). Branch width was typically consistent from base towards apices within an individual but variable between individuals (1-5 mm wide); apices were blunt, rounded or at times acute (Fig. F.3). In transverse section, blades were 100-200 um thick with (1-) 2 (-3) layers of rounded or ellipsoid, colourless medullary cells ranging in size (100-200 um long x 50-150 um wide) and surrounded by filaments of small, elongated cells (Fig. F.4). Cortex comprised of a single outer layer of small, oblong, darkly staining cells and incomplete inner layer (Fig.

F.4). Tetrasporangia produced from apex towards base, predominantly on one thallus surface, lagging in development on the opposite surface. Mature tetrasporangia cruciately divided, 18-25 um long x 13-15 um wide, scattered in the cortex (Fig. F.5).

HOLOTYPE: GWS020798 deposited in Connell Memorial Herbarium (UNB). Images of the holotype shown in Figs F.1-F.2. Collected from Telephone Point, Skidegate, Haida

Gwaii, British Columbia, Canada (53.24233, -132.01566), 7 June 2010; from 6 m on

401

rock; leg. G.W. Saunders and K. Dixon. Holotype DNA barcode: GenBank HQ545092,

COI-5P.

ISOTYPES: GWS020789, GWS020793, GWS020798, GWS020808, GWS020824 and

GWS020838 deposited in Connell Memorial Herbarium (UNB).

ETYMOLOGY: Named for the highly variable nature of this species with the gross morphology of individuals variously reminiscent of species in the genera Callophyllis,

Leptofauchea and Rhodymenia.

DISTRIBUTION: Thus far reported only from British Columbia, Canada (Table F.S1).

Additional comments: For detailed anatomical and reproductive observations see

Clarkston & Saunders (2013; as C. radula). Representatives of C. variforma were most closely related (3.62% interspecific divergence) to another Northeast Pacific species, C. heanophylla Setchell (1923), which was described from Shaw Island, Washington and also reported from British Columbia (Scagel et al. 1989; Clarkston & Saunders 2013).

These two species may be distinguished based on thallus texture (C. heanophylla features a distinct soft and silky texture vs. C. variforma, which is more cartilaginous) and individual branch width, as C. variforma is narrower than C. heanophylla (up to 8 mm wide).

Another Far East Russian species of Callophyllis with taxonomic confusion in the

North Pacific is C. rhynchocarpa Ruprecht (1850), which was described from Ayan, Sea of Ochotsk. In their review of red algal species from the Bering Sea, Wynne & Heine

(1992) suggested that Callophyllis flabellulata Harvey (1862), with a type locality of

Vancouver Island, British Columbia, Canada was conspecific with C. rhynchocarpa.

Although some subsequent authors adopted Wynne & Heine’s (1992) perspective

402

of C. rhynchocarpa as a pan-North Pacific species (e.g., Harrer et al. 2013), others continued to regard the Northeast C. flabellulata as distinct (e.g., Spalding et al. 2003;

Gabrielson et al. 2012; Clarkston & Saunders 2013). Our COI-5P data for a specimen of

C. rhynchocarpa from Southern Sakhalin, Sea of Okhotsk (Fig. F.1) was unequivocally divergent (4.07-4.96%) from our Northeast Pacific collections (n= 55; including topotype material of C. flabellulata) and refuted the previously proposed synonymy. These populations should be regarded as two genetically (Fig. F.2) and biogeographically distinct species. Although the full distributional range of C. rhynchocarpa is unknown, we have yet to encounter that species in our extensive collections from the Northeast

Pacific. According to our data, C. flabellulata is widely distributed in the Northeast

Pacific; however, future studies are needed to determine if its distribution extends to the

Northwest Pacific.

Another species described from Kamchatka is Polysiphonia bipinnata Postels &

Ruprecht (1840), which was later transferred to Pterosiphonia by Falkenberg (1901).

This species has been widely reported in the North Pacific from Japan, Russia,

Commander Islands, Alaska, British Columbia, Washington, Oregon, and California [see

Guiry & Guiry (2015) for full references]. The rbcL sequence for a collection of P. bipinnata from Kamchatka (Fig. F.1) was a match (3 – 4 bp different over 1363 bp) to two collections from northern British Columbia. However, both rbcL and COI-5P from

P. bipinnata were strongly divergent (5.65% and 7.2%, respectively) from sequence data for most Northeast Pacific specimens (including collections from Pigeon Point, San

Mateo, California) previously identified as P. bipinnata (n= 74). Northeast Pacific plants thus need a new specific assignment for which the resurrected epithet P. robusta,

403

which was described from Moss Beach, San Mateo, California by N.L. Gardner (1927), reduced to synonymy with P. bipinnata by Kylin (1941) and later recognized at the varietal level within P. bipinnata by Doty (1947), is available. Interestingly, phylogenetic analysis of the tribe Pterosiphonieae failed to resolve both P. bipinnata and P. robusta in a monophyletic lineage with the generitype of Pterosiphonia, P. cloiophylla (C. Agardh)

Falkenberg. This issue, as well as other outstanding taxonomic problems in the

Pterosiphonieae, will be addressed in a future manuscript (Savoie & Saunders 2016).

Sparlingia pertusa was originally described from Kamchatka as Porphyra pertusa

Postels & Ruprecht (1840), which was transferred by Agardh (1851) to Rhodymenia where it was long recognized as an atypical member of the genus owing to its unbranched and typically perforate blades (Sparling 1957). Molecular phylogenetic analyses established that R. pertusa was distantly related to the generitype R. pseudopalmata (J.V.

Lamouroux) P.C. Silva and the novel genus Sparlingia was established (Saunders et al.

1999).

Currently, S. pertusa is reportedly widespread in the North Pacific [see Guiry &

Guiry (2015) for full references]. Our COI-5P data for a specimen of S. pertusa from

Kamchatka (Fig. F.1) when compared to data from Northeast Pacific individuals (n= 81) indicated two closely related (2.20% interspecific divergence) genetic clusters with a

Northwest versus Northeast biogeographical split. Owing to the low divergence between the two genetic groups, we assessed a shortened part of the nuclear ITS2, which resolved the collections as a single genetic species group [1 bp difference across 262 bp (Saunders

& McDevit 2012b)]. Atypically high divergence in COI-5P corresponding with low divergence in the ITS is consistent with past isolation, potentially during the

404

Pleistocene glaciation in this case, followed by genetic mixing of the formally isolated populations with retention of ancestral mitochondrial types in the populations (e.g.,

Saunders & McDonald 2010; Filloramo & Saunders unpublished). Thus, we take the conservative approach and recognize S. pertusa as a single pan-North Pacific species.

Recently, Rhodymenia stipitata Kylin (1925), described from Friday Harbor,

Washington, was resurrected and transferred to Sparlingia to accommodate specimens from Kamchatka that were considered morphologically distinct from S. pertusa

(Klochkova et al. 2009). Molecular assessment of specimens assigned to S. stipitata from

Kamchatka is necessary to confirm their distinction from S. pertusa. Regardless, it is unlikely that this name is correctly assigned to an additional species of Sparlingia from

Kamchatka should one be uncovered. We have sampled Sparlingia extensively in the

Northeast Pacific (n= 87), including a collection from the type locality, and have uncovered only a single genetic group, which based on the data here was correctly referred to as S. pertusa with its type locality in Kamchatka, Far East Russia. We thus retain Sparlingia stipitata as a synonym of S. pertusa.

Rhodymenia wilkesii Harvey & Bailey is another species in need of consideration.

Described from the Strait of St. Juan de Fuca (Harvey & Bailey 1851; Bailey & Harvey

1862) as part of the first American-sponsored scientific exploration, the United States

Exploring Expedition (1838-1842) under the command of Charles Wilkes, U.S.N, R. wilkesii has been orphaned in the taxonomic literature, likely owing to the delayed publication and limited production of the Wilkes’ expedition reports (Collins 1912;

Wynne 2009). When R. wilkesii was described, Harvey and Bailey (1851) accidentally overlooked the previous description of R. pertusa and upon recognition that these

405

two species were morphologically indistinguishable, Harvey (1853) reduced R. wilkesii to synonymy with R. pertusa. We have tracked the type specimen (Catalogue #:

ABRU00002068) of R. wilkesii to the Brown University Herbarium where it was sent by

J.W. Bailey to his son, W.W. Bailey, who was head of the botany department (Collins

1912; Wynne 2003). We have also discovered that the Connell Memorial Herbarium at the University of New Brunswick, Fredericton houses a collection sent from J.W. Bailey to his son, L.W. Bailey (with the apparently incorrect provenance of California associated with the specimen; l.c. Saunders & McDevit 2012b). Owing to the proper publication of

R. wilkesii by Harvey & Bailey (1851) in addition to the existence of a type specimen, R. wilkesii should be regarded as a valid species, one that would take priority over R. stipitata, should a distinct Northeast Pacific species ever be uncovered matching this concept.

In conclusion, this project investigated the taxonomic identifications of red algal species with reportedly wide distributions in the North Pacific by comparing genetic data for specimens of species with their type locality in Far East Russia to collections from the

Northeast Pacific. Owing to logistical issues, this study was limited in the number of Far

Eastern Russian collections included in molecular analyses and comprehensive sampling from that area is warranted to determine if the Northeast Pacific species recognized herein are also present in the Northwest Pacific. The taxonomic conclusions herein are the best that can be devised based on the available data and represent steps forward from the current taxonomic knowledge for these species.

406

Acknowledgements

Without the collection assistance of N. Klochkova and G. Zhigadlova in Far East Russia, this project would not have been possible. We thank the following collectors for their contributions to this project: M. Bruce, B. Clarkston, S. Clayden, K. Dixon, S. Hamsher,

H. Kucera, A. Lamb, D. McDevit and K. Roy. Thank you to T. Bringloe, M. Bruce, K.

Dixon, B. Herman, L. Kraft, L. Le Gall, D. McDevit and T. Moore for generating some of the sequence data used in analyses. We thank C.W. Schneider for his assistance with

Latin nomenclature. We also thank J. Parnell from the Trinity College Dublin Herbarium for his information regarding Sparlingia wilkesii. This research was supported through funding to GWS from the Natural Sciences and Engineering Research Council of Canada

(NSERC), the Canada Foundation for Innovation, and the New Brunswick Innovation

Foundation.

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411

Figure F.1 Map of the North Pacific showing sites of recent collections (indicated by square) of Callophyllis radula (1), C. rhynchocarpa (2), Pterosiphonia bipinnata (3) and Sparlingia pertusa (4). The type locality of those species is also indicated

(circle), excepting P. bipinnata and S. pertusa, which were described from Kamchatka without reference to a specific location.

412

Figure F.2 Phylogenetic tree resulting from RAxML analysis of COI-5P data from the Callophyllis spp. with bootstrap values above 50% appended. Bold taxa are species emphasized in the current study. Country abbreviations indicate where each Callophyllis species has been collected (AUS= Australia; CE= Chile;

NEP= Northeast Pacific; RUS= Russia). 413

Figure F.3 Callophyllis variforma sp. nov. from British Columbia, Canada. a)

Habit of vegetative holotype (GWS020798). Scale bar = 25 mm. b) Transverse section at midthallus of the vegetative holotype (GWS020798). Scale bar = 50 µm. c) Transverse section at midthallus of tetrasporophyte showing cruciate tetrasporangia embedded in a single cortex (GWS012627). Scale bar = 85 µm. 414

Table F.S1 Collection details and GenBank Accession numbers for specimens used in our molecular analyses (newly determined data indicated by bold type). ND refers to genetic data not determined for this study.

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession Callophyllis GWS000580 Bamfield, Dixon 06-Apr- ABMMC3239-08 JX034295 ND ND flabellulata I., BC, CAN 1999 Harvey GWS001420 Bamfield, 08-Oct- ABMMC1151-07 JX034287 ND ND Wizard I., BC, 2002 CAN GWS001651 Bamfield, 09-May- ABMMC354-06 JX034303 ND ND Wizard I., BC, 2003 CAN GWS002185 Bear Cove Park, 17-Jun-04 ABMMC1831-07 JX034308 ND ND Port Hardy, Vancouver Island, BC, CAN GWS002271 Bamfield, 25-Jun- ABMMC1156-07 JX034302 ND ND Wizard I., BC, 2004 CAN GWS003067 Bamfield, 15-Jun- ABMMC3240-08 JX034292 ND ND Seapool Rock, 2005 BC, CAN GWS003076 Satellite Reef, 15-Jun- ABMMC1159-07 JX034291 ND ND Bamfield, BC, 2005 CAN

415

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS003077 Satellite Reef, 15-Jun- ABMMC1160-07 JX034299 ND ND Bamfield, BC, 2005 CAN GWS003079 Satellite Reef, 15-Jun- ABMMC1161-07 JX034301 ND ND Bamfield, BC, 2005 CAN GWS003149 Bamfield, 14-Sep- ABMMC9049-10 HM918552 ND ND Seapool Rock, 2005 BC, CAN GWS003266 Bamfield, 17-Sep- ABMMC3242-08 JX034298 ND ND Wizard I., BC, 2005 CAN GWS004133 Bamfield, Scotts 18-Jun- ABMMC19593-14 KU873104 ND ND Bay, BC, CAN 2006 GWS004135 Bamfield, Scotts 18-Jun- ABMMC3243-08 JX034300 ND ND Bay, BC, CAN 2006 GWS004253 Saxe Pt., 21-Jun- ABMMC9241-10 HM918641 ND ND Victoria, BC, 2006 CAN GWS004569 Bamfield, 27-Jun- ABMMC9303-10 HM918670 ND ND Satellite Passage 2006 Reef, BC, CAN GWS004572 Satellite Reef, 27-Jun- ABMMC1832-07 JX034309 ND ND Bamfield, BC, 2006 CAN

416

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS004573 Satellite Reef, 27-Jun- ABMMC1833-07 JX034280 ND ND Bamfield, BC, 2006 CAN GWS004574 Satellite Reef, 27-Jun- ABMMC1834-07 JX034281 ND ND Bamfield, BC, 2006 CAN GWS004575 Satellite Reef, 27-Jun- ABMMC1835-07 JX034285 ND ND Bamfield, BC, 2006 CAN GWS004577 Bamfield, 27-Jun- ABMMC9304-10 HM918671 ND ND Satellite Passage 2006 Reef, BC, CAN GWS004739 Browning Wall, 04-Jul-2006 ABMMC9332-10 HM918688 ND ND north of Port Hardy, BC, CAN GWS004744 Browning Wall, 04-Jul-2006 ABMMC1200-07 JX034293 ND ND north of Port Hardy, BC, CAN GWS004746 Browning Wall, 04-Jul-2006 ABMMC9335-10 HM918691 ND ND north of Port Hardy, BC, CAN

417

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS004747 Browning Wall, 04-Jul-2006 ABMMC1836-07 JX034289 ND ND north of Port Hardy, BC, CAN GWS004774 Browning Wall, 04-Jul-2006 ABMMC1837-07 JX034296 ND ND north of Port Hardy, BC, CAN GWS006318 Pier near statue 24-May- ABMMC6183-10 HM916741 ND ND of diver, Sidney, 2007 BC, CAN GWS006518 Tahsis Narrows, 29-May- ABMMC6238-10 JX034304 ND ND BC, CAN 2007 GWS006536 Tahsis Narrows, 29-May- ABMMC2021-07 JX034297 ND ND BC, CAN 2007 GWS008998 Bamfield, Scotts 21-Sep- ABMMC9654-10 JX441891 ND ND Bay, BC, CAN 2007 GWS009001 Bamfield, Scotts 21-Sep- ABMMC3258-08 JX034284 ND ND Bay, BC, CAN 2007 GWS009035 Mears Bluff, 22-Sep- ABMMC14553-11 KU873112 ND ND Broken Group, 2007 BC, CAN GWS009086 Bamfield, 23-Sep- ABMMC3259-08 JX034288 ND ND Wizard I., BC, 2007 CAN

418

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS009516 Nine Mile Point, 16-May- ABMMC10978-10 KU873106 ND ND Sechelt, BC, 2008 CAN GWS010154 Tahsis, Flower 24-May- ABMMC6824-10 HM917201 ND ND Islet, Esperanza 2008 Channel, BC, CAN GWS010161 Tahsis, Flower 24-May- ABMMC6829-10 HM917206 ND ND Islet, Esperanza 2008 Channel, BC, CAN GWS010172 Tahsis, Flower 24-May- ABMMC6833-10 HM917210 ND ND Islet, Esperanza 2008 Channel, BC, CAN GWS010411 Savoie Rocks, 29-May- ABMMC6904-10 HM917272 ND ND Hornby Island, 2008 BC, CAN GWS019359 Bamfield, 31-May- ABMMC12685-10 HQ919308 ND ND Seapool Rock, 2010 BC, CAN

419

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS021018 Between Wiah 08-Jun- ABMMC14051-10 HQ919392 ND ND Point & Cape 2010 Edensaw (#2), NW of Masset, Haida Gwaii, BC, CAN GWS022096 Stillwater Cove, 20-May- ABMMC12259-10 JX034294 ND ND Pebble Beach, 2010 CA, USA GWS022127 Stillwater Cove, 20-May- ABMMC12288-10 HQ544199 ND ND Pebble Beach, 2010 CA, USA GWS022140 Stillwater Cove, 20-May- ABMMC12301-10 JX034305 ND ND Pebble Beach, 2010 CA, USA GWS022177 Stillwater Cove, 20-May- ABMMC12331-10 JX034282 ND ND Pebble Beach, 2010 CA, USA GWS022180 Stillwater Cove, 20-May- ABMMC12334-10 JX034286 ND ND Pebble Beach, 2010 CA, USA GWS027009 Bamfield, 28-Jun- ABMMC15679-11 KM254828 ND ND opening of 2011 Grappler Inlet, BC, CAN

420

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS027016 Bamfield, 28-Jun- ABMMC15681-11 KM254785 ND ND opening of 2011 Grappler Inlet, BC, CAN GWS027327 Beaver Pt., Salt 02-May- ABMMC15127-11 JX034306 ND ND Spring I., BC, 2011 CAN GWS027342 Beaver Pt., Salt 02-May- ABMMC15130-11 JX034307 ND ND Spring I., BC, 2011 CAN GWS027385 Victoria Shoal, 03-May- ABMMC15133-11 JX034283 ND ND Salt Spring I., 2011 BC, CAN GWS027403 Panther Pt., 03-May- ABMMC15136-11 JX034290 ND ND Wallace I., BC, 2011 CAN GWS030811 Macrocystis bed 11-Jun- ABMMC17337-12 KM254429 ND ND at entrance to 2012 George Bay, Gwaii Haanas, BC, CAN GWS030872 Beach south of 12-Jun- ABMMC17341-12 KM254921 ND ND Kiju Point, 2012 Gwaii Haanas, BC, CAN

421

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS034834 Halkett Wall, 16-May- ABMMC17953-13 KU873114 ND ND Gambier Isl., 2013 BC, CAN GWS035637 Lagoon Inlet 14-Aug- ABMMC18372-13 KU873099 ND ND Tidal Rapids 2013 (inside), Haida Gwaii, BC, CAN GWS035643 Lagoon Inlet 14-Aug- ABMMC18378-13 KU873101 ND ND Tidal Rapids 2013 (inside), Haida Gwaii, BC, CAN Callophyllis KAM0006 Eastern 10-Jun- KAMCH020-13 KU873098 ND ND radula Kamchatka, 2013 Perestenko Avacha Gulf, Starichkov Island, KAM, RUS Callophyllis GWS0362402 Southern 1-Aug-1993 ABMMC20738-14 KU873108 ND ND rhynchocarpa Sakhalin, Sea of Ruprecht Okhotsk, RUS

422

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession Callophyllis GWS012627 Mazarredo 17-Jun- ABMMC3963-09 HM915313 ND ND variforma Islands, NW of 2009 Filloramo, Masset, Haida Selivanova & Gwaii, BC, G.W. Saunders CAN GWS012642 Mazarredo 17-Jun- ABMMC3977-09 HM915323 ND ND Islands, NW of 2009 Masset, Haida Gwaii, BC, CAN GWS012651 Mazarredo 17-Jun- ABMMC3984-09 HM915330 ND ND Islands, NW of 2009 Masset, Haida Gwaii, BC, CAN GWS019666 Bamfield, Scotts 03-Jun- ABMMC12976-10 HQ544564 ND ND Bay, BC, CAN 2010 GWS019671 Bamfield, Scotts 03-Jun- ABMMC12981-10 HQ544568 ND ND Bay, BC, CAN 2010 GWS019697 Bamfield, Scotts 03-Jun- ABMMC13006-10 HQ544588 ND ND Bay, BC, CAN 2010 GWS020789 Telephone 07-Jun- ABMMC13840-10 HQ545084 ND ND Point, 2010 Skidegate, Haida Gwaii, BC, CAN

423

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS020793 Telephone 07-Jun- ABMMC13844-10 HQ545088 ND ND Point, 2010 Skidegate, Haida Gwaii, BC, CAN GWS020798 Telephone 07-Jun- ABMMC13849-10 HQ545092 ND ND Point, 2010 Skidegate, Haida Gwaii, BC, CAN GWS020808 Telephone 07-Jun- ABMMC13859-10 HQ545099 ND ND Point, 2010 Skidegate, Haida Gwaii, BC, CAN GWS020824 Telephone 07-Jun- ABMMC13875-10 HQ545111 ND ND Point, 2010 Skidegate, Haida Gwaii, BC, CAN GWS020838 Telephone 07-Jun- ABMMC13889-10 HQ545123 ND ND Point, 2010 Skidegate, Haida Gwaii, BC, CAN

424

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS027010 Bamfield, 28-Jun- ABMMC15972-11 KM254365 ND ND opening of 2011 Grappler Inlet, BC, CAN GWS027300 Beaver Pt., Salt 02-May- ABMMC15125-11 JX034391 ND ND Spring I., BC, 2011 CAN GWS027305 Beaver Pt., Salt 02-May- ABMMC15214-11 JX034383 ND ND Spring I., BC, 2011 CAN GWS027322 Beaver Pt., Salt 02-May- ABMMC14993-11 JX034382 ND ND Spring I., BC, 2011 CAN GWS027330 Beaver Pt., Salt 02-May- ABMMC15128-11 JX034380 ND ND Spring I., BC, 2011 CAN GWS027344 Beaver Pt., Salt 02-May- ABMMC15215-11 JX034381 ND ND Spring I., BC, 2011 CAN GWS027368 Victoria Shoal, 03-May- ABMMC15188-11 JX034385 ND ND Salt Spring I., 2011 BC, CAN GWS027369 Victoria Shoal, 03-May- ABMMC15189-11 JX034392 ND ND Salt Spring I., 2011 BC, CAN

425

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS027376 Victoria Shoal, 03-May- ABMMC15123-11 JX034386 ND ND Salt Spring I., 2011 BC, CAN GWS027391 Panther Pt., 03-May- ABMMC15134-11 JX034384 ND ND Wallace I., BC, 2011 CAN GWS027412 Panther Pt., 03-May- ABMMC15205-11 JX034390 ND ND Wallace I., BC, 2011 CAN GWS027416 Panther Pt., 03-May- ABMMC15137-11 JX034389 ND ND Wallace I., BC, 2011 CAN GWS027443 South Galiano 04-May- ABMMC15139-11 JX034388 ND ND Wall, Galiano 2011 Is., BC, CAN GWS027450 South Galiano 04-May- ABMMC15140-11 JX034387 ND ND Wall, Galiano 2011 Is., BC, CAN GWS028181 Murchison Is., 07-Jul-2011 ABMMC15694-11 KM254683 ND ND Northwest Beach, Gwaii Haanas, BC, CAN GWS034806 South Wall 16-May- ABMMC17903-13 KU873107 ND ND Bowyer Isl., BC, 2013 CAN

426

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS035672 Burnaby 16-Aug- ABMMC18407-13 KU873110 ND ND Narrows (S 2013 opening, in Macrocystis bed), Gwaii Haanas, BC, CAN GWS035676 Burnaby 16-Aug- ABMMC18411-13 KU873102 ND ND Narrows (S 2013 opening, in Macrocystis bed), Gwaii Haanas, BC, CAN GWS035693 Burnaby 16-Aug- ABMMC18428-13 KU873103 ND ND Narrows (S 2013 opening, in Macrocystis bed), Gwaii Haanas, BC, CAN

427

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS035694 Burnaby 16-Aug- ABMMC18429-13 KU873105 ND ND Narrows (S 2013 opening, in Macrocystis bed), Gwaii Haanas, BC, CAN GWS035695 Burnaby 16-Aug- ABMMC18430-13 KU873109 ND ND Narrows (S 2013 opening, in Macrocystis bed), Gwaii Haanas, BC, CAN GWS035718 Burnaby 16-Aug- ABMMC18453-13 KU873111 ND ND Narrows (S 2013 opening, in Macrocystis bed), Gwaii Haanas, BC, CAN

428

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS035726 Burnaby 16-Aug- ABMMC18460-13 KU873113 ND ND Narrows (S 2013 opening, in Macrocystis bed), Gwaii Haanas, BC, CAN Pterosiphonia KAM0009 Eastern 10-Jun- KAMCH013-13 ND ND KU564490 binnata (Postels Kamchatka, 2013 & Ruprecht) Avacha Gulf, Falkenberg Starichkov Island, KAM, RUS GWS004966 Ridley Island 11-Jul-2006 ABMMC9386-10 ND ND KU5644533 (south of coal terminal), Prince Rupert, BC, CAN GWS030845 Southern Bay at 12-Jun- ABMMC17192-12 KU564325 ND KU564455 Benjamin Point, 2012 Gwaii Haanas, BC Pterosiphonia GWS002717 Pachena Beach, 04-Jun- ABMMC8976-10 HQ919602 ND ND robusta N.L. Bamfield, BC, 2005 Gardner CAN

429

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS002809 Bamfield, Dixon 06-Jun- ABMMC8989-10 HM918517 ND ND I., BC, CAN 2005

GWS002853 Bamfield, 07-Jun- ABMMC8996-10 KU564417 ND ND Wizard I., BC, 2005 CAN GWS002939 Bamfield, 09-Jun- ABMMC9008-10 HM918530 ND ND Seppings I., BC, 2005 CAN GWS003368 Pachena Beach, 12-Jun- ABMMC9080-10 KU564412 ND ND Bamfield, BC, 2006 CAN GWS003369 Pachena Beach, 12-Jun- ABMMC9081-10 ND ND KU8765613 Bamfield, BC, 2006 CAN GWS003976 Bamfield, 14-Jun- ABMMC9189-10 KU564347 ND ND Seppings I., BC, 2006 CAN GWS004815 Ridley Island 08-Jul-2006 ABMMC9359-10 KU564371 ND ND (south of coal terrminal), Prince Rupert, BC, CAN

430

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS004965 Ridley Island 11-Jul-2006 ABMMC9385-10 HQ919609 ND ND (south of coal terrminal), Prince Rupert, BC, CAN GWS004981 Ridley Island 11-Jul-2006 ABMMC9390-10 KU564400 ND ND (south of coal terrminal), Prince Rupert, BC, CAN GWS005084 Ridley Island 11-Jul-2006 ABMMC9408-10 HM918740 ND ND (north of grain terrminal), Prince Rupert, BC, CAN GWS005085 Ridley Island 11-Jul-2006 ABMMC9409-10 HM918741 ND ND (north of grain terrminal), Prince Rupert, BC, CAN GWS005166 Butze Rapids, 12-Jul-2006 ABMMC9414-10 KU564393 ND ND Prince Rupert, BC, CAN

431

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS006383 Otter Point, near 25-May- ABMMC6208-10 HM916762 ND ND Sooke, 2007 Vancouver Island, BC, CAN GWS006625 Tahsis, Island 30-May- ABMMC6270-10 HM916810 ND ND #40 on 2007 Esperenza Inlet Chart, BC, CAN GWS006689 Tahsis, Island 30-May- ABMMC6282-10 KU564376 ND ND #40 on 2007 Esperenza Inlet Chart, BC, CAN GWS006826 `Lands End` up 02-Jun- ABMMC6307-10 HM916839 ND ND the left side of 2007 Pachena Bay where it opens to the ocean, Bamfield, BC, CAN GWS008176 Bamfield, Scotts 03-Jun- ABMMC6567-10 HQ919565 ND ND Bay, BC, CAN 2007 GWS008205 Bamfield, 04-Jun- ABMMC6572-10 HM916998 ND ND Blowhole at 2007 Bradys Beach, BC, CAN

432

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS008358 Ridley Island 08-Jun- ABMMC5859-09 HM916524 ND ND (south of coal 2007 terrminal), Prince Rupert, BC, CAN GWS008360 Ridley Island 08-Jun- ABMMC5871-09 HM916532 ND ND (south of coal 2007 terrminal), Prince Rupert, BC, CAN GWS008436 Pier at Davis 12-Jun- ABMMC5907-09 KU564363 ND ND Bay Sunshine 2007 Coast, BC, CAN GWS009067 Bamfield, 23-Sep- ABMMC5903-09 HM916551 ND ND Bradys Beach, 2007 BC, CAN GWS009148 Waypoint #44 24-Sep- ABMMC5855-09 HM916521 ND ND from Land`s 2007 End to Pachena Beach, Bamfield, BC, CAN GWS009568 Halfway Beach, 17-May- ABMMC5914-09 HM916559 ND ND near Sechelt, 2008 BC, CAN

433

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS009629 Tahsis, Island 20-May- ABMMC5902-09 HM916550 ND ND #40 on 2008 Esperenza Inlet Chart, BC, CAN GWS010349 Point Holmes, 27-May- ABMMC5875-09 HM916533 ND ND Comox, BC, 2008 CAN GWS010879 Bamfield, 06-Jun- ABMMC5898-09 HM916547 ND ND Blowhole at 2008 Bradys Beach, BC, CAN GWS012517 Park at end of 15-Jun- ABMMC3912-09 KU564362 ND ND Honna Road, 2009 Queen Charlotte City, Haida Gwaii, BC, CAN GWS012520 Park at end of 15-Jun- ABMMC3915-09 KU564415 ND ND Honna Road, 2009 Queen Charlotte City, Haida Gwaii, BC, CAN

434

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS012770 Burnaby Island 19-Jun- ABMMC4143-09 KU564402 ND ND near Saw Reef, 2009 Gwaii Haanas, BC, CAN GWS012777 Burnaby Island 19-Jun- ABMMC4133-09 HM915450 ND ND near Saw Reef, 2009 Gwaii Haanas, BC, CAN GWS012998 Scudder Point, 20-Jun- ABMMC4185-09 KU564403 ND ND Burnaby Island, 2009 Gwaii Haanas, BC, CAN GWS013003 Scudder Point, 20-Jun- ABMMC4155-09 HM915469 ND ND Burnaby Island, 2009 Gwaii Haanas, BC, CAN GWS013096 Ramsey Island 21-Jun- ABMMC4215-09 HM915513 ND ND (point adjacent 2009 Kloo Rock), Gwaii Haanas, BC, CAN

435

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS013236 Hot Spring 21-Jun- ABMMC4326-09 KU564429 ND ND Island at 2009 Watchmen Station (in hot water seepage), Gwaii Haanas, BC, CAN GWS019713 Alder Island, 11-Jun- ABMMC13037-10 KU564350 ND ND Gwaii Haanas, 2010 BC, CAN GWS019791 Alder Island, 11-Jun- ABMMC13094-10 KU564318 ND ND Gwaii Haanas, 2010 BC, CAN GWS019971 Warden Station 12-Jun- ABMMC13222-10 KU564357 ND ND Huxley Island, 2010 Gwaii Haanas, BC, CAN GWS020085 Saw Reef, 13-Jun- ABMMC13317-10 KU564381 ND ND Gwaii Haanas, 2010 BC, CAN GWS020139 Saw Reef, 13-Jun- ABMMC13358-10 HQ544771 ND ND Gwaii Haanas, 2010 BC, CAN

436

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS020216 East Copper 14-Jun- ABMMC13409-10 KU564398 ND ND Island (easterly 2010 point), Gwaii Haanas, BC, CAN GWS020251 East Copper 14-Jun- ABMMC13442-10 HQ544823 ND ND Island (easterly 2010 point), Gwaii Haanas, BC, CAN GWS020413 Hot Spring 15-Jun- ABMMC13564-10 HQ544900 ND ND Island (east 2010 `back` side), Gwaii Haanas, BC, CAN GWS020523 Hot Spring 15-Jun- ABMMC13643-10 HQ544957 ND ND Island (north 2010 side), Gwaii Haanas, BC, CAN GWS020524 Hot Spring 15-Jun- ABMMC13644-10 HQ544958 ND ND Island (north 2010 side), Gwaii Haanas, BC, CAN

437

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS020619 North Beach 17-Jun- ABMMC13723-10 HQ545015 ND ND (near Naval 2010 Station), Haida Gwaii, BC, CAN GWS020628 North Beach 17-Jun- ABMMC13730-10 KU564396 ND ND (near Naval 2010 Station), Haida Gwaii, BC, CAN GWS021094 West of Juskatla 09-Jun- ABMMC14119-10 HQ545302 ND ND Narrows, Masset 2010 Inlet, Haida Gwaii, BC, CAN GWS021155 Warden Station 10-Jun- ABMMC14170-10 KU564422 ND ND Huxley Island, 2010 Gwaii Haanas, BC, CAN GWS021159 Warden Station 10-Jun- ABMMC14173-10 HQ919432 ND ND Huxley Island, 2010 Gwaii Haanas, BC, CAN

438

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS021302 Pigeon Point 15-May- ABMMC11359-10 KM254385 ND ND Lighthouse, San 2010 Mateo, CA, USA GWS021377 Pigeon Point 15-May- ABMMC11421-10 KM254727 ND ND Lighthouse, San 2010 Mateo, CA, USA GWS022424 600m N/W of 23-May- ABMMC14212-10 HQ545315 ND ND Roberts Creek 2010 Rivermouth, Roberts Creek, Sunshine Coast, BC, CAN GWS022431 600m N/W of 23-May- ABMMC14216-10 HQ545318 ND KU564466 Roberts Creek 2010 Rivermouth, Roberts Creek, Sunshine Coast, BC, CAN GWS022432 600m N/W of 23-May- ABMMC14217-10 HQ545319 ND KU564477 Roberts Creek 2010 Rivermouth, Roberts Creek, Sunshine Coast, BC, CAN

439

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS022444 600m N/W of 23-May- ABMMC14227-10 HQ545324 ND ND Roberts Creek 2010 Rivermouth, Roberts Creek, Sunshine Coast, BC, CAN GWS027475 End of Mount 05-May- ABMMC15055-11 KU564368 ND ND Taum Road, Salt 2011 Spring, BC, CAN GWS027525 Walkers Hook 06-May- ABMMC15060-11 KU564379 ND ND (rocky point), 2011 Salt Spring, BC, CAN GWS028693 East side of 11-Jul-2011 ABMMC15573-11 KU564327 ND ND Powrivco Bay, Gwaii Haanas, BC, CAN GWS030308 Blackburn Point, 05-Jun- ABMMC17164-12 KU564315 ND ND Gwaii Haanas, 2012 BC, CAN GWS030345 Blackburn Point, 05-Jun- ABMMC17167-12 KU564339 ND ND Gwaii Haanas, 2012 BC, CAN

440

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS030346 Blackburn Point, 05-Jun- ABMMC17168-12 KU564360 ND ND Gwaii Haanas, 2012 BC, CAN GWS030575 Trevan Rock 08-Jun- ABMMC17179-12 KU564335 ND ND (Rose Harbour), 2012 Gwaii Haanas, BC, CAN GWS031334 Ferry Terminal, 03-Jun- ABMMC17222-12 KU564338 ND ND Skidegate, 2012 Haida Gwaii, BC, CAN GWS031358 Gordon Islands 04-Jun- ABMMC17159-12 KU564332 ND ND (East), Gwaii 2012 Haanas, BC, CAN GWS031359 Gordon Islands 04-Jun- ABMMC17160-12 KU564333 ND ND (East), Gwaii 2012 Haanas, BC, CAN GWS034830 Halkett Wall, 16-May- ABMMC17918-13 KU564341 ND ND Gambier Isl., 2013 BC, CAN GWS034863 Worlcombe Isl. 17-May- ABMMC17919-13 KU564423 ND ND (east point), BC, 2013 CAN

441

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS034871 Worlcombe Isl. 17-May- ABMMC17920-13 KU564387 ND ND (east point), BC, 2013 CAN GWS036734 West Point, 14-May- ABMMC19666-14 KU564416 ND ND Deception Pass, 2014 WA, USA GWS038504 South side of 10-Jul-2014 ABMMC19888-14 KU564410 ND ND Bischoff Main Is., Gwaii Haanas, BC, CAN GWS038633 Murchison 14-Jul-2014 ABMMC20012-14 KU564395 ND ND Island Lagoon, Gwaii Haanas, BC, CAN GWS038635 Murchison 14-Jul-2014 ABMMC20014-14 KU564321 ND ND Island Lagoon, Gwaii Haanas, BC, CAN Sparlingia GWS000581 Bamfield, Dixon 06-May- ABMMC5637-09 HM916393 ND ND pertusa (Postels I., BC, CAN 1999 & Ruprecht) G.W. Saunders, I.M. Strachan & Kraft

442

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS000581 Bamfield, Dixon 06-May- ARCHD017-11 JN021133 JN021189 ND H I., BC, CAN 1999 GWS001123 San Juan I., 16-May- ARCHD018-11 ND JN021188 ND H Cattle Pt., WA, 2001 USA GWS002227 Browning Wall, 18-Jun- ABMMC5707-09 HQ545351 ND ND north of Port 2004 Hardy, BC, CAN GWS002227 Browning Wall, 18-Jun- ARCHD019-11 JN021131 JN021187 ND H north of Port 2004 Hardy, BC, CAN GWS002876 Bamfield, 08-Jun- ABMMC9001-10 HM918524 ND ND `Sparlingia Pt.`, 2005 Bradys Beach, BC, CAN GWS002876 Bamfield, 08-Jun- ARCHD020-11 JN021130 JN021186 ND H `Sparlingia Pt.`, 2005 Bradys Beach, BC, CAN GWS002886 Bamfield, 08-Jun- ABMMC2522-08 KU687747 ND ND `Sparlingia Pt.`, 2005 Bradys Beach, BC, CAN

443

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS004009 Bamfield, 15-Jun- ABMMC5688-09 KU687527 ND ND `Sparlingia Pt.`, 2006 Bradys Beach, BC, CAN GWS004009 Bamfield, 15-Jun- ARCHD021-11 JN021129 JN021185 ND H `Sparlingia Pt.`, 2006 Bradys Beach, BC, CAN GWS004010 Bamfield, 15-Jun- ABMMC2539-08 KU687754 ND ND `Sparlingia Pt.`, 2006 Bradys Beach, BC, CAN GWS004150 Otter Point, near 20-Jun- ABMMC2541-08 KU687512 ND ND Sooke, 2006 Vancouver Island, BC, CAN GWS004156 Otter Point, near 20-Jun- ABMMC5653-09 HM916402 ND ND Sooke, 2006 Vancouver Island, BC, CAN GWS004234 Saxe Pt., 21-Jun- ABMMC5665-09 HM916409 ND ND Victoria, BC, 2006 CAN

444

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS004370 Botanical 26-Jun- ABMMC5689-09 HM916420 ND ND Beach, Port 2006 Renfrew, Vancouver I., BC, CAN GWS004400 Botanical 26-Jun- ABMMC5701-09 HQ919542 ND ND Beach, Port 2006 Renfrew, Vancouver I., BC, CAN GWS004521 Bamfield, 27-Jun- ABMMC2085-08 KU687498 ND ND Seapool Rock, 2006 BC, CAN GWS004754 Browning Wall, 04-Jul-2006 ABMMC2092-08 KU687779 ND ND north of Port Hardy, BC, CAN GWS004769 Browning Wall, 04-Jul-2006 ABMMC1464-07 KU687510 ND ND north of Port Hardy, BC, CAN GWS004865 Tree Knob 09-Jul-2006 ABMMC2098-08 KU687767 ND ND Islands, Prince Rupert, BC, CAN

445

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS004929 Stenhouse Reef, 09-Jul-2006 ABMMC5655-09 HM916403 ND ND Prince Rupert, BC, CAN GWS005042 Ridley Island 11-Jul-2006 ABMMC2100-08 KU687780 ND ND (south of coal terrminal), Prince Rupert, BC, CAN GWS005043 Ridley Island 11-Jul-2006 ABMMC5715-09 HM916433 ND ND (south of coal terrminal), Prince Rupert, BC, CAN GWS005045 Ridley Island 11-Jul-2006 ABMMC2101-08 KU687652 ND ND (south of coal terrminal), Prince Rupert, BC, CAN GWS005156 Butze Rapids, 12-Jul-2006 ABMMC5680-09 HM916415 ND ND Prince Rupert, BC, CAN GWS006340 Pier near statue 24-May- ABMMC5704-09 HM916428 ND ND of diver, Sidney, 2007 BC, CAN GWS006414 Spring Bay, BC, 26-May- ABMMC2125-08 KU687704 ND ND CAN 2007

446

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS008423 Martin Rd., 12-Jun- ABMMC2149-08 KU687729 ND ND Beaver I., near 2007 Sechelt, Sunshine Coast, BC, CAN GWS008427 Martin Rd., 12-Jun- ABMMC2150-08 KU687749 ND ND Beaver I., near 2007 Sechelt, Sunshine Coast, BC, CAN GWS008449 Tuwanek, near 13-Jun- ABMMC2151-08 KU687488 ND ND Sechelt, BC, 2007 CAN GWS008450 Tuwanek, near 13-Jun- ABMMC6618-10 HM917032 ND ND Sechelt, BC, 2007 CAN GWS008451 Tuwanek, near 13-Jun- ABMMC2152-08 KU687701 ND ND Sechelt, BC, 2007 CAN GWS008483 Tuwanek, near 13-Jun- ABMMC1822-07 KU687795 ND ND Sechelt, BC, 2007 CAN GWS008633 Point Holmes, 15-Jun- ABMMC2155-08 KU687590 ND ND Comox, BC, 2007 CAN

447

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS008672 Bamfield, 16-Jun- ABMMC2159-08 KU687550 ND ND Seapool Rock, 2007 BC, CAN GWS009458 Tuwanek, near 15-May- ABMMC3033-08 KU687630 ND ND Sechelt, BC, 2008 CAN GWS009459 Tuwanek, near 15-May- ABMMC5662-09 HM916408 ND ND Sechelt, BC, 2008 CAN GWS009476 Tuwanek, near 15-May- ABMMC5698-09 HQ919541 ND ND Sechelt, BC, 2008 CAN GWS009518 Nine Mile Point, 16-May- ABMMC6071-10 HQ545359 ND ND Sechelt, BC, 2008 CAN GWS009542 Nine Mile Point, 16-May- ABMMC6083-10 HM916685 ND ND Sechelt, BC, 2008 CAN GWS010166 Tahsis, Flower 24-May- ABMMC6076-10 HM916679 ND ND Islet, Esperanza 2008 Channel, BC, CAN GWS010431 Bamfield, 01-Jun- ABMMC6101-10 HM916698 ND ND Seapool Rock, 2008 BC, CAN

448

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS010504 Bamfield, 02-Jun- ABMMC6031-10 HM916645 ND ND Edward King 2008 Island, BC, CAN GWS010507 Bamfield, 02-Jun- ABMMC6043-10 HM916652 ND ND Edward King 2008 Island, BC, CAN GWS010799 Bamfield, 05-Jun- ABMMC6104-10 HM916700 ND ND Seppings I., BC, 2008 CAN GWS012643 Mazarredo 17-Jun- ABMMC3978-09 HM915324 ND ND Islands, NW of 2009 Masset, Haida Gwaii, BC, CAN GWS013007 Scudder Point, 20-Jun- ABMMC4199-09 HM915501 ND ND Burnaby Island, 2009 Gwaii Haanas, BC, CAN GWS013624 Warden Station 25-Jun- ABMMC5533-09 HM916307 ND ND Huxley Island, 2009 Gwaii Haanas, BC, CAN

449

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS019484 Bamfield, 01-Jun- ABMMC12806-10 HQ544425 ND ND Taylor Islet, BC, 2010 CAN GWS019635 Bamfield, 03-Jun- ABMMC12947-10 HQ544545 ND ND opening of 2010 Grappler Inlet, BC, CAN GWS019824 Alder Island, 11-Jun- ABMMC13122-10 HQ544639 ND ND Gwaii Haanas, 2010 BC, CAN GWS020030 Saw Reef, 13-Jun- ABMMC13265-10 HQ544720 ND ND Gwaii Haanas, 2010 BC, CAN GWS020201 East Copper 14-Jun- ABMMC13394-10 HQ544790 ND ND Island (easterly 2010 point), Gwaii Haanas, BC, CAN GWS020235 East Copper 14-Jun- ABMMC13426-10 HQ544810 ND ND Island (easterly 2010 point), Gwaii Haanas, BC, CAN

450

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS020618 North Beach 17-Jun- ABMMC13722-10 HQ545014 ND ND (near Naval 2010 Station), Haida Gwaii, BC, CAN GWS020721 Kwuna Island, 06-Jun- ABMMC13773-10 HQ545040 ND ND Haida Gwaii, 2010 BC, CAN GWS020763 Kwuna Island, 06-Jun- ABMMC13814-10 HQ545069 ND ND Haida Gwaii, 2010 BC, CAN GWS020845 Indian Head, 07-Jun- ABMMC13895-10 HQ545126 KU68791 ND Skidegate, 2010 2 Haida Gwaii, BC, CAN GWS020913 Indian Head, 07-Jun- ABMMC13962-10 KU687777 ND ND Skidegate, 2010 Haida Gwaii, BC, CAN GWS021051 Juskatla 09-Jun- ABMMC14080-10 HQ545274 KU68789 ND Narrows, Masset 2010 5 Inlet, Haida Gwaii, BC, CAN

451

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS021070 West of Juskatla 09-Jun- ABMMC14098-10 HQ545284 ND ND Narrows, Masset 2010 Inlet, Haida Gwaii, BC, CAN GWS021071 West of Juskatla 09-Jun- ABMMC14099-10 HQ545285 ND ND Narrows, Masset 2010 Inlet, Haida Gwaii, BC, CAN GWS021072 West of Juskatla 09-Jun- ABMMC14100-10 HQ545286 KU68789 ND Narrows, Masset 2010 1 Inlet, Haida Gwaii, BC, CAN GWS021084 West of Juskatla 09-Jun- ABMMC14111-10 HQ545296 ND ND Narrows, Masset 2010 Inlet, Haida Gwaii, BC, CAN GWS021127 Cowley Islands, 09-Jun- ABMMC14146-10 KU687658 ND ND Masset Inlet, 2010 Haida Gwaii, BC, CAN

452

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS027549 Beddis Beach 06-May- ABMMC15223-11 KU687628 ND ND (left point & 2011 seagrass bed), Salt Spring, BC, CAN GWS027905 Lost Islands, SE 06-Jul-2011 ABMMC15990-11 KU687711 ND ND wall, Gwaii Haanas, BC, CAN GWS027937 Lost Islands, SE 06-Jul-2011 ABMMC16134-11 KU687718 KU68791 ND wall, Gwaii 4 Haanas, BC, CAN GWS028509 Ramsey Island 10-Jul-2011 ABMMC15452-11 KU687646 ND ND (N Bay w anchorage), Gwaii Haanas, BC, CAN GWS028619 Point S of 11-Jul-2011 ABMMC16136-11 KU687556 ND ND Dodge Point, Gwaii Haanas, BC, CAN GWS028620 Point S of 11-Jul-2011 ABMMC16137-11 KU687537 ND ND Dodge Point, Gwaii Haanas, BC, CAN

453

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS030590 Louscoone 08-Jun- ABMMC17596-12 KU687658 ND ND Point, Gwaii 2012 Haanas, BC, CAN GWS030715 East corner of 10-Jun- ABMMC17597-12 KU687547 ND ND entrance to Slim 2012 Inlet, Gwaii Haanas, BC, CAN GWS030720 East corner of 10-Jun- ABMMC16994-12 KU687775 ND ND entrance to Slim 2012 Inlet, Gwaii Haanas, BC, CAN GWS030795 Macrocystis bed 11-Jun- ABMMC17598-12 KU687544 ND ND at entrance to 2012 George Bay, Gwaii Haanas, BC, CAN GWS030798 Macrocystis bed 11-Jun- ABMMC17599-12 KU687545 ND ND at entrance to 2012 George Bay, Gwaii Haanas, BC, CAN

454

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS034814 South Wall 16-May- ABMMC18035-13 KU687642 ND ND Bowyer Isl., BC, 2013 CAN GWS034833 Halkett Wall, 16-May- ABMMC18036-13 KU687518 ND ND Gambier Isl., 2013 BC, CAN GWS034885 Bird Islet, 18-May- ABMMC17997-13 KU687541 ND ND Horseshoe Bay, 2013 BC, CAN GWS035389 Marble Island 24-Aug- ABMMC18315-13 KU687558 XXXXX ND (NW reef below 2013 `Killer-Whale- Fin Rock`), Haida Gwaii, BC, CAN GWS035391 Marble Island 24-Aug- ABMMC18317-13 KU687670 ND ND (NW reef below 2013 `Killer-Whale- Fin Rock`), Haida Gwaii, BC, CAN GWS035773 SGang Gwaay 18-Aug- ABMMC18505-13 KU687678 ND ND (rocks to NW), 2013 Gwaii Haanas, BC, CAN

455

COI-5P ITS rbcL Collection GenBank GenBank GenBank Species Sample ID Location1 Date BOLD ID Accession Accession Accession GWS036718 Rosario Beach, 13-May- ABMMC19651-14 KU687740 ND ND WA, USA 2014 GWS036752 Rosario Beach 14-May- ABMMC19684-14 KU687557 ND ND Point, WA, USA 2014 GWS038909 Tartu Point (E 11-Jul-2014 ABMMC20033-14 KU687499 ND ND corner), Rennell Sound, Haida Gwaii, BC, CAN GWS038969 Cape Knox, 13-Jul-2014 ABMMC20093-14 KU687641 ND ND Haida Gwaii, BC, CAN KAM0012 Eastern 10-Jun- KAMCH026-13 KU873100 XXXXX ND Kamchatka, 2013 Avacha Gulf, Starichkov Island, KAM, RUS

1 Province/State and Country abbreviations: “BC”= British Columbia; “CA”= California; “WA”= Washington; “CAN”= Canada; “USA”= United States of America.

2 Material for specimen GWS036240 was subsampled from press MICH 644907, which is deposited in the University of Michigan Herbarium (MICH).

3For samples GWS003369 and GWS004966 approximately 800 bp from the 3P end of the rbcL was sequenced rather than full-length

456 rbcL.

Curriculum Vitae

Candidate’s full name: Gina Victoria Filloramo

Universities attended: B.Sc., Trinity College, Hartford, Connecticut, USA, 2010

Publications:

Filloramo, G.V., Saunders, G.W. 2015. A re-examination of the genus Leptofauchea (Faucheaceae, Rhodymeniales) with clarification of species in Australia and the northwest Pacific. Phycologia, 54: 375-384.

Filloramo, G.V., Saunders, G.W. 2016. Application of multigene phylogenetics and site- stripping to resolve intraordinal relationships in the Rhodymeniales (Rhodophyta). Journal of Phycology, 52: 339-335.

Filloramo, G.V., Saunders, G.W. 2016. Molecular-assisted alpha taxonomy of the genus Rhodymenia (Rhodymeniaceae, Rhodymeniales) from Australia reveals overlooked species diversity. European Journal of Phycology, http://dx.doi.org/10.1080/09670262.2016.1170886.

Filloramo, G.V., Saunders, G.W. Assessment of the order Rhodymeniales (Rhodophyta) from British Columbia using an integrative taxonomic approach reveals overlooked and cryptic species diversity. in preparation for Botany.

Filloramo, G.V., Savoie, A.M., Selivanova, O.N., Wynne, M.J., Saunders, G.W. 2016. Key Kamchatkan collections provide new taxonomic and distributional insights for reportedly pan-North Pacific species of Rhodymeniophycidae (Rhodophyta). Phycologia, accepted May 2016.

Fleming, R.J., Hori, K., Sen, A., Filloramo, G.V., Langer, J.M., Obar, R.A., Artavanis- Tsakonas, S., Maharaj-Best, A.C. 2013. An extracellular region of Serrate is essential for ligand-induced cis-inhibition of Notch signaling. Development, 140: 2039-2049.

Saunders, G.W., Filloramo, G., Dixon, K., Le Gall, L., Maggs, C.A., Kraft, G.T. Multigene analyses resolve early diverging lineages in the Rhodymeniophycidae (Florideophyceae, Rhodophyta). Journal of Phycology, doi:10.1111/jpy.12426.

Conference Presentations:

Filloramo, G.V., Saunders, G.W. 2016. Molecular assisted alpha taxonomy of the order Rhodymeniales (Rhodophyta) from British Columbia reveals overlooked and cryptic species diversity. Northeast Algal Symposium, Westfield, Massachusetts. (International, poster presentation).

Filloramo, G.V., Saunders, G.W. 2015. DNA barcode assessment of the genus Rhodymenia (Rhodymeniaceae, Rhodymeniales) from Australia reveals cryptic and overlooked species diversity. Annual Meeting of the Phycological Society of America, Philadelphia, Pennsylvania. (International, oral presentation).

Filloramo, G.V., Saunders, G.W. 2015. DNA barcode assessment of the genus Rhodymenia (Rhodymeniaceae, Rhodymeniales) from Australia reveals cryptic and overlooked species diversity. Northeast Algal Symposium, Syracuse, New York. (International, oral presentation).

Filloramo, G.V., Saunders, G.W. 2014. Using multigene phylogenetics and novel reconstruction techniques to improve suprageneric resolution in Rhodymeniales. Joint Aquatic Sciences Meeting, Portland, Oregon. (International, oral presentation).

Filloramo, G.V., Saunders, G.W. 2014. Using multigene phylogenetics and novel reconstruction techniques to improve suprageneric resolution in Rhodymeniales. Northeast Algal Symposium, Newport, Rhode Island. (International, oral presentation).

Filloramo, G.V., Saunders, G.W. 2013. Assessment of the red algal order Rhodymeniales using a multigene phylogenetic approach. Northeast Algal Symposium, Mystic, Connecticut. (International, oral presentation).

Filloramo, G.V., Saunders, G.W. 2012. Assessment of cryptic Rhodymenia spp. (Rhodymeniaceae, Rhodophyta) in British Columbia, Canada: an integrative taxonomic approach. Annual Meeting of the Phycological Society of America, Charleston, South Carolina. (International, oral presentation).

Filloramo, G.V., Saunders, G.W. 2012. Assessment of cryptic Rhodymenia spp. (Rhodymeniaceae, Rhodophyta) in British Columbia, Canada: an integrative taxonomic approach. Northeast Algal Symposium, Schoodic Point, Maine. (International, oral presentation).

Filloramo, G.V., Saunders, G.W. 2011. Establishment of species diversity of the Rhodymeniales (Rhodophyta) in British Columbia using molecular and morphological techniques. Northeast Algal Symposium, Woods Hole, Massachusetts. (International, poster presentation).

Filloramo, G.V., Schneider, C.W. 2010. Morphological assessment of the genus Gayliella. Northeast Algal Symposium, Bristol, Rhode Island. (International, poster presentation).